Cell factory for production of sulfurol

Abstract

The present disclosure relates to a genetically modified host cell having improved production of sulfurol and / or sulfurol precursors, wherein the host cell expresses one or more heterologous thiaminase I, thiaminase II and / or phosphatase enzymes and / or one or more heterologous thiamine pathway enzymes converting thiamine, 5-(2-hydroxyethyl)-4-methyl-1,3-thiazole-2-carboxylic phosphate acid (cTHZ-P), 5-(2-hydroxyethyl)-4-methyl-1,3-thiazole-2-carboxylic acid (cTHZ) and / or THZ-P to sulfurol and / or 5-(2-hydroxyethyl)-4-methyl-1,3-thiazole-2-carboxylic acid (cTHZ), whereby the production of the sulfurol in the genetically modified host cell is improved compared to an unmodified parent host cell. Chemical methods for production of sulfurol are also provided.

Description

Case Ref. P180WO IPTector® Cell factory for production of sulfurol Technical Field

[0001] The present disclosure relates to genetically modified host cells having increased production of sulfurol through providing for increased production of thiamine and carboxy sulfurol phosphate, to genetic constructs for expression of such mutants; to cultures of the genetically modified host cells and its use to produce the said products; to fermentation liquids comprising the said products resulting from such production; to compositions comprising the fermentation liquid; to dietary, flavour or pharmaceutical preparations made from such compositions and to the uses of such compositions and preparations. Background

[0002] Sulfurol or thiamine thiazole (THZ) is an aromatic, sulfurous compound that can be used as an additive to enhance meaty or milky flavors in foodstuffs. Biosynthetic pathways for the de novo biosynthesis of sulfurol are unknown, but bacteria can naturally produce a THZ related compound that has a carboxylic and phosphate group called carboxy-thiazole phosphate (5-(2-hydroxyethyl)-4- methyl-1,3-thiazole-2-carboxylic phosphate acid (cTHZ)-P), which is part of the biosynthetic pathway of thiamine or vitamin B1. This compound, however, has different organoleptic properties than sulfurol.

[0003] The use of microorganism-based cell factories is a potential route for the biosynthetic production of B vitamins and derivates such as sulfurol (Acevedo-Rocha, et al.2019). The advantages of recombinant microorganisms such as Escherichia coli as a microbial cell factory for the production of bio-products are widely recognized due to the fact that: (i) E. coli has unparalleled fast growth kinetics; with a doubling time of about 20 minutes when cultivated in glucose-salts media and under optimal environmental conditions, (ii) it easily achieves a high cell density; where the theoretical density limit of an E. coli liquid culture is estimated to be about 200 g dry cell weight / L or roughly 1X1013viable bacteria / mL. Additionally, there are many molecular tools and protocols at hand for genetic optimization of E. coli; as well as it being an organism that is amenable to the expression of heterologous proteins; both of which may be essential for obtaining high-level production of desired bioproducts. Summary

[0004] Herein, the present inventors have engineered production strains for the biosynthesis ofCase Ref. P180WO IPTector® sulfurol, or the precursors of sulfurol such as cTHZ-P, THZ-P, cTHZ and thiamine, which can be converted to sulfurol in subsequent chemical or enzymatic step(s). The production of sulfurol via the thiamine route requires not only a thiaminase that is active and specific in the host cell for converting thiamine into sulfurol, but also a cell factory that can efficiently produce thiamine at high levels.

[0005] The de novo biosynthesis of thiamine is a highly complex and regulated process present in microbes and plants. It starts with the biosynthesis of the precursors 5-(2-hydroxyethyl)-4-methyl-1,3- thiazole-2-carboxylic phosphate acid (cTHZ-P) and hydroxymethylpyrimidine diphosphate (HMP-PP), which are combined to form thiamine-mono-phosphate (TMP), which in turn can be dephosphorylated to thiamine by a phosphatase to thiamine (THI) or phosphorylated to thiamine di- phosphate (TPP) by a kinase to form an enzymatic cofactor. In a second enzymatic step, a thiaminase II converts THI into sulfurol. Both technical problems are solved in the current invention.

[0006] The first technical challenge is to generate thiamine and its precursors: the thiazole moiety and the pyridine moiety (HMP). The most complex part is the thiazole moiety, as it involves the action of at least 7 genes (thiF, thiS, thiG, thiI, thiO_BSU or thiH, iscS and dxs) to generate 5-(2-hydroxyethyl)-4- methyl-1,3-thiazole-2-carboxylic phosphate acid (cTHZ)-P. On the other hand, hydroxymethylpyrimidine (HMP) can be obtained from a commercial source, and it can be converted in vivo to HMP-P and HMP-PP (hydroxymethylpyrimidine phosphate and diphosphate, respectively) by thiD (hydroxymethylpyrimidine / phosphomethyl pyrimidine kinase). ThiE (thiamine-phosphate synthase) condenses both HMP-PP and 5-(2-hydroxyethyl)-4-methyl-1,3-thiazole-2-carboxylic phosphate acid (cTHZ-P) to form thiamine monophosphate (TMP), which is dephosphorylated by Arabidopsis thaliana phosphatase TH2_ATH to produce thiamine (Figure 1).

[0007] In control strain BS07312, the genes above are over-expressed from a plasmid (thiF, thiS, thiG, thiO, thiI, thiE) or chromosome (thiD, thiE, phosphatase) to produce 30 mg / L thiamine in small scale fed-batch cultivations (Figure 2).

[0008] The second technical challenge is to find a suitable thiaminase that can convert thiamine into sulfurol. Such enzymes are part of the so-called thiamine salvage pathway present in only a limited number of bacterial species and eukaryotic organisms. For example, Thiaminase I from Paenibacillus thiamineolyticus (Bacillus thiamineolyticus) has been expressed previously in E. coli, however, we were unable to see sulfurol production in our experiments. Thus, we did genome mining, cloned, and tested 8 additional thiaminases from different species: Cyanothece, Megasphaera elsdenii DSM 20460, Naegleria gruberi, Burkholderia glumae BGR1, Haliangium ochraceum DSM14365, Oleidesulfovibrio alaskensis G20, Stigmatella aurantiaca DW4 and Acinetobacter nectaris. Only 3 out of 9 thiaminases did produce 2-5 mg / L THZ. The most active thiaminase is from strain BS07309, followed by BS07310 and BS07329. Thiaminases are enzymes that are prone to substrate inhibitionCase Ref. P180WO IPTector® and the nucleophile needed for catalysis is unknown in vivo.

[0009] In addition, the enzyme must be specific for thiamine and not TMP and TPP, otherwise the enzyme activity would be toxic to the host. Thus, these 3 challenges need to be overcome for optimal performance. Further over-expression and engineering of the thiaminase could improve titers. Lastly, a de novo pathway that can produced THZ directly from 5-(2-hydroxyethyl)-4-methyl-1,3-thiazole-2- carboxylic phosphate acid (cTHZ-P) would be a more elegant approach by combining the over- expression of a phosphatase and a decarboxylase in 2 different configurations (Figure 1). Interestingly, while Begley et al. reported the use of alkaline phosphatase from calf intestine to remove the phosphate group of 5-(2-hydroxyethyl)-4-methyl-1,3-thiazole-2-carboxylic phosphate acid (cTHZ-P) to general 5-(2-hydroxyethyl)-4-methyl-1,3-thiazole-2-carboxylic acid (cTHZ) in vitro, Leys et al. reported that decarboxylases are very versatile enzymes with a large substrate specificity that could be used for many applications in biocatalysis. For example, pyrrole-2-carboxylate decarboxylase can accept heterocycles, a compound structurally related to THZ. Protein engineering could help tailor the binding pocket of such enzymes to convert 5-(2-hydroxyethyl)-4-methyl-1,3-thiazole-2-carboxylic acid (cTHZ) into THZ.

[0010] In the THZ branch, there are 3 sub-branches that form the thiazole ring. The first, second and third sub-branches, respectively, encompass a sugar backbone coming from pyruvate and glyceraldehyde 3-phosphate catalyzed by dxs, from a sulfur transfer step coming from cysteine catalyzed by IscS, ThiI, ThiF, ThiS, and from dehydroglycine (DHG) that can be generated from tyrosine by ThiH or glycine by B. subtilis ThiO. These 3 components are combined by the enzyme ThiG, leading to the production of carboxy-thiazole phosphate (5-(2-hydroxyethyl)-4-methyl-1,3-thiazole-2- carboxylic phosphate acid (cTHZ-P)). Finally, ThiE joins the pyrimidine (HMP-PP) and thiazole (5-(2- hydroxyethyl)-4-methyl-1,3-thiazole-2-carboxylic phosphate acid (cTHZ-P)) moieties to generate thiamine monophosphate (TMP), which can be dephosphorylated by Arabidopsis thaliana phosphatase to form thiamine (Figure 1).

[0011] Accordingly, in a first aspect, provided for herein is a genetically modified host cell having improved production of 5-(2-hydroxyethyl)-4-methyl-1,3-thiazole-2-carboxylic phosphate acid (cTHZ- P), wherein the host cell expresses one or more heterologous ThiO enzymes converting glycine into dehydroglycine (DHG) in the host cell, whereby the production of the thiamine and / or cTHZ in the genetically modified host cell is improved compared to an unmodified parent host cell.

[0012] In a further aspect, provided for herein is a genetically modified host cell having improved production of thiamine and / or cTHZ, wherein the host cell expresses one or more native or heterologous ThiI enzymes catalyzing the transfer of sulfur from IscS to the sulfur carrier protein ThiS in the host cell, whereby the production of the thiamine and / or cTHZ in the genetically modified hostCase Ref. P180WO IPTector® cell is improved compared to an unmodified parent host cell.

[0013] In a further aspect, provided for herein is a method for producing sulfurol comprising: a) culturing the cell culture at conditions allowing the host cells to produce the thiamine and / or 5-(2-hydroxyethyl)-4-methyl-1,3-thiazole-2-carboxylic phosphate acid (cTHZ-P) and / or cTHZ and / or THZ-P; b) optionally recovering and / or isolating the thiamine and / or 5-(2-hydroxyethyl)-4-methyl-1,3- thiazole-2-carboxylic phosphate acid (cTHZ-P) and / or cTHZ and / or THZ-P; c) thiaminase converts thiamine to sulfurol (THZ).

[0014] In a further aspect, provided for herein is a chemical conversion of thiamine and / or 5-(2- hydroxyethyl)-4-methyl-1,3-thiazole-2-carboxylic phosphate acid (cTHZ-P) and / or cTHZ and / or THZ-P to sulfurol.

[0015] In a further aspect, provided for herein is a composition comprising the fermentation composition and one or more carriers, agents, adjuvants, additives and / or excipients.

[0016] In a further aspect, provided for herein is a method for recovering sulfurol for dietary supplement, a cosmetic, a food preparation, a flavor preparation, a feed preparation and / or an analytical or diagnostic reagent.

[0017] In a further aspect, a genetically modified host cell is provided having improved production of sulfurol and / or one or more sulfurol precursors, wherein the host cell expresses one or more heterologous and / or genetically modified native genes encoding enzymes selected from a thiaminase I, a thiaminase II and / or a phosphatase and / or a decarboxylase whereby the production of the sulfurol and / or one or more sulfurol precursors in the genetically modified host cell is improved compared to an unmodified parent host cell.

[0018] In a further aspect, a genetically modified host cell is provided having improved production of sulfurol and / or one or more sulfurol precursors, wherein the host cell expresses one or more heterologous and / or genetically modified native genes encoding enzymes selected from a thiaminase I, a thiaminase II and / or a phosphatase and / or a decarboxylase.

[0019] In a further aspect, a method is provided for preparing sulfurol from a sulfurol precursor comprising: a) Obtaining the sulfurol precursor by culturing the host cell of any preceding claims in a growth medium to provide the sulfurol precursor; b) Recovering the sulfurol precursor from the growth medium, and c) Converting the sulfurol precursor to sulfurol in vitro, optionally by chemical synthesis.Case Ref. P180WO IPTector® Description of drawings and figures

[0020] The figures included herein are illustrative and simplified for clarity, and they merely show details which are essential to the understanding of the invention, while other details may have been left out.

[0021] Figure 1 shows the modules of the pathway for microbial production of sulfurol in E. coli. Sulfurol (THZ) can be produced by several different pathways. Some pathways start from the thiazole branch of the thiamine pathway whose main product is carboxy-thiazole phosphate (5-(2- hydroxyethyl)-4-methyl-1,3-thiazole-2-carboxylic phosphate acid (cTHZ-P)). This compound is formed when the protein ThiG combines the sugar moiety DXP (1-deoxy-D-xylulose 5-phosphate) coming from pyruvate and glyceraldehyde 3-phosphate with a dehydroglycine (DHG) from glycine and a sulfur originating from cysteine. All precursors are abundant, but it is necessary to over- express a glycine oxidase enzyme (ThiO) for generating DHG as well as ThiI, ThiS and ThiF for sulfur activation and efficient formation of 5-(2-hydroxyethyl)-4-methyl-1,3-thiazole-2-carboxylic phosphate acid (cTHZ-P). In one pathway, THZ can be produced by action of a decarboxylase to decarboxylate first 5-(2-hydroxyethyl)-4-methyl-1,3-thiazole-2-carboxylic phosphate acid (cTHZ-P) into thiazole-phosphate (THZ-P) followed by dephosphorylation of the latter compound into THZ (upper route in 1). Alternatively, it is possible to remove first the phosphate group of 5-(2- hydroxyethyl)-4-methyl-1,3-thiazole-2-carboxylic phosphate acid (cTHZ-P) to produce carboxy- thiazole (5-(2-hydroxyethyl)-4-methyl-1,3-thiazole-2-carboxylic acid (cTHZ)), followed by a decarboxylation step to form THZ. Some strategies could be approached by discovering and / or engineering an enzyme pair of phosphatase / decarboxylase because no enzymes are known to catalyse such reactions. In a further pathway, firstly, it is necessary not only to feed HMP (4-amino-5- hydroxymethyl-2-methylpyrimidine) in the cell factory, but also to phosphorylate it to HMP-P and HMP-PP by the co-expression of HMP kinase thiD together with the co-expression of ligase thiE and phosphatase to form thiamine. Finally, the co-expression of thiaminase cleaves thiamine into THZ.

[0022] The figure further details how chemistry steps can be employed from different sulfurol precursors to facilitate formation of sulfurol.

[0023] Figure 2 shows bar graphs of ThiM KO which allows for secretion of 5-(2-hydroxyethyl)-4- methyl-1,3-thiazole-2-carboxylic acid (cTHZ) into the production media in a ThiM knock-out strain.

[0024] Figure 3 shows the BioLector®Pro fed-batch fermentation of BS07312 and BS07309. BS07309 expresses Thi1_CYA.

[0025] Figure 4 shows chemical conversion of 5-(2-hydroxyethyl)-4-methyl-1,3-thiazole-2-carboxylic acid (cTHZ) 1 to sulfurol 2.Case Ref. P180WO IPTector®

[0026] Figure 5 shows chemical conversion of 5-(2-hydroxyethyl)-4-methyl-1,3-thiazole phosphate (THZ-P) 6 to sulfurol 2.

[0027] Figure 6 shows chemical reaction of the conversion of thiamine 4 or thiamine phosphate 5 to sulfurol 2.

[0028] Figure 7 shows sulfurol 2 formation through degradation of THI 4.10 mM of 4 was dissolved in a solution containing 2.0 % NaHSO3 at pH 6.5 for 50 minutes. Product formation was analyzed by HPLC at 254 nM by taking aliquots of the reaction mixture at 10, 20 and 50 minutes starting from t = 0.

[0029] Figure 8 shows the chemical conversion of thiamine to sulfurol.

[0030] Figure 9 shows the chemical conversion of carboxy-sulfurol to sulfurol.

[0031] Figure 10 shows GCMS analysis of recovered sulfurol from carboxysulfurol producing strains.

[0032] Figure 11 shows H -NMR spectra of recovered sulfurol from thiamine producing strains in methanol.

[0033] Figure 12 shows GCMS used for analyzing purity and identity of sulfurol generated from carboxysulfurol producing strains in the downstream process of example 9.

[0034] Figure 13 shows NMR used for analyzing purity and identity of sulfurol generated from carboxysulfurol producing strains in the downstream process of example 9.^

[0035] Figure 14 shows general overview of the purification of sulfurol from carboxsulfurol producing strains using resins.

[0036] Figure 15 shows NMR used for analyzing purity and identity of sulfurol generated from carboxysulfurol producing strains in a downstream process of example 10.^

[0037] Figure 16 shows the conversion of chemical conversion of 5-(2-hydroxyethyl)-4-methyl-1,3- thiazolecarboxylic acid phosphate (cTHZ-P) 3 to sulfurol 2.

[0038] Figure 17 shows the overall scheme for the chemical conversion of the fermentation products to sulfurol. Incorporation by reference

[0039] All publications, patents, and patent applications referred to herein are incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. In the event of a conflict between a term herein and a term in an incorporated reference, the term herein prevails and controls. Detailed Description

[0040] Provided for herein are genetically modified host cells having increased production of sulfurol and / or thiamine and / or cTHZ and / or THZ-P and / or cTHZ-P achieved by expressing one or moreCase Ref. P180WO IPTector® heterologous Thiamine pathway enzymes. The production of sulfurol requires not only a thiaminase that is active and specific in the host cell for converting thiamine into THZ, but also a cell factory that can efficiently produce thiamine at high levels. The de novo biosynthesis of thiamine is a highly complex and regulated process present in microbes and plants. It starts with the biosynthesis of the precursors 5-(2-hydroxyethyl)-4-methyl-1,3-thiazole-2-carboxylic phosphate acid (cTHZ-P) and hydroxymethylpyrimidine diphosphate (HMP-PP), which are combined to form thiamine-mono- phosphate (TMP), which in turn can be dephosphorylated to thiamine (THI) by a phosphatase or phosphorylated to thiamine di-phosphate (TPP) by a kinase to form an enzymatic cofactor. In a second step, a thiaminase converts THI into sulfurol. Both technical problems are solved in the current invention. Alternatively, the host cell is engineered to produce either thiamine (THI) and / or the sulfurol precursors cTHZ-P and / or cTHZ and / or THZ-P, and these molecules are then converted to sulfurol by chemical reactions. Definitions

[0041] Any EC numbers used herein refer to Enzyme Nomenclature 1992 from NC-IUBMB, Academic Press, San Diego, California, including 30 supplements 1-5 published in Eur. J. Bio-chem.1994, 223, 1- 5; Eur. J. Biochem.1995, 232, 1-6; Eur. J. Biochem.1996, 237, 1-5; Eur. J. Biochem.1997, 250, 1-6; and Eur. J. Biochem.1999, 264, 610-650; respectively. The nomenclature is regularly supplemented and updated; see e.g., http: / / enzyme.expasy.org / . The term “PEP” as used herein refers to phosphoenolpyruvate.

[0042] The term “thiamine” as used herein may be used interchangeably about the compound thiamine as such, but also where appropriate thiamine mono phosphate (TMP) and / or thiamine pyrophosphate (TPP).

[0043] The term “THZ” as used herein may be used interchangeably about the compound sulfurol as such or the IUPAC name 4-methyl-5-beta-hydroxyethylthiazole.

[0044] The term “cTHZ” as used herein may be used interchangeably about the compound carboxysulfurol as such or the IUPAC name 5-(2-hydroxyethyl)-4-methyl-1,3-thiazole-2-carboxylic acid.

[0045] The term “cTHZ-P” as used herein may be used interchangeably about the compound carboxysulfurol-phosphate as such or the IUPAC name 5-(2-hydroxyethyl)-4-methyl-1,3-thiazole-2- carboxylic phosphate acid.

[0046] The term “THZ-P” as used herein may be used interchangeably about the compound sulfurol- phosphate as such or the IUPAC name 4-methyl-5-beta-hydroxyethylthiazole phosphate.

[0047] The term “TMP-phosphatase” as used herein refers to a thiamine monophosphateCase Ref. P180WO IPTector® phosphatase dephosphorylating thiamine monophosphate to thiamine. It has been shown that the bifunctional TH2 protein from Arabidopsis thaliana has this activity (see also WO2017103221).

[0048] The term “ThiK” as used herein refers to a thiamine kinase that catalyzes the phosphorylation of thiamine to thiamine-monophosphate (TMP).

[0049] The term “ThiL” as used herein refers to a thiamine-monophosphate kinase. It catalyzes the ATP-dependent phosphorylation of thiamine-monophosphate (TMP) to form thiamine- pyrophosphate (TPP), the active form of vitamin B1. It cannot use thiamine as a substrate. Is highly specific for ATP as a phosphate donor.

[0050] The term “ThiM” as used herein refers to a hydroxyethylthiazole kinase that catalyzes the phosphorylation of the hydroxyl group of 4-methyl-5-beta-hydroxyethylthiazole.

[0051] The term “ThiD” as used herein refers to a hydroxymethylpyrimidine or phosphomethylpyrimidine kinase that catalyzes the phosphorylation of hydroxymethylpyrimidine phosphate (HMP-P) to HMP-PP, and of HMP to HMP-P. ThiD shows no activity with pyridoxal, pyridoxamine or pyridoxine.

[0052] The term “ThiC” as used herein refers to a phosphomethylpyrimidine synthase protein that catalyzes the synthesis of the hydroxymethylpyrimidine phosphate (HMP-P) moiety of thiamine from aminoimidazole ribotide (AIR) in a radical S-adenosyl-L-methionine (SAM)-dependent reaction.

[0053] The term “ThiE” as used herein refers to a thiamine-phosphate synthase protein that condenses 4-methyl-5-[2-(phosphonatooxy)ethyl]-1,3-thiazole-2-carboxylate (5-(2-hydroxyethyl)-4- methyl-1,3-thiazole-2-carboxylic phosphate acid (cTHZ-P)) and 2-methyl-4-amino-5-hydroxymethyl pyrimidine pyrophosphate (HMP-PP) to form thiamine monophosphate (TMP).

[0054] The term “ThiF” as used herein refers to a sulfur carrier protein ThiS adenylyltransferase. ThiF catalyzes the adenylation of the carboxy terminus of ThiS and the subsequent displacement of AMP catalyzed by ThiI-persulfide to give a ThiS-ThiI acyl disulfide ThiS.

[0055] The term “ThiS” as used herein refers to a sulfur carrier protein in which a C-terminal thiocarboxylation occurs in 2 steps: First, it is acyl-adenlyated by ThiF and then thiocarboxylated by ThiI.

[0056] The term “ThiG” as used herein refers to a Thiazole synthase protein that catalyzes the rearrangement of 1-deoxy-D-xylulose 5-phosphate (DXP) to produce the thiazole phosphate moiety of thiamine. Sulfur is provided by the thiocarboxylate moiety of the carrier protein ThiS.

[0057] The term “ThiH” as used herein refers to a 2-iminoacetate synthase protein that catalyzes the radical SAM-mediated cleavage of tyrosine to 2-iminoacetate and 4-cresol.

[0058] The term “ThiI” as used herein refers to a tRNA sulfurtransferase protein that catalyzes the ATP-dependent transfer of sulfur to tRNA to produce 4-thiouridine in position 8 of tRNAs, whichCase Ref. P180WO IPTector® functions as a near-UV photosensor. Also catalyzes the transfer of sulfur to the sulfur carrier protein ThiS, forming ThiS-thiocarboxylate. This is a step in the synthesis of thiazole, in the thiamine biosynthesis pathway. The sulfur is donated as persulfide by IscS.

[0059] The term “dxs protein” as used herein refers to a deoxy-D-xylose-5-phosphat synthase that catalyses the acyloin condensation reaction between C atoms 2 and 3 of pyruvate and glyceraldehyde 3-phosphate to yield 1-deoxy-xylulose-5-phosphate (DXP).

[0060] The term “ThiO” as used herein refers to a glycine oxidase that catalyzes the FAD-dependent oxidative deamination of various amines and D-amino acids to yield the corresponding alpha-keto acids, ammonia / amine, and hydrogen peroxide including the formation of dehydroglycine from glycine. It is essential for thiamine biosynthesis in organisms that generally lack ThiH since the oxidation of glycine catalyzed by ThiO also generates the glycine imine intermediate (dehydroglycine) required for the biosynthesis of the thiazole ring of thiamine pyrophosphate.

[0061] The term “TenI” as used herein refers to thiazole tautomerase involved in thiamine thiazole biosynthesis. While TenI contains sequence regions that are highly conserved between thiamine phosphate synthase thiE and tenI, the purified protein has no thiamine phosphate synthase activity. TenI catalyzes the aromatization of the cTHZ-P* tautomer (formed by the condensation reaction of thiS / thiG) to the aromatic cTHZ-P.

[0062] The term “iscR” as used herein refers to HTH-type transcriptional regulator IscR that regulates the transcription of several operons and genes involved in the biogenesis of Fe-S clusters and Fe-S- containing proteins. Transcriptional repressor of the iscRSUA operon, which is involved in the assembly of Fe-S clusters into Fe-S proteins.

[0063] The term “sodA” as used herein refers to the superoxide dismutase that converts superoxide radicals, optionally into oxygen and hydrogen peroxide. Superoxide radicals often appear during environmental stress.

[0064] The term “iscS” as used herein refers to a cysteine desulfurase which provides sulfur for Fe-S cluster synthesis in vitro.

[0065] The term “Thi1” as used herein refers to thiaminase I (Thiamine pyridinylase) which degrades thiamine by catalyzing the base-exchange substitution of thiazole with a nucleophile.

[0066] The term “PHOS” as used herein refers to phosphatases to dephosphorylate cTHZ-P or THZ-P.

[0067] The term “deCARB” as used herein refers to cTHZ / cTHZ-P decarboxylase that can decarboxylate the carboxy group on cTHZ to yield THZ.

[0068] The term “TenA” as used herein refers to thiaminase II or TH2, a thiamine (mono)phosphate phosphatase which either degrades thiamine by catalyzing the base-exchange substitution of thiazole with a nucleophile, or which can carry out the hydrolysis of aminomethylpyrimidine to HMP.Case Ref. P180WO IPTector®

[0069] The term “ThiP” as used herein refers to thiamine ABC transporter membrane subunit facilitating thiamine import.

[0070] The term “CyaA” as used herein refers to a adenylate cyclase that can catalyzes the synthesis of cyclic AMP (cAMP) by an intramolecular transfer of the adenylyl group of ATP to the 3'-hydroxy group, releasing pyrophosphate.The term "host cell" refers to any cell type that is susceptible to transformation, transfection, transduction, or the like with a nucleic acid construct or expression vector comprising a polynucleotide of the present invention. The host cell encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication.

[0071] The term "polynucleotide construct" refers to a polynucleotide, either single- or double- stranded, which is isolated from a naturally occurring gene or is modified to contain segments of nucleic acids in a manner that would not otherwise exist in nature, or which is synthetic, and which comprises a polynucleotide encoding a polypeptide and one or more control sequences.

[0072] The terms “nucleotide sequence” and “polynucleotide” are used herein interchangeably.

[0073] The term "coding sequence" refers to a nucleotide sequence, which directly specifies the amino acid sequence of a polypeptide. The boundaries of the coding sequence are generally determined by an open reading frame, which begins with a start codon such as ATG, GTG, or TTG and ends with a stop codon such as TAA, TAG, or TGA. The coding sequence may be a genomic DNA, cDNA, synthetic DNA, or a combination thereof.

[0074] The term "control sequence" as used herein refers to a nucleotide sequence necessary for expression of a polynucleotide encoding a polypeptide. A control sequence may be native (i.e., from the same gene or organism) or heterologous or foreign (i.e., from a different or organism) to the polynucleotide encoding the polypeptide. Control sequences include, but are not limited to leader sequences, polyadenylation sequences, pro-peptide coding sequences, promoter sequences, signal peptide coding sequences, translation terminator (stop) sequences and transcription terminator (stop) sequences. To be operational control sequences usually must include promoter sequences and transcriptional and translational stop signals. Control sequences may be provided with linkers for the purpose of introducing specific restriction sites facilitating ligation of the control sequences with a coding region of a polynucleotide encoding a polypeptide.

[0075] The term "expression vector" refers to a DNA molecule, either single- or double-stranded, either linear or circular, which comprises a polynucleotide encoding a polypeptide and is operably linked to control sequences that provide for its expression. Expression vectors include expression cassettes for the integration of genes into a host cell as well as plasmids and / or chromosomes comprising such genes.

[0076] The term "expression" includes any step involved in the production of a polypeptide including,Case Ref. P180WO IPTector® but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0077] The term "over-expression" includes a situation when one or more components of the cell may be present at a higher-than-normal cellular level (i.e., higher than the concentration known to usually be present in the cell type exhibiting the gene and / or protein complex of interest). For example, the gene encoding a protein may begin to be overexpressed, or may be amplified (i. e., its gene copy number may be increased) in certain cells, leading to an increased number of component molecules within these cells.

[0078] The terms "heterologous" or “recombinant” or “genetically modified” and their grammatical equivalents as used herein interchangeably refer to entities "derived from a different species or cell”. For example, a heterologous or recombinant polynucleotide gene is a gene in a host cell not naturally containing that gene, i.e., the gene is from a different species or cell type than the host cell. The terms as used herein about microbial host cells refer to microbial host cells comprising and expressing heterologous or recombinant polynucleotide genes.

[0079] The term “metabolic pathway” as used herein is intended to mean two or more enzymes acting sequentially in a live cell to convert chemical substrate(s) into chemical product(s). Enzymes are characterized by having catalytic activity, which can change the chemical structure of the substrate(s). An enzyme may have more than one substrate and produce more than one product. The enzyme may also depend on cofactors, which can be inorganic chemical compounds or organic compounds such as proteins for example enzymes (co-enzymes). A CPR that reduces certain cytochrome P450 enzymes is an example of an enzymatic co-factor. The term “operative biosynthetic metabolic pathway” refers to a metabolic pathway that occurs in a live recombinant host, as described herein.

[0080] The term "in vivo", as used herein refers to within a living cell or organism, including, for example, animal, a plant or a microorganism.

[0081] The term "in vitro", as used herein refers to outside a living cell or organism, including, without limitation, for example, in a microwell plate, a tube, a flask, a beaker, a tank, a reactor and the like.

[0082] The term "substrate" or “precursor”, as used herein refers to any compound that can be converted into a different compound. For example, AIR (aminoimidazole ribotide) can be a substrate for phosphomethylpyrimidine synthase and can be converted into hydroxymethylpyrimidine phosphate (HMP-P), a precursor of thiamine. For clarity, substrates and / or precursors include both compounds generated in situ by an enzymatic reaction in a cell or exogenously provided compounds, such as exogenously provided organic molecules which the host cell can metabolize into a desired compound.

[0083] The term "endogenous" or “native” as used herein refers to a gene or a polypeptide in a hostCase Ref. P180WO IPTector® cell which originates from the same host cell.

[0084] The term “deletion” as used herein refers to the manipulation of a gene so that it is no longer expressed in a host cell.

[0085] The term “attenuation” as used herein refers to the manipulation of a gene or any of the machinery participating in the expression of the gene so that the expression of the gene is reduced as compared to expression without the manipulation.

[0086] The terms "substantially" or "approximately" or "about", as used herein refer to a reasonable deviation around a value or parameter such that the value or parameter is not significantly changed. These terms of deviation from a value should be construed as including a deviation of the value where the deviation would not negate the meaning of the value deviated from. For example, in relation to a reference numerical value, the terms of degree can include a range of values plus or minus 10% from that value. For example, deviation from a value can include a specified value plus or minus a certain percentage from that value, such as plus or minus 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% from the specified value.

[0087] The term "and / or" as used herein is intended to represent an inclusive "or". The wording X and / or Y is meant to mean both X or Y and X and Y. Further, the wording X, Y and / or Z is intended to mean X, Y and Z alone or any combination of X, Y, and Z.

[0088] The term "isolated" as used herein about a compound, refers to any compound, by means of human intervention, has been put in a form or environment that differs from the form or environment in which it is found in nature. Isolated compounds include but is no limited to compounds disclosed herein for which the ratio of the compounds relative to other constituents with which they are associated in nature is increased or decreased. In an important embodiment, the amount of compound is increased relative to other constituents with which the compound is associated in nature. In an embodiment, the compound disclosed herein may be isolated into a pure or substantially pure form. In this context, a substantially pure compound means that the compound is separated from other extraneous or unwanted material present from the onset of producing the compound or generated in the manufacturing process. Such a substantially pure compound preparation contains less than 10%, such as less than 8%, such as less than 6%, such as less than 5%, such as less than 4%, such as less than 3%, such as less than 2%, such as less than 1 %, such as less than 0.5% by weight of other extraneous or unwanted material usually associated with the compound when expressed natively or recombinantly. In an embodiment, the isolated compound is at least 90% pure, such as at least 91% pure, such as at least 92% pure, such as at least 93% pure, such as at least 94% pure, such as at least 95% pure, such as at least 96% pure, such as at least 97% pure, such as at least 98% pure, such as at least 99% pure, such as at least 99.5% pure, such as 100 % pure by weight.Case Ref. P180WO IPTector®

[0089] The term "% identity" is used herein about the relatedness between two amino acid sequences or between two nucleotide sequences. For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. "% identity" as used herein about amino acid sequences refers to the degree of identity in percent between two amino acid sequences obtained when using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet.16: 276-277), preferably version 5.0.0 or later. The parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The output of Needle labeled "longest identity" (obtained using the -nobrief option) is used as the percent identity and is calculated as follows: ^^^^^^^^^ ^^^^^ ^^^^ ^^^^^^^^ Length of alignment − total number of gaps in alignment! 100

[0090] The term "% identity" as used herein about nucleotide sequences refers to the degree of identity in percent between two deoxyribonucleotide sequences obtained when using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, supra), preferably version 5.0.0 or later. The parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The output of Needle labelled "longest identity" (obtained using the -nobrief option) is used as the percent identity and is calculated as follows: ^^^^^^^^^ ^^^^^ ^^^^ ^^^^^^^^ Length of alignment − total number of gaps in alignment! 100

[0091] Other examples of algorithms that are suitable for determining percent sequence identity and sequence similarity include the BLAST and BLAST 2.0 algorithms, which are described in Altschul, et al. (1977) Nuc. Acids Res.25: 3389-3402 and Altschul, et al. (1990) J.Mol. Biol.215: 403-410, or the WU- BLAST-2 program (Altschul et al., Meth. Enzymol., 266: 460-480 (1996).

[0092] The protein sequences of the present invention can further be used as a "query sequence" toCase Ref. P180WO IPTector® perform a search against sequence databases, for example, to identify other family members or related sequences. Such searches can be performed using the BLAST programs. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov). BLASTP is used for amino acid sequences and BLASTN for nucleotide sequences. The BLAST program uses as defaults: Cost to open gap: default= 5 for nucleotides / 11 for proteins Cost to extend gap: default = 2 for nucleotides / 1 for proteins Penalty for nucleotide mismatch: default = -3 Reward for nucleotide match: default= 1 Expect value: default = 10 Wordsize: default = 11 for nucleotides / 28 for megablast / 3 for proteins.

[0093] Furthermore, the degree of local identity between the amino acid sequence query or nucleic acid sequence query and the retrieved homologous sequences is determined by the BLAST program. However, only those sequence segments are compared that give a match above a certain threshold. Accordingly, the program calculates the identity only for these matching segments. Therefore, the identity calculated in this way is referred to as local identity.

[0094] The term "mature polypeptide" or "mature enzyme" as used herein refers to a polypeptide in its final active form following translation and any post-translational modifications, such as N-terminal processing, C-terminal truncation, glycosylation, phosphorylation, etc. It is known in the art that a host cell may produce a mixture of two or more different mature polypeptides (i.e., with a different C- terminal and / or N-terminal amino acid) expressed by the same polynucleotide.

[0095] The term "cDNA" refers to a DNA molecule that can be prepared by reverse transcription from a mature, spliced, mRNA molecule obtained from a eukaryotic or prokaryotic cell. cDNA lacks intron sequences that may be present in the corresponding genomic DNA. The initial, primary RNA transcript is a precursor to mRNA that is processed through a series of steps, including splicing, before appearing as mature spliced mRNA.

[0096] The terms "comprise" and "include" as used throughout the specification and the accompanying items as well as variations such as "comprises", "comprising", "includes" and "including" are to be interpreted inclusively. These words are intended to convey the possible inclusion of other elements or integers not specifically recited, where the context allows.

[0097] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to one or at least one) of the grammatical object of the article. By way of example, "an element" may mean oneCase Ref. P180WO IPTector® element or more than one element.

[0098] Terms like "preferably", "commonly", "particularly", and "typically" are not utilized herein to limit the scope of the item’s invention or to imply that certain features are critical, essential, or even important to the structure or function of the item invention. Rather, these terms are merely intended to highlight alternative or additional features that can or cannot be utilized in a particular embodiment of the present invention.

[0099] The term "cell culture" as used herein refers to a culture medium comprising a plurality of host cells disclosed herein. A cell culture may comprise a single strain of host cells or may comprise two or more distinct host cell strains. The culture medium may be any medium that may comprise a recombinant host, e.g., a liquid medium (i.e., a culture broth) or a semi-solid medium, and may comprise additional components, e.g., one or more of (i) trace metals; (ii) vitamins; (iii) salts (such as salts of phosphate, magnesium, potassium, zinc, iron); (iv) nitrogen sources (such as YNB, ammonium sulphate, urea, yeast extracts, ammonium nitrate, ammonium chloride, malt extract, peptone and / or amino acids); (v) carbon source (such as dextrose, sucrose, glycerol, glucose, maltose, molasses, starch, cellulose, xylan, pectin, lignocellolytic biomass hydrolysate, and / or acetate); (vi) nucleobases; (vii) aminoglycosides; and / or (viii) antibiotics (such as tetracyclin and spectinomycin).

[0100] The term "radical SAM" as used herein refers to a superfamily of enzymes that use a [4Fe-4S]+cluster to reductively cleave S-adenosyl-L-methionine (SAM) to generate a radical, usually a 5’- deoxyadenosyl radical, as a critical intermediate. The vast majority of known radical SAM enzymes have a cysteine-rich motif that matches or resembles CxxxCxxC or CX3CX3.

[0101] The term “pathway element” as used herein refers to a biological component that contributes to the function of a metabolic pathway, such as an operative metabolic pathway. A pathway element typically comprises a polypeptide having enzymatic activity that catalyzes a biochemical reaction within the pathway toward a target compound, e.g. toward sulfurol or one of the sulfurol precursors disclosed herein. In some embodiments, “a pathway element” may also describe a gene encoding such a polypeptide, or regulatory sequences or cofactor functionally associated with the enzymatic activity. Pathway elements may be naturally occurring, i.e. native or heterologous to the host cell, and may be expressed recombinantly or introduced by other means. In some embodiments, the “pathway element” is a polypeptide, such as a polypeptide which is part of the pathway producing cTHZ, THZ-P, cTHZ-P and / or sulfurol as disclosed herein. Genetically modified host cells

[0102] The genetically modified host cells having increased production of sulfurol (THZ), carboxy- sulfurol (c-THZ), cTHZ-P, THZ-P or thiamine (THI) provided for herein, express in some embodimentsCase Ref. P180WO IPTector® one or more heterologous Thiamine pathway enzymes converting thiamine, 5-(2-hydroxyethyl)-4- methyl-1,3-thiazole-2-carboxylic phosphate acid (cTHZ-P), 5-(2-hydroxyethyl)-4-methyl-1,3-thiazole- 2-carboxylic acid (cTHZ) and / or THZ-P into sulfurol in the host cell, whereby the production of the sulfurol in the genetically modified host cell is improved compared to an unmodified parent host cell. The genetically modified host cells having increased production of sulfurol, provided for herein, expresses in other one or more heterologous phosphatase enzymes catalyzing the transfer of from IscS to the sulfur carrier protein ThiS in the host cell, whereby the production of the thiamine in the genetically modified host cell is improved compared to an unmodified parent host cell.

[0103] The increase in the genetically modified host cell's capacity to produce sulfurol can in some embodiments be at least 5%, such as at least 10%, such as least 15%, such as at least 20%.

[0104] In one embodiment, the genetically modified host cell having improved production of 5-(2- hydroxyethyl)-4-methyl-1,3-thiazole-2-carboxylic phosphate acid (cTHZ-P), THZ-P, 5-(2-hydroxyethyl)- 4-methyl-1,3-thiazole-2-carboxylic acid (cTHZ), and / or sulfurol (THZ), wherein the host cell expresses one or more heterologous and / or genetically modified native genes encoding enzymes selected from thiaminase I and / or thiaminase II and / or decarboxylase and / or phosphatase whereby the production of the THZ-P, 5-(2-hydroxyethyl)-4-methyl-1,3-thiazole-2-carboxylic acid (cTHZ), 5-(2-hydroxyethyl)-4- methyl-1,3-thiazole-2-carboxylic phosphate acid (cTHZ-P) and / or sulfurol in the genetically modified host cell is improved compared to an unmodified parent host cell.

[0105] In some embodiments, the host cell expresses: a) thiaminase I or thiaminase II which converts thiamine to sulfurol; and / or b) phosphatase (PHOS) which dephosphorylates THZ-P to sulfurol and / or 5-(2-hydroxyethyl)-4- methyl-1,3-thiazole-2-carboxylic phosphate acid (cTHZ-P) to 5-(2-hydroxyethyl)-4-methyl-1,3- thiazole-2-carboxylic acid (cTHZ).

[0106] In some embodiments, the thiaminase I, thiaminase II and / or phosphatase of the host cell are fungal enzymes.

[0107] In some embodiments, the thiaminase I, thiaminase II and / or phosphatase of the host cell are bacterial enzymes.

[0108] In some embodiments, the host cell expresses thiaminase I and / or thiaminase II from a genus selected from Gloeothece (Cyanothece), Megasphaera, Naegleria, Burkholderia, Haliangium, Oleidesulfovibrio, Stigmatella and / or Acinetobacter, Helicobacter, Bacillus, Candida, Halaalkalibacterium, Pyrobaculum, Pyrococcus Staphylococcus and / or Saccharomyces.

[0109] In some embodiments, the host cell expresses thiaminase I and / or thiaminase II from a species selected from Gloeothece citriformis, Megasphaera elsdenii DSM 20460, Naegleria gruberi, Burkholderia glumae BGR1, Haliangium ochraceum DSM14365, Oleidesulfovibrio alaskensis G20,Case Ref. P180WO IPTector® Stigmatella aurantiaca DW4 and / or Acinetobacter nectaris, Helicobacter pylori, Bacillus anerurinolyticus, Bacilluc migulanus, Bacillus subtilis, Candida parapsilosis, Halaalkalibacterium halodurans, Pyrobaculum aerophilum, Pyrococcus furiosus, Pyrococcus horikoshii, Staphylococcus aureus, Staphylococcus epidermidis, Saccharomyces solfataricus and / or Saccharomyces cerevisiae.

[0110] In some embodiments the phosphatase is from a species selected from Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Pseudomonas Putida KT2440, Shigella sonnei (strain Ss046), Pseudomonas asiatica, Hirschfeldia incana, and / or Citrus sinensis.

[0111] In some embodiments the host cell comprises: a) thiaminase I (Thi1) has an amino acid sequence which is at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the thiaminase I comprised in SEQ ID NO: 29, 31, 33, 35, 37, 39, 41 and / or 43. a) thiaminase II (TenA) has an amino acid sequence which is at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the thiaminase II comprised in SEQ ID NO: 45, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137 and / or 139; b) phosphatase (PHOS) has an amino acid sequence which is at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the phosphatases comprised in SEQ ID NO: 25, 47,-49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103 and / or 105.

[0112] In some embodiments, the bacterial thiaminase I of the host cell has an amino acid sequence as set forth in SEQ ID NO: 29, 31, 33, 35, 37, 39, 41 and / or 43.

[0113] In some embodiments, the bacterial thiaminase II (TenA) of the host cell has an amino acid sequence as set forth in SEQ ID NO: 45, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137 and / or 139.

[0114] In some embodiments, the phosphatase of the host cell has an amino acid sequence as set forth in SEQ ID NO: 25, 4749, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103 and / or 105.

[0115] In some embodiments, the thiaminase I of the host cell is from a genus or species selected from Cyanothece, Megasphaera elsdenii DSM 20460, Naegleria gruberi, Burkholderia glumae BGR1, Haliangium ochraceum DSM14365, Oleidesulfovibrio alaskensis G20, Stigmatella aurantiaca DW4 and / or Acinetobacter nectaris having SEQ ID NO: 29, 31, 33, 35, 37, 39, 41 and / or 43.

[0116] In some embodiments, the host cell comprises a gene encoding:Case Ref. P180WO IPTector® a) the thiaminase I is least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 30, 32, 34, 36, 38, 40, 42 and / or 44 or genomic DNA thereof encoding the thiaminase I enzyme comprised in SEQ ID NO: 29, 31, 33, 35, 37, 39, 41 and / or 43; b) the thiaminase II (TenA) is least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 45, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138 and / or 140 or genomic DNA thereof encoding the thiaminase II enzyme comprised in SEQ ID NO: 45, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137 and / or 139; and / or c) the phosphatase is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 26, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68,70, 72, 74, 76, 78, 80, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104 and / or 106 or genomic DNA thereof encoding the phosphatase enzyme comprised in SEQ ID NO: 25, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 83, 85, 87, 89,91, 93, 95, 97, 99, 101, 103 and / or 105..

[0117] In some embodiments, the host cell comprises the gene encoding: a) thiaminase I gene is at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the thiaminase I gene comprised in SEQ ID NO: 30, 32, 34, 36, 38, 40, 42 and / or 44; b) phosphatase gene is at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the phosphatase gene comprised in SEQ ID NO: 26, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68,70, 72, 74, 76, 7880, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104 and / or 106.

[0118] In some embodiments, the thiaminase I gene of the host cell is a truncated thiaminase I gene and / or the thiaminase II gene is a truncated thiaminase II gene.

[0119] In some embodiments, the phosphatase gene of the host cell is a truncated phosphatase gene.

[0120] In some embodiments the host cell further comprises an operative metabolic pathway comprising one or more native or heterologous pathway elements producing 5-(2-hydroxyethyl)- 4-methyl-1,3-thiazole-2-carboxylic acid (cTHZ), THZ-P, 5-(2-hydroxyethyl)-4-methyl-1,3-thiazole-Case Ref. P180WO IPTector® 2-carboxylic phosphate acid (cTHZ-P) and / or sulfurol.

[0121] In some embodiments the host cell comprises one or more native or heterologous pathway elements comprise one or more radical SAM enzymes.

[0122] In some embodiments the host cell comprises one or more native or heterologous pathway elements selected from: a) a phosphomethylpyrimidine synthase (ThiC); that catalyzes the synthesis of the hydroxymethylpyrimidine phosphate (HMP-P) moiety of thiamine from aminoimidazole ribotide (AIR) in a radical S-adenosyl-L-methionine (SAM)-dependent reaction; b) a hydroxymethylpyrimidine / phosphomethylpyrimidine kinase (ThiD) that catalyzes the phosphorylation of hydroxymethylpyrimidine phosphate (HMP-P) to HMP-PP, and of HMP to HMP-P; c) an adenylyltransferase (ThiF) that catalyzes the adenylation of the carboxy terminus of the sulfur carrier protein ThiS and the subsequent displacement of AMP catalyzed by ThiI- persulfide to give a ThiS-ThiI acyl disulfide ThiS; d) a sulfur carrier protein (ThiS) in which its C-terminal thiocarboxylation occurs in 2 steps: first, it is acyl-adenylated and then thiocarboxylated by ThiL; e) a 2-iminoacetate synthase (ThiH) converting that catalyzes the radical-mediated cleavage of tyrosine to 2-iminoacetate and 4-cresol; f) a thiazole synthase (ThiG) that catalyzes the rearrangement of 1-deoxy-D-xylulose 5- phosphate (DXP) to produce the thiazole phosphate moiety of thiamine (5-(2-hydroxyethyl)- 4-methyl-1,3-thiazole-2-carboxylic phosphate acid (cTHZ-P)); g) a hydroxyethylthiazole kinase (ThiM) that catalyzes the phosphorylation of the hydroxyl group of 4-methyl-5-beta-hydroxyethylthiazole (THZ); h) a thiamine-monophosphate (TMP) phosphatase (TH2) that dephosphorylates thiamine monophosphate to thiamine; i) a thiamine kinase (ThiK) that catalyzes the phosphorylation of thiamine to thiamine- monophosphate (TMP); j) a thiamine-monophosphate kinase (ThiL) which catalyzes the ATP-dependent phosphorylation of thiamine-monophosphate (TMP) to form thiamine-pyrophosphate (TPP); k) a thiamine-monophosphate synthase (ThiE) that condenses carboxy-4-methyl-5-(beta- hydroxyethyl)thiazole monophosphate (5-(2-hydroxyethyl)-4-methyl-1,3-thiazole-2- carboxylic phosphate acid (cTHZ-P)) and 2-methyl-4-amino-5-hydroxymethyl pyrimidine pyrophosphate (HMP-PP) to form thiamine-monophosphate (TMP); l) an HTH-type transcriptional regulator (IscR) that regulates the transcription of several operonsCase Ref. P180WO IPTector® and genes involved in the biogenesis of Fe-S clusters and Fe-S-containing proteins; m) a tRNA sulfotransferase (ThiI) which catalyzes the transfer of sulfur to the sulfur carrier protein ThiS, forming ThiS-thiocarboxylate; n) a glycine oxidase (ThiO) converting glycine into dehydroglycine (DHG); o) a cysteine desulfurase (IscS) which provides sulfur for Fe-S cluster synthesis in vitro; p) a thiazole tautomerase (TenI) which catalyzes the aromatization of cThz*-P to cThz-P; q) a thiaminase I (Thi1) which degrades thiamine by catalyzing the base-exchange substitution of thiazole with a nucleophile; r) a 2-(2-Carboxy-4-methylthiazol-5-yl)ethyl phosphate (5-(2-hydroxyethyl)-4-methyl-1,3- thiazole-2-carboxylic phosphate acid (cTHZ-P)) phosphatase (PHOS) that dephosphorylates 5- (2-hydroxyethyl)-4-methyl-1,3-thiazole-2-carboxylic phosphate acid (cTHZ-P) to 5-(2- hydroxyethyl)-4-methyl-1,3-thiazole-2-carboxylic acid (cTHZ); s) a thiaminase II (tenA) which either degrades thiamine by catalyzing the base-exchange substitution of thiazole with a nucleophile, or which can carry out the hydrolysis of aminomethylpyrimidine to HMP; t) a deCARB decarboxylase that can decarboxylate the carboxy group on cTHZ to yield THZ; u) a thiamine transporter (ThiP) that is a thiamine ABC transporter membrane subunit facilitating thiamine import; v) a DNA-binding transcriptional dual regulator (fur) that can lead to higher Fe2+ availability when mutated to fur-K14I; w) an adenylate cyclase (cyaA) that can catalyzes the synthesis of cyclic AMP (cAMP) by an intramolecular transfer of the adenylyl group of ATP to the 3'-hydroxy group, releasing pyrophosphate; and / or x) a superoxide dismutase (sodA) as defined herein;

[0123] In some embodiments the host cell comprises one or more native or heterologous pathway elements selected from: b) ThiC has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the ThiC comprised in SEQ ID NO: 1; c) ThiD has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the ThiD comprised in SEQ ID NO: 3; d) ThiE has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the ThiECase Ref. P180WO IPTector® comprised in SEQ ID NO: 17; e) ThiF has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the ThiF comprised in SEQ ID NO: 5; f) ThiS has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the ThiS comprised in SEQ ID NO: 7; g) ThiO has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the ThiO comprised in SEQ ID NO: 27; h) ThiH has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the ThiH comprised in SEQ ID NO: 9; i) ThiG has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the ThiG comprised in SEQ ID NO: 11; j) ThiM has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the ThiM comprised in SEQ ID NO: 13; k) ThiL has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the ThiL comprised in SEQ ID NO: 15; l) ThiI has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the ThiI comprised in SEQ ID NO: 19; m) TenI has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the Ten I comprised in SEQ ID NO: 23; n) Thiaminase I (Thi1) has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the Thi1 comprised in SEQ ID NO: 29, 31, 33, 35, 37, 39, 41 and / or 43; o) ThiK has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the ThiK comprised in SEQ ID NO: 141;Case Ref. P180WO IPTector® p) IscR has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the IScR comprised in SEQ ID NO: 109; q) IscS has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the Thi1 comprised in SEQ ID NO: 145; r) TenA has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the TenA comprised in SEQ ID NO: 45, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137 and / or 139; s) deCARB has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the deCARB comprised in SEQ ID NO: 81; t) ThiP has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the ThiP comprised in SEQ ID NO: 107; u) fur-k14I has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the fur-k14I comprised in SEQ ID NO: 111; v) CyaA has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the CyaA comprised in SEQ ID NO: 113; w) SodA has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the SodA comprised in SEQ ID NO: 143; x) TH2 has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the TH2 comprised in SEQ ID NO: 25; y) PHOS has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the PHOS comprised in SEQ ID NO: 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 83, 85, 87, 89,91, 93, 95, 97, 99, 101, 103 and / or 105; and / or z) dxs has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the dxsCase Ref. P180WO IPTector® comprised in SEQ ID NO: 147.

[0124] In some embodiments the host cell comprises a mutation which is a deletion, disruption, and / or an attenuation of the gene.

[0125] In some embodiments the host cell comprises a mutation which is or a deletion through complete removal of the gene or a translational knockout by introducing one more stop codons or frameshift mutations preventing expression of an active peptide.

[0126] In some embodiments the host cell comprises a deletion, disruption and / or attenuation or translational knockout of ThiP, CyaA, Fur-K14I iscR-H107Y and / orThiM.

[0127] In some embodiments the host cell comprises one or more pathway elements encoding by one or more genes selected from the group of: a) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 2 or genomic DNA thereof encoding the ThiC comprised in SEQ ID NO: 1; b) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 4 or genomic DNA thereof encoding the ThiD comprised in SEQ ID NO: 3; c) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 18 or genomic DNA thereof encoding the ThiE comprised in SEQ ID NO: 17; d) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 6 or genomic DNA thereof encoding the ThiF comprised in SEQ ID NO: 5; e) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 8 or genomic DNA thereof encoding the ThiS comprised in SEQ ID NO: 7; f) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 28 or genomic DNA thereof encoding the ThiO comprised in SEQ ID NO: 27;Case Ref. P180WO IPTector® g) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 12 or genomic DNA thereof encoding the ThiG comprised in SEQ ID NO: 11; h) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 14 or genomic DNA thereof encoding the ThiM comprised in SEQ ID NO: 13; i) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 16 or genomic DNA thereof encoding the ThiL comprised in SEQ ID NO: 15; j) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 20 or genomic DNA thereof encoding the ThiI comprised in SEQ ID NO: 19; k) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 10 or genomic DNA thereof encoding the ThiH comprised in SEQ ID NO: 9; l) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 24 or genomic DNA thereof encoding the TenI comprised in SEQ ID NO: 23; m) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: : 30, 32, 34, 36, 38, 40, 42 and / or 44 or genomic DNA thereof encoding the Thiaminase I comprised in SEQ ID NO: 29, 31, 33, 35, 37, 39, 41 and / or 43; n) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: : 142 or genomic DNA thereof encoding the thiK comprised in SEQ ID NO: 141; o) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, suchCase Ref. P180WO IPTector® as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 110 or genomic DNA thereof encoding the IscR comprised in SEQ ID NO: 109; p) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 146 or genomic DNA thereof encoding the IscS comprised in SEQ ID NO: 145; q) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 82 or genomic DNA thereof encoding the deCARB comprised in SEQ ID NO: 81; r) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 108 or genomic DNA thereof encoding the ThiP comprised in SEQ ID NO: 107; s) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 112 or genomic DNA thereof encoding the fur-k14I comprised in SEQ ID NO: 111; and / or t) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138 and / or 140 or genomic DNA thereof encoding the TenA Thiaminase II comprised in SEQ ID NO: 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137 and / or 139; u) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 114 or genomic DNA thereof encoding the CyaA comprised in SEQ ID NO: 113; v) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68,70, 72, 74, 76, 7880, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104 and / or 106 or genomic DNA thereof encoding the PHOS comprised in SEQ ID NO: 47, 49, 51, 53,Case Ref. P180WO IPTector® 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 83, 85, 87, 89,91, 93, 95, 97, 99, 101, 103 and / or 105; w) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 144 or genomic DNA thereof encoding the SodA comprised in SEQ ID NO: 143 x) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 26 or genomic DNA thereof encoding the TH2 TMP phosphatase comprised in SEQ ID NO: 25. y) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 148 or genomic DNA thereof encoding the dxs comprised in SEQ ID NO: 147.

[0128] In another aspect the genetically modified host cells having increased production of thiamine compound provided for herein, expresses in some embodiments one or more thiamine pathway enzymes converting into thiamine, in the host cell, whereby the production of the thiamine in the genetically modified host cell is improved compared to an unmodified parent host cell.

[0129] In yet another aspect the genetically modified host cells having increased production of caboxy-sulfurol compound provided for herein, expresses in some embodiments one or more heterologous thiamine pathway enzymes converting into caboxy-sulfurol compound in the host cell, whereby the production of the caboxy-sulfurol compound in the genetically modified host cell is improved compared to an unmodified parent host cell.

[0130] In some embodiments the host cell further comprises an operative metabolic pathway comprising one or more native or heterologous pathway elements producing the sulfurol compound.

[0131] In some embodiments the host cell comprises one or more pathway elements comprise one or more FeS cluster dependent enzymes.

[0132] In some embodiments the host comprises one or more pathway elements comprise one or more radical SAM enzymes.

[0133] In some embodiments the host cell comprises one or more pathway enzymes carry N-terminal signal peptides directing newly synthesized proteins toward the secretory pathway.

[0134] In some embodiments the host cell comprises one or more pathway elements selected from: a) ThiC has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identityCase Ref. P180WO IPTector® to the ThiC comprised in SEQ ID NO: 1; b) ThiD has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the ThiD comprised in SEQ ID NO: 3; c) ThiE has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the ThiE comprised in SEQ ID NO: 17; d) ThiF has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the ThiF comprised in SEQ ID NO: 5; e) ThiS has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the ThiS comprised in SEQ ID NO: 7; f) ThiO_PPT has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the ThiO_PPT comprised in SEQ ID NO: 27; g) ThiH has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the ThiH comprised in SEQ ID NO: 9; h) ThiG has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the ThiG comprised in SEQ ID NO: 11; i) TenI_BSU has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the TenI_BSU comprised in SEQ ID NO: 23; j) TMP phosphatase (TH2_ATH) has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the TH2_ATH comprised in SEQ ID NO: 25; k) Thiaminase I (Thi1_CYA) has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the Thi1_CYA comprised in SEQ ID NO: 29; l) Thiaminase I (Thi1_SAU) has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the Thi1_SAU comprised in SEQ ID NO: 31;Case Ref. P180WO IPTector® m) Thiaminase I (Thi1_ANE) has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the Thi1_ANE comprised in SEQ ID NO: 33; n) Thiaminase I (Thi1_MEL) has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the Thi1_MEL comprised in SEQ ID NO: 35; o) Thiaminase I (Thi1_NGR) has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the Thi1_NGR comprised in SEQ ID NO: 37; p) Thiaminase I (Thi1_BGL) has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the Thi1_BGL comprised in SEQ ID NO: 39; q) Thiaminase I (Thi1_HOC) has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the Thi1_HOC comprised in SEQ ID NO: 41; r) Thiaminase I (Thi1_OAL) has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the Thi1_OAL comprised in SEQ ID NO: 43; and / or s) Thiaminase II (TenA_BSU) has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the TenA_BSU comprised in SEQ ID NO: 45.

[0135] In some embodiments the host cell comprises one or more pathway elements are selected from: a) ThiC has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the ThiC comprised in SEQ ID NO: 1; b) ThiD has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the ThiD comprised in SEQ ID NO: 3; c) ThiE has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the ThiE comprised in SEQ ID NO: 17; d) ThiF has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identityCase Ref. P180WO IPTector® to the ThiF comprised in SEQ ID NO: 5; e) ThiS has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the ThiS comprised in SEQ ID NO: 7; f) ThiO_PPT has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the ThiO_PPT comprised in SEQ ID NO: 27; g) ThiH has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the ThiH comprised in SEQ ID NO: 9; h) ThiG has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the ThiG comprised in SEQ ID NO: 11; i) TMP phosphatase (TH2_ATH) has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the TH2_ATH comprised in SEQ ID NO: 25; and / or j) TenI_BSU has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the TenI_BSU comprised in SEQ ID NO: 23.

[0136] In some embodiments the host cell further comprises an operative metabolic pathway comprising one or more native or heterologous pathway elements producing the sulfurol, cTHZ-P, THZ-P, cTHZ and / or thiamine.

[0137] In some embodiments the host cell further comprises an operative metabolic pathway comprising one or more native or heterologous pathway elements producing the sulfurol compound.

[0138] In some embodiments the host cell comprises one or more pathway elements which are selected from: a) ThiF has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the ThiF comprised in SEQ ID NO: 5; b) ThiS has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the ThiS comprised in SEQ ID NO: 7; c) ThiO_PPT has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100%Case Ref. P180WO IPTector® identity to the ThiO_PPT comprised in SEQ ID NO: 28; d) ThiH has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the ThiH comprised in SEQ ID NO: 9; e) ThiG has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the ThiG comprised in SEQ ID NO: 11; f) TMP phosphatase (TH2_ATH) has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the TH2_ATH comprised in SEQ ID NO: 25; g) phosphatase PHOS_PhoA has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the phosphatase PHOS_PhoA comprised in SEQ ID NO: 47; h) Phosphatase PHOS_NudJ has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the PHOS_NudJ comprised in SEQ ID NO: 49; i) Phosphatase PHOS_Cof has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the PHOS_Cof comprised in SEQ ID NO: 51; j) Phosphatase PHOS_YbhA has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the PHOS_YbhA comprised in SEQ ID NO: 53; k) Phosphatase PHOS_KdsC has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the PHOS_KdsC comprised in SEQ ID NO: 55; l) Phosphatase PHOS_RsgA_SAE has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the PHOS_RsgA_SAE comprised in SEQ ID NO: 57; m) Phosphatase PHOS_PhoS_PAE has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the PHOS_PhoS_PAE comprised in SEQ ID NO: 59; n) Phosphatase PHOS_NudJ_PPU has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the PHOS_NudJ_PPU comprised in SEQ ID NO: 61;Case Ref. P180WO IPTector® o) Phosphatase PHOS_AlkP_PPU has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the PHOS_AlkP_PPU comprised in SEQ ID NO: 63; p) Phosphatase PHOS_ygiF_PPU has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the PHOS_ygiF_PPU comprised in SEQ ID NO: 65; q) Phosphatase PHOS_upxB_PPU has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the PHOS_upxB_PPU comprised in SEQ ID NO: 67; r) Phosphatase PHOS_PhoD_PPU has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the PHOS_PhoD_PPU comprised in SEQ ID NO: 69; s) Phosphatase PHOS_AphA has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the PHOS_AphA comprised in SEQ ID NO: 71; t) Phosphatase PHOS_NudJ_SSO has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the PHOS_NudJ_SSO comprised in SEQ ID NO: 73; u) Phosphatase PHOS_PhoD_PAS has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the PHOS_PhoD_PAS comprised in SEQ ID NO: 75; v) Phosphatase PHOS_AH2_HIN has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the PHOS_AH2_HIN comprised in SEQ ID NO: 77; and / or w) Phosphatase PHOS_AH2_CSI has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the PHOS_AH2_CSI comprised in SEQ ID NO: 79.

[0139] In some embodiments the host cell comprises one or more pathway elements encoded by one or more genes selected from the group of: a) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 2 or genomic DNA thereof encoding the ThiC comprised in SEQ ID NO: 1; b) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at leastCase Ref. P180WO IPTector® 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 4 or genomic DNA thereof encoding the ThiD comprised in SEQ ID NO: 3; c) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 18 or genomic DNA thereof encoding the ThiE comprised in SEQ ID NO: 17; d) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 6 or genomic DNA thereof encoding the ThiF comprised in SEQ ID NO: 5; e) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 8 or genomic DNA thereof encoding the ThiS comprised in SEQ ID NO: 7; f) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 28 or genomic DNA thereof encoding the ThiO_PPT comprised in SEQ ID NO: 27; g) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 12 or genomic DNA thereof encoding the ThiG comprised in SEQ ID NO: 11; h) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 10 or genomic DNA thereof encoding the ThiH comprised in SEQ ID NO: 9; i) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 24 or genomic DNA thereof encoding the TenI_BSU comprised in SEQ ID NO: 23; j) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at leastCase Ref. P180WO IPTector® 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: : 26 or genomic DNA thereof encoding the TH2_ATH comprised in SEQ ID NO: 25; k) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 30 or genomic DNA thereof encoding the Thiaminase I Thi1_CYA comprised in SEQ ID NO: 29; l) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 32 or genomic DNA thereof encoding the Thiaminase I Thi1_SAU comprised in SEQ ID NO: 31; m) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 34 or genomic DNA thereof encoding the Thiaminase I Thi1_ANE comprised in SEQ ID NO: 33; n) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 36 or genomic DNA thereof encoding the Thiaminase I Thi1_MEL comprised in SEQ ID NO: 35; o) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 38 or genomic DNA thereof encoding the Thiaminase I Thi1_NGR comprised in SEQ ID NO: 37; p) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 40 or genomic DNA thereof encoding the Thiaminase I Thi1_BGL comprised in SEQ ID NO: 39;Case Ref. P180WO IPTector® q) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 42 or genomic DNA thereof encoding the Thiaminase I Thi1_HOC comprised in SEQ ID NO: 41; r) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 44 or genomic DNA thereof encoding the Thiaminase I Thi1_OAL comprised in SEQ ID NO: 43; and / or s) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 46 or genomic DNA thereof encoding the Thiaminase II TenA_BSU comprised in SEQ ID NO: 45.

[0140] In some embodiments the host cell comprises one or more pathway elements encoded by one or more genes selected from the group of: a) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 2 or genomic DNA thereof encoding the ThiC comprised in SEQ ID NO: 1; b) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 4 or genomic DNA thereof encoding the ThiD comprised in SEQ ID NO: 3; c) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 18 or genomic DNA thereof encoding the ThiE comprised in SEQ ID NO: 17; d) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 6 or genomic DNA thereof encoding the ThiF comprised in SEQ ID NO: 5; e) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, suchCase Ref. P180WO IPTector® as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 8 or genomic DNA thereof encoding the ThiS comprised in SEQ ID NO: 7; f) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 28 or genomic DNA thereof encoding the ThiO_PPT comprised in SEQ ID NO: 27; g) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 12 or genomic DNA thereof encoding the ThiG comprised in SEQ ID NO: 11; h) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 10 or genomic DNA thereof encoding the ThiH comprised in SEQ ID NO: 9; i) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 26 or genomic DNA thereof encoding the TH2_ATH comprised in SEQ ID NO: 25; and / or j) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 24 or genomic DNA thereof encoding the TenI_BSU comprised in SEQ ID NO: 23.

[0141] In some embodiments the host cell comprises one or more pathway elements encoded by one or more genes selected from the group of: a) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 6 or genomic DNA thereof encoding the ThiF comprised in SEQ ID NO: 5; b) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 8 or genomic DNA thereof encoding the ThiS comprised in SEQ ID NO: 7; c) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at leastCase Ref. P180WO IPTector® 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 28 or genomic DNA thereof encoding the ThiO_PPT comprised in SEQ ID NO: 27; d) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 12 or genomic DNA thereof encoding the ThiG comprised in SEQ ID NO: 11; e) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 10 or genomic DNA thereof encoding the ThiH comprised in SEQ ID NO: 9; f) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 26 or genomic DNA thereof encoding the phosphatase TH2_ATH comprised in SEQ ID NO: 25; g) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 48 or genomic DNA thereof encoding the phosphatase PHOS_PhoA comprised in SEQ ID NO: 47; h) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 50 or genomic DNA thereof encoding the phosphatase PHOS_NudJ comprised in SEQ ID NO: 49; i) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 52 or genomic DNA thereof encoding the phosphatase PHOS_Cof comprised in SEQ ID NO: 51; j) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%,Case Ref. P180WO IPTector® such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 54 or genomic DNA thereof encoding the phosphatase PHOS_YbhA comprised in SEQ ID NO: 53; k) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 56 or genomic DNA thereof encoding the phosphatase PHOS_KdsC comprised in SEQ ID NO: 55; l) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 58 or genomic DNA thereof encoding the phosphatase PHOS_RsgA_SAE comprised in SEQ ID NO: 57; m) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 60 or genomic DNA thereof encoding the phosphatase PHOS_PhoS_PAE comprised in SEQ ID NO: 59; n) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 62 or genomic DNA thereof encoding the phosphatase PHOS_NudJ_PPU comprised in SEQ ID NO: 61; o) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 64 or genomic DNA thereof encoding the phosphatase PHOS_AlkP_PPU comprised in SEQ ID NO: 63; p) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 66 or genomic DNA thereof encoding the phosphatase PHOS_ygiF_PPU comprised in SEQ ID NO: 65; q) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at leastCase Ref. P180WO IPTector® 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 68 or genomic DNA thereof encoding the phosphatase PHOS_upxB_PPU comprised in SEQ ID NO: 67; r) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 70 or genomic DNA thereof encoding the phosphatase PHOS_PhoD_PPU comprised in SEQ ID NO: 69; s) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 72 or genomic DNA thereof encoding the phosphatase PHOS_AphA comprised in SEQ ID NO: 71; t) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 74 or genomic DNA thereof encoding the phosphatase PHOS_NudJ_SSO comprised in SEQ ID NO: 73; u) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 76 or genomic DNA thereof encoding the phosphatase PHOS_PhoD_PAS comprised in SEQ ID NO: 75; v) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 78 or genomic DNA thereof encoding the phosphatase PHOS_AH2_HIN comprised in SEQ ID NO: 77; w) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 80 or genomic DNA thereof encoding the phosphatase PHOS_AH2_CSI comprised in SEQ ID NO: 79; and / orCase Ref. P180WO IPTector® x) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 82 or genomic DNA thereof encoding the deCARB_CadA comprised in SEQ ID NO: 81.

[0142] In some embodiments the host cell comprises one or more pathway elements encoded by one or more genes selected from the group of: a) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 116 or genomic DNA thereof encoding the tenA_HP1287_HPY comprised in SEQ ID NO: 115; b) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 118 or genomic DNA thereof encoding the tenA_AAN comprised in SEQ ID NO: 117; c) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 120 or genomic DNA thereof encoding the tenA_AMI comprised in SEQ ID NO: 119; d) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 122 or genomic DNA thereof encoding the TenA_BSU comprised in SEQ ID NO: 121; e) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 124 or genomic DNA thereof encoding the tenA_CPA comprised in SEQ ID NO: 123; f) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 126 or genomic DNA thereof encoding the tenA_HHA comprised in SEQ ID NO: 125; g) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 128 or genomicCase Ref. P180WO IPTector® DNA thereof encoding the tenA_PAE comprised in SEQ ID NO: 127; h) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 130 or genomic DNA thereof encoding the tenA_PFU comprised in SEQ ID NO: 129; i) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 132 or genomic DNA thereof encoding the tenA_PHO comprised in SEQ ID NO: 131; j) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 134 or genomic DNA thereof encoding the tenA_SAU comprised in SEQ ID NO: 133; k) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 136 or genomic DNA thereof encoding the tenA_SEP comprised in SEQ ID NO: 135; l) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 138 or genomic DNA thereof encoding the tenA_SSO comprised in SEQ ID NO: 137; m) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 140 or genomic DNA thereof encoding the tenA_THI20_SCE comprised in SEQ ID NO: 139; n) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 142 or genomic DNA thereof encoding the thiK comprised in SEQ ID NO: 141; o) gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 144 or genomic DNA thereof encoding the SodA comprised in SEQ ID NO: 143; p) gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%,Case Ref. P180WO IPTector® such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 146 or genomic DNA thereof encoding the iscS comprised in SEQ ID NO: 145; and / or q) gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 148 or genomic DNA thereof encoding the dxs comprised in SEQ ID NO: 147.

[0143] In some embodiments the host cell comprises one or more pathway elements encoded by one or more genes selected from the group of: a) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 84 or genomic DNA thereof encoding the PHOS_At4g29530_ATH comprised in SEQ ID NO: 83; b) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 86 or genomic DNA thereof encoding the PHOS_TMPPH_APA comprised in SEQ ID NO: 85; c) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 88 or genomic DNA thereof encoding the PHOS_HADPH_ACO comprised in SEQ ID NO: 87; d) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 90 or genomic DNA thereof encoding the PHOS_HADPH_DLO comprised in SEQ ID NO: 89; e) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 92 or genomic DNA thereof encoding the PHOS_HADPH_EVE comprised in SEQ ID NO: 91;Case Ref. P180WO IPTector® f) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 92 or genomic DNA thereof encoding the PHOS_HADPH_EVE comprised in SEQ ID NO: 91; g) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 94 or genomic DNA thereof encoding the PHOS_PSPH_SWO comprised in SEQ ID NO: 93; h) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 96 or genomic DNA thereof encoding the PHOS_HADPH_SEQ comprised in SEQ ID NO: 95; i) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 98 or genomic DNA thereof encoding the PHOS_HADPH_RUM comprised in SEQ ID NO: 97; j) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 100 or genomic DNA thereof encoding the PHOS_HAD_OBA comprised in SEQ ID NO: 99; k) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 102 or genomic DNA thereof encoding the PHOS_HADH_TSP comprised in SEQ ID NO: 101; l) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 104 or genomic DNA thereof encoding the PHOS_HADPH_ROS comprisedCase Ref. P180WO IPTector® in SEQ ID NO: 103; m) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 106 or genomic DNA thereof encoding the PHOS_HADPH_CBA comprised in SEQ ID NO: 105;

[0144] In some embodiments the host cell comprises one or more pathway elements encoded by one or more genes selected from the group of: a) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 108 or genomic DNA thereof encoding the ThiP comprised in SEQ ID NO: 107; b) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 110 or genomic DNA thereof encoding the iscR-H107Y comprised in SEQ ID NO: 109; c) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 112 or genomic DNA thereof encoding the fur-K14I comprised in SEQ ID NO: 111; d) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 114 or genomic DNA thereof encoding the cyaA comprised in SEQ ID NO: 113;

[0145] In some embodiments the host cell comprises one or more pathway elements encoded by one or more genes selected from the group of: a) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 116 or genomic DNA thereof encoding the tenA_HP1287_HPY comprised inCase Ref. P180WO IPTector® SEQ ID NO: 115; b) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 118 or genomic DNA thereof encoding the tenA_AAN comprised in SEQ ID NO: 117; c) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 120 or genomic DNA thereof encoding the tenA_AMI comprised in SEQ ID NO: 119; d) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 122 or genomic DNA thereof encoding the TenA_BSU comprised in SEQ ID NO: 121; e) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 124 or genomic DNA thereof encoding the tenA_CPA comprised in SEQ ID NO: 123; f) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 126 or genomic DNA thereof encoding the tenA_HHA comprised in SEQ ID NO: 125; g) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 128 or genomic DNA thereof encoding the tenA_PAE comprised in SEQ ID NO: 127; h) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprisedCase Ref. P180WO IPTector® in SEQ ID NO: 130 or genomic DNA thereof encoding the tenA_PFU comprised in SEQ ID NO: 129; i) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 132 or genomic DNA thereof encoding the tenA_PHO comprised in SEQ ID NO: 131; j) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 134 or genomic DNA thereof encoding the tenA_SAU comprised in SEQ ID NO: 133; k) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 136 or genomic DNA thereof encoding the tenA_SEP comprised in SEQ ID NO: 135; l) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 138 or genomic DNA thereof encoding the tenA_SSO comprised in SEQ ID NO: 137; m) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 140 or genomic DNA thereof encoding the tenA_THI20_SCE comprised in SEQ ID NO: 139; n) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 142 or genomic DNA thereof encoding the thiK comprised in SEQ ID NO: 141; o) gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, suchCase Ref. P180WO IPTector® as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 144 or genomic DNA thereof encoding the SodA comprised in SEQ ID NO: 143; p) gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 146 or genomic DNA thereof encoding the iscS comprised in SEQ ID NO: 145; and / or q) gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 148 or genomic DNA thereof encoding the dxs comprised in SEQ ID NO: 147.

[0146] In some embodiments the host cell comprises one or more genes and / or polypeptides of the pathway for the sulfurol which are heterologous to the host cell.

[0147] In some embodiments the host cell comprises one or more genes and / or polypeptides of the pathway for the thiamine which are heterologous to the host cell.

[0148] In some embodiments the host cell comprises one or more genes and / or polypeptides of the pathway for the carboxy- sulfurol which are heterologous to the host cell.

[0149] In some embodiments the host cell comprising at least 2 copies of one or more genes and / or polypeptides of the pathway for the sulfurol compound.

[0150] In some embodiments the host cell further comprising a transporter molecule facilitating transport of a precursor for or a product of the pathway for the sulfurol compound.

[0151] In some embodiments the host cell is further genetically modified to provide an increased amount of a substrate in the pathway for the sulfurol compound.

[0152] In some embodiments the host cell is further genetically modified to exhibit increased tolerance towards one or more substrates, intermediates, or products in the pathway for the sulfurol compound.

[0153] In some embodiments the host cell comprising one or more deleted, disrupted and / or attenuated additional native or endogenous genes of the host cell.

[0154] In some embodiments the host cell comprises one or more genes in the pathway for the sulfurol compound which is overexpressed.

[0155] In some embodiments the host cell is prokaryotic or eukaryotic.

[0156] In some embodiments the host cell is prokaryotic and of a genus selected from the group consisting of Escherichia, Bacillus, Brevibacterium, Burkholderia, Campylobacter, Corynebacterium, Serratia, Lactobacillus, Lactococcus, Acinetobacter, Acetobacter and Pseudomonas.Case Ref. P180WO IPTector®

[0157] In some embodiments the host cell is of the genus Escherichia, Corynebacterium, Bacillus, Serratia, Pseudomonas.

[0158] In some embodiments the host cell is selected from the species Escherichia coli, Corynebacterium glutamicum, Bacillus subtilis, Serratia marcescens, pseudomonas putida and / or Pseudomonas mutabilis.

[0159] In some embodiments the host cell is eukaryotic and selected from the group consisting of mammalian, insect, plant, fungal or archaeal cells.

[0160] In some embodiments the host cell is a fungal cell of a genus selected from Saccharomyces, Pichia and / or Ashbya.

[0161] In some embodiments the host cell is selected from the species Saccharomyces cerevisiae, Pichia pastoris and / or Ashbya gossypii.

[0162] In some embodiments a mutated a polypeptide selected from the group consisting of a polypeptide which is at least 90% identical to: a) the mutant phosphatase having a sequence comprised in SEQ ID NO: 25, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 77, 79, 83, 85, 87, 89,91, 93, 95, 97, 99, 101, 103 and / or 105; b) the mutant thiaminase I having a sequence comprised in SEQ ID NO: 29, 31, 33, 35, 37, 39, 41 and / or 43; and / or c) the mutant thiaminase II having a sequence comprised in SEQ ID NO: 45, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137 and / or 139.

[0163] In some embodiments the polynucleotide construct of the invention comprising a polynucleotide sequence encoding a phosphatase, thiaminase I and / or thiaminase II operably linked to one or more control sequences, wherein the polynucleotide construct comprises one or more mutations deleting, disrupting and / or attenuating the phosphatase and / or thiaminase I.

[0164] In some embodiments the polynucleotide construct is an expression vector.

[0165] In some embodiments the host cell comprising the polynucleotide construct of disclosed above.

[0166] In some embodiments the host cell comprising a cell culture and a growth medium.

[0167] In some embodiments the host cell is configured for producing cTHZ-P and comprises or expresses genes encoding: a. thiF, such as the ThiF having an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the ThiF comprised in SEQ ID NO: 5; b. thiS, such as the ThiS having an amino acid sequence which is at least 70%, such at least 75%,Case Ref. P180WO IPTector® such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the ThiS comprised in SEQ ID NO: 7; c. thiG, such as the ThiG having an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the ThiG comprised in SEQ ID NO: 11; d. thiI, such as the ThiI having an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the ThiI comprised in SEQ ID NO: 19; e. thiO, such as the ThiO having an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the ThiO comprised in SEQ ID NO: 27; f. thiH, such as the ThiH having an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the ThiH comprised in SEQ ID NO: 9; g. iscS, such as the IscS having an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the IscS comprised in SEQ ID NO: 145; and / or h. dxs, such as the dxs having an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the dxs comprised in SEQ ID NO: 147.

[0168] In some embodiments the host cell is configured for producing thiamine and comprises or expresses genes encoding: a. optionally ThiC, such as ThiC having an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the ThiC comprised in SEQ ID NO: 1; b. ThiD, such as ThiD having an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the ThiD comprised in SEQ ID NO: 3; c. ThiE, such as ThiE having an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the ThiE comprised in SEQ ID NO: 17;ThiF has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the ThiF comprised in SEQ ID NO: 5; d. TH2, such as the TH2 having an amino acid sequence which is at least 70%, such at least 75%,Case Ref. P180WO IPTector® such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the TH2 comprised in SEQ ID NO: 25; and / or e. optionally a thiaminase, such as Thi1 or TenA, for example wherein the Thi1 has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the Thi1 comprised in SEQ ID NO: 29, 31, 33, 35, 37, 39, 41 and / or 43, or wherein the TenA has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the TenA comprised in SEQ ID NO: 45, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137 and / or 139.

[0169] In some embodiments the host cell is configured for producing carboxy sulfurol (cTHZ) and comprises or expresses genes encoding a phosphatase capable of dephosphorylating cTHZ-P to cTHZ, such as wherein the phosphatase has an amino acid sequence which is at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the phosphatase comprised in SEQ ID NO: 47, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103 and / or 105.

[0170] In some embodiments of the host cell the substrate in the pathway for the sulfurol compound is selected from the group consisting of: cTHZ-P, THZ-P, cTHZ and thiamine. Method of production

[0171] In some aspects, a method for producing sulfurol is provided comprising: a) culturing the cell culture described herein at conditions allowing the host cells to produce the thiamine compound; b) optionally recovering and / or isolating the thiamine broth; c) subjecting the thiamine broth to thiaminase I; and / or d) optionally recovering and / or isolating the sulfurol compound.

[0172] In some aspects a method for producing sulfurol is provided comprising: a) culturing the cell culture described herein at conditions allowing the host cells to produce the 5-(2-hydroxyethyl)-4-methyl-1,3-thiazole-2-carboxylic acid (cTHZ) compound; b) optionally recovering and / or isolating the 5-(2-hydroxyethyl)-4-methyl-1,3-thiazole-2- carboxylic acid (cTHZ) compound; c) performing chemical decarboxylation of 5-(2-hydroxyethyl)-4-methyl-1,3-thiazole-2-carboxylic acid (cTHZ) (1) to sulfurol (2):Case Ref. P180WO IPTector®and / or d. optionally recovering and / or isolating the sulfurol compound.

[0173] In some aspects, a method for producing sulfurol is provided comprising: a) culturing the cell culture described herein at conditions allowing the host cells to produce the 5-(2-hydroxyethyl)-4-methyl-1,3-thiazole-2-carboxylic phosphate acid (cTHZ-P) compound; b) optionally recovering and / or isolating the 5-(2-hydroxyethyl)-4-methyl-1,3-thiazole-2- carboxylic phosphate acid (cTHZ-P) compound; c) chemical dephosphorylation yielding carboxy-sulfurol (1) via basic hydrolysis of 5-(2- hydroxyethyl)-4-methyl-1,3-thiazole-2-carboxylic phosphate acid (cTHZ-P) (3):and the subsequent chemical decarboxylation of 5-(2-hydroxyethyl)-4-methyl-1,3-thiazole-2- carboxylic acid (cTHZ) (1) to sulfurol (2) as shown in (a); and / or d. optionally recovering and / or isolating the sulfurol compound.

[0174] In some embodiments the polynucleotide construct of the invention comprising a polynucleotide sequence encoding a phosphatase, thiaminase I and / or thiaminase II operably linked to one or more control sequences, wherein the polynucleotide construct comprises one or more mutations deleting, disrupting and / or attenuating the phosphatase, thiaminase I and / or thiaminase II.

[0175] In some aspects, a method for producing sulfurol is provided comprising: culturing the cell culture as defined herein at conditions allowing the host cells to produce the sulfurol compound; optionally recovering and / or isolating the sulfurol compound.

[0176] In some aspects a method of producing sulfurol is provided comprising a) culturing the cell culture described herein at conditions allowing the host cells to produce the thiamine compound; b) optionally recovering and / or isolating the thiamine compound;Case Ref. P180WO IPTector® c) chemical conversion of thiamine to sulfurol byand / or d) optionally recovering and / or isolating the sulfurol compound.

[0177] In another aspect a method of producing sulfurol compound is provided comprising: a. culturing the cell culture at conditions allowing the host cells to produce the thiamine compound; b. chemically converting the thiamine compound to sulfurol; c. optionally recovering and / or isolating the sulfurol compound.

[0178] In some embodiments, the method comprises a step of recovering thiamine from the cell culture prior to step b.

[0179] In some embodiments the method for producing a sulfurol compound comprising a chemical conversion of thiamine with bisulphite in a concentration from 0,2 to 4,0 w / v such as 1,0, 1,5, 2,0, 2,5, 3,0, 3,5 or 4,0% w / v.

[0180] In some embodiments the method for producing a sulfurol compound comprising a bisulphite selected from Potassium bisulphite, Sodium metabisulphite and / or Sodium bisulphite (NaHSO3).

[0181] In some embodiments the method for producing a sulfurol compound wherein the bisulphite is Sodium bisulphite (NaHSO3).

[0182] In some embodiments the method for producing a sulfurol compound wherein the Sodium bisulphite (NaHSO3) is in a concentration of 2,0 w / v.

[0183] In some embodiments the method for producing a sulfurol compound comprising a chemical conversion of thiamine with bisulphite at a pH from 3.0 to 8.0, such as 3,0, 3,5, 4,0, 4,5, 5,0, 5,5, 6,0, 6,5, 7,0 and / or 8,0.

[0184] In some embodiments the method for producing a sulfurol compound comprising a chemical conversion of thiamine with bisulphite at a pH of 4.5 to 6.0 such as pH 5,5.

[0185] In some embodiments the method for producing a sulfurol compound comprising a chemical conversion of thiamine with bisulphite at a temperature from 5 to 30 degree Celsius, such as 5, 10, 15, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 to 30 degree Celsius or at room temperature such as 15 to 25Case Ref. P180WO IPTector® degree Celsius.

[0186] In some embodiments the method for producing a sulfurol compound comprising a chemical conversion of thiamine with bisulphite at room temperature such as 15 to 25 degree Celsius such as 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25 degree Celcius.

[0187] In some embodiments the method for producing sulfurol at conditions allowing the host cells to produce a thiamine compound; a. chemically converting the thiamine compound to sulfurol by adding 2,0, % w / v of NaHSO3at pH 5,5 at room temperature such as 15 to 25 degree Celsius: b. optionally recovering and / or isolating the sulfurol compound.

[0188] In some embodiments the method for producing a sulfurol compound comprising: a. culturing the cell culture as defined herein at conditions allowing the host cells to produce the carboxy-sulfurol compound; b. recovering and / or isolating the carboxy-sulfurol compound broth by a resin and / or an organic solvent; c. chemically converting the carboxy-sulfurol compound to sulfurol; d. optionally recovering and / or isolating the sulfurol compound.

[0189] In some other aspects the method further comprising feeding one or more exogenous sulfurol precursor therefore to the host cell culture, optionally selected from the group of O-methylpimeloyl- acyl carrier protein, pimeloyl-acyl carrier protein, KAPA, DAPA, DTB, amino-HMP, and pimelate.

[0190] In another aspect a method of producing sulfurol is provided wherein recovering and / or isolating the carboxy-sulfurol compound broth comprises a strong anionic or weak anionic resin such as AmberLite FPA66 OH, AmberLite IRN78 OH, AmberLyst A26 OH, Ambersep 900 and / or AmberLite IRA67.

[0191] In some embodiments the method for producing a sulfurol compound wherein recovering and / or isolating the carboxy-sulfurol compound broth comprises an organic solvent such as Ethyl acetate (EtOAc / CH3COOC2H5), Ethanol (C2H6O), n-Butanol (C2H6O), and / or methylethyl ketone for extraction.

[0192] In some embodiments the method for producing a sulfurol compound wherein recovering and / or isolating the carboxy-sulfurol compound broth comprises a resin and Ethyl acetate (EtOAc / CH3COOC2H5) for extraction.

[0193] In some embodiments the method for producing a sulfurol compound comprising a chemical conversion of carboxy-sulfurol with an acid.

[0194] In some embodiments the method for producing a sulfurol compound comprising a chemical conversion of carboxy-sulfurol with an acid such as HCl, Acetic acid (HC2H3O2), Phosphoric acid (H3PO4),Case Ref. P180WO IPTector® nitric acid (HNO3) and / or sulfuric acid (H2SO4).

[0195] In some embodiments of the method, the carboxy-sulfurol (cTHZ) yield and / or titer is at least 4 mg / L, optionally at least 8 mg / L, optionally at least 10mg / L, optionally at least 15 mg / L, optionally at least 20 mg / L, optionally at least 30mg / L, optionally at least 50 mg / L, optionally at least 100 mg / L, optionally at least 150mg / L, optionally at least 200 mg / L, 300mg / L, optionally at least 500 mg / L, optionally at least 1 g / L, optionally at least 1.5 g / L, optionally at least 2 g / L, for example at least 10 g / L, such as at least 15 g / L.

[0196] In some embodiments of the method, the thiamine yield and / or titer is at least 4 mg / L, optionally at least 8 mg / L, optionally at least 10mg / L, optionally at least 15 mg / L, optionally at least 20 mg / L, optionally at least 30mg / L, optionally at least 50 mg / L, optionally at least 100 mg / L, optionally at least 150mg / L, optionally at least 200 mg / L, 300mg / L, optionally at least 500 mg / L, optionally at least 1 g / L, optionally at least 1.5 g / L, optionally at least 2 g / L, for example at least 10 g / L, such as at least 15 g / L.

[0197] In some embodiments the method, one or more steps of producing the sulfurol compound is performed in vitro, such as by chemical synthesis.

[0198] In some embodiments the method of preparing sulfurol from a sulfurol precursor comprising : a. Obtaining the sulfurol precursor by culturing the host cell of any preceding claims in a growth medium to provide the sulfurol precursor; b. Recovering the sulfurol precursor from the growth medium, c. Converting the sulfurol precursor to sulfurol in vitro, optionally by chemical synthesis.

[0199] In some embodiments the method wherein the sulfurol precursor is selected from the group consisting of: thiamine (THI), carboxy-sulfurol (cTHZ), 4-methyl-5-beta-hydroxyethylthiazole phosphate (THZ-P), and 5-(2-hydroxyethyl)-4-methyl-1,3-thiazole-2-carboxylic phosphate acid (cTHZ- P).

[0200] In some embodiments the method wherein the sulfurol precursor is thiamine and step c) is conducted using a bisulfite, such as NaHSO3.

[0201] In some embodiments the method wherein the sulfurol precursor is cTHZ and step c) is conducted using a base.

[0202] In some embodiments the method wherein the sulfurol precursor is THZ-P and step c) is conducted using an acid and optionally a metal cation.

[0203] In some embodiments the method wherein the sulfurol precursor is cTHZ-P and step c) is conducted using an acid, for example hydrochloric acid (HCl), and optionally a metal cation, such as Zn2+.

[0204] In some embodiments of the method wherein a substrate in the pathway for producingCase Ref. P180WO IPTector® sulfurol, THI, cTHZ, THZ-P, and / or cTHZ-P is added to the host cell.

[0205] In some embodiments of the method the substrate is amino-HMP.

[0206] In some embodiments of the method the host cell does not comprise or express ThiC.

[0207] In some embodiments of the method the host cell comprises or expresses a gene encoding: a thiaminase, such as a thiaminase defined in any one of the preceding claims, for example Thi1 or TenA.

[0208] In some embodiments the method for producing a sulfurol compound comprising a chemical conversion of carboxy-sulfurol with an acid at an acidic pH from 0.1 to 6.0, such as 0.1, 0.5, 1.0, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 6.0 and / or an acid in the molarity from 0.05 to 2 such as 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 2.0. In some embodiments the method for producing a sulfurol compound comprising a chemical conversion of carboxy-sulfurol with an acid at an acidic pH of 1.0 to 4.0 and / or molarity of 0.1 to 1.0.

[0209] In some embodiments the method for producing a sulfurol compound comprising a chemical conversion of carboxy-sulfurol with an acid at a temperature from 50 to 150 degrees Celsius, such as a temperature from 50, 60, 70, 80, 90, 100, 110, 120, 130, 140 and / or 150 degrees Celsius.

[0210] In some embodiments the method for producing a sulfurol compound comprising a chemical conversion of carboxy-sulfurol with an acid at 100 degrees Celsius.

[0211] In some embodiments the method of producing a sulfurol compound at conditions allowing the host cells to produce a carboxy-sulfurol compound comprising: a. recovering and / or isolating the carboxy-sulfurol compound broth by a strong anionic or weak anionic resin such as AmberLite FPA66 OH, AmberLite IRN78 OH, AmberLyst A26 OH, Ambersep 900 and / or AmberLite IRA67 and / or EtOAc extraction; b. chemically converting the carboxy-sulfurol compound to sulfurol with CH3COOH and / or HCl at an acidic pH at 100 degree Celsius: c. optionally recovering and / or isolating the sulfurol compound.

[0212] In some other aspects the method of producing sulfurol further comprising one or more elements selected from: a) culturing the cell culture under aerobic or anaerobic conditions b) cultivating the host cells under mixing; c) cultivating the host cells at a temperature of between 25°C to 50°C; d) cultivating the host cells at a pH of between 3-9; and / or e) cultivating the host cells for between 10 hours to 120 days.

[0213] In some embodiments the method of producing sulfurol comprising recovering and / or isolation step comprises separating a liquid phase of the cell culture from a solid phase of the cell culture to obtain a supernatant comprising the sulfurol compound and subjecting the supernatant toCase Ref. P180WO IPTector® one or more steps selected from: a) contacting the supernatant with one or more adsorbent resins in order to obtain at least a portion of the produced sulfurol compound, then optionally recovering the sulfurol compound from the resin in a concentrated solution prior to isolation of the sulfurol compound by crystallisation or solvent evaporation; b) contacting the supernatant with one or more ion exchange or reversed-phase chromatography columns to obtain at least a portion of the sulfurol compound, then optionally recovering the sulfurol compound from the resin in a concentrated solution prior to isolation of the sulfurol compound by crystallisation or solvent evaporation; and / or c) extracting the thiamine compound from the supernatant, such as by liquid-liquid extraction into an immiscible solvent, then optionally isolating the sulfurol compound by crystallisation or solvent evaporation; and thereby recovering and / or isolating the sulfurol compound.

[0214] In some embodiments of the method of producing sulfurol, the sulfurol compound yield is at least 10% higher such as at least 50%, such as at least 100%, such as least 150%, such as at least 200% higher than production by a host cell by a) expressing one or more heterologous phosphatase and / or thiaminase genes capable of converting 5-(2-hydroxyethyl)-4-methyl-1,3-thiazole-2-carboxylic phosphate acid (cTHZ-P) or thiamine into sulfurol, THZ-P and / or 5-(2-hydroxyethyl)-4-methyl-1,3-thiazole-2-carboxylic acid (cTHZ) in the host cell; and / or b) over-expressing one or more ThiO genes capable of catalysing the glycine to DHG in the host cell.

[0215] In some embodiments of the method, the sulfurol yield and / or titer is at least 4 mg / L, optionally at least 8 mg / L, optionally at least 10mg / L, optionally at least 15 mg / L, optionally at least 20 mg / L, optionally at least 30mg / L, optionally at least 50 mg / L, optionally at least 100 mg / L, optionally at least 150mg / L, optionally at least 200 mg / L, 300mg / L, optionally at least 500 mg / L, optionally at least 1 g / L, optionally at least 1.5 g / L, optionally at least 2 g / L, optionally at least 3g / L, optionally at least 5 g / L, optionally at least 8 g / L, optionally at least 10 g / L, optionally at least 20 g / L.

[0216] In some embodiments the method, one or more steps of producing the sulfurol compound is performed in vitro.

[0217] In some embodiments the method further comprising mixing the sulfurol compound with one or more carriers, agents, adjuvants, additives and / or excipients, optionally pharmaceutical grade carriers, agents, adjuvants, additives and / or excipients.

[0218] In some embodiments is disclosed a fermentation composition comprising the cell culture from the sulfurol production method and the sulfurol compound.Case Ref. P180WO IPTector®

[0219] In some embodiments is disclosed a fermentation composition, wherein at least 50%, such as at least 75%, such as at least 95%, such as at least 99% of the genetically modified host cells are lysed and / or disintegrated.

[0220] In some embodiments is disclosed a fermentation composition wherein at least 50%, such as at least 75%, such as at least 95%, such as at least 99% of solid cellular material has been separated from the composition.

[0221] In some embodiments the fermentation composition further comprising one or more compounds selected from: a) precursor or products of the operative metabolic pathway producing the sulfurol compound; b) supplemental nutrients; and wherein the concentration of the sulfurol compound is at least 1 mg / L composition.

[0222] In some embodiments is disclosed a composition comprising the fermentation composition and one or more carriers, agents, adjuvants, additives and / or excipients and at least trace amounts of one or more metabolites of the cell culture, optionally signature metabolites for the genetically modified host cell.

[0223] In some embodiments is disclosed a fermentation composition and one or more carriers, agents, adjuvants, additives and / or excipients which are in a dry solid form.

[0224] In some embodiments the composition comprising the fermentation composition and the one or more carriers, agents, adjuvants, additives and / or excipients are in a liquid stabilized form.

[0225] In some embodiments the composition is refined into a pharmaceutical preparation, a dietary supplement, a cosmetic, a food preparation, a flavor preparation, a feed preparation and / or an analytical or diagnostic reagent.

[0226] In some embodiments the composition preparation is in form of a powder, tablet, capsule, hard chewable and or soft lozenge, liposomes or a gum.

[0227] In some embodiments the pharmaceutical preparation is in form of a liquid pharmaceutical solution.

[0228] In some embodiments, the genetically modified host cell is a cell wherein the endogenous native ThiH gene is deleted, disrupted and / or attenuated. In a further embodiment, the deletion, disruption and / or attenuation is caused by introducing a deletion through complete removal of the gene or a translational knockout by introducing one or more stop codons or frameshift mutations preventing expression of an active peptide. In another embodiment, the deletion, disruption and / or attenuation comprises a translational knockout or a frameshift mutation. In another embodiment, the deletion, disruption and / or attenuation comprises a translational knockout or a frameshift mutation. In a further embodiment, the deletion, disruption and / or attenuation is a point mutation in aCase Ref. P180WO IPTector® promoter for the protein-encoding sequence, in the RBS region and / or in a protein-encoding sequence.

[0229] In some embodiments, the gene is disrupted or deleted, wherein the gene is: a) thiC, thiE, thiF, thiS, thiG, and / or thiH; b) thiE, thiF, thiS, thiG, and / or thiH; or c) thiM, and / or thiD.

[0230] In some embodiments, one or more genes and / or polypeptides of the pathway for the sulfurol are heterologous to the host cell. In another embodiment, the host cell further comprises at least 2 copies of one or more genes and / or polypeptides of the pathway for the sulfurol.

[0231] In a further embodiment, the host cell further comprises a transporter molecule facilitating transport of a precursor for or a product of the pathway for the sulfurol. In one embodiment, the host cell is further genetically modified to provide an increased amount of a substrate in the pathway for the sulfurol. In another embodiment, the host cell is further genetically modified to exhibit increased tolerance towards one or more substrates, intermediates, or products in the pathway for the sulfurol. In further embodiment, the one or more additional native or endogenous genes of the host cell are deleted, disrupted and / or attenuated. In a still further embodiment, one or more genes in the pathway for the sulfurol are overexpressed. In another embodiment, the host cell is further genetically modified to provide an increased amount of a substrate in the pathway for the sulfurol. Host cells

[0232] The host cell disclosed herein may be any host cell suitable for hosting and expressing the [pathway for the sulfurol. Such cell may be a prokaryotic or eukaryotic cell. Suitable prokaryotic host cells can be of a genus selected from Escherichia, Bacillus, Brevibacterium, Burkholderia, Campylobacter, Corynebacterium, Serratia, Lactobacillus, Lactococcus, Acinetobacter, Acetobacter, archae, or Pseudomonas. Particularly useful prokaryotic host cells are of the genus Escherichia, Corynebacterium, Bacillus, Serratia, or Pseudomonas, such as the species Escherichia coli, Corynebacterium glutamicum, B. subtilis, Serratia marcescens, P. putida and / or Pseudomonas mutabilis. Useful eukaryotic host cells include mammalian, insect, plant, or fungal cells. Among the eukaryotic host cells fungal cells of the genus Saccharomyces, Kluveromyces, Candida, Pichia, Debaromyces, Hansenula, Yarrowia, Zygosaccharomyces, Schizosaccharomyces and Ashbya are particularly useful, such as the species Kluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces bensis, Saccharomyces oviformis, Yarrowia lipolytica, Pichia pastoris or Ashbya gossypii.Case Ref. P180WO IPTector® Polynucleotide constructs and expression vectors

[0233] Also provided for herein are polynucleotide constructs harboring gene(s) encoding native or mutated ThiC, ThiD, ThiF, ThiS, ThiH, ThiG, ThiM, ThiE, TMP phosphatase, ThiK, ThiL, ThiI, IscR, TenI, IscS, tenA, Thi1, CARB, deCARB, ThiP, fur, cyaA, dxs, sodA, PHOS, and / or ThiO genes operably linked to one or more control sequences, said polynucleotide constructs comprising mutations to delete, disrupt, and / or an attenuate the gene transcription or translation or the activity and / or function of the encoded genes. The control sequences direct the expression of the encoded ThiC, ThiD, ThiF, ThiS, ThiH, ThiG, ThiM, ThiE, TMP phosphatase, ThiK, ThiL, ThiI, IscR, TenI, IscS, tenA, Thi1, CARB, deCARB, ThiP, fur, cyaA, dxs or ThiO genes in the host cell harboring the polynucleotide construct. Conditions for the expression should be compatible with the control sequences. The control sequence may be heterologous or native to the gene(s) encoding the ThiC, ThiD, ThiF, ThiS, ThiH, ThiG, ThiM, ThiE, TMP phosphatase, ThiK, ThiL, ThiI, IscR, TenI, IscS, tenA, Thi1, CARB, deCARB, ThiP, fur, cyaA, dxs or ThiO genes and / or to the host cell. In some embodiments, both the control sequence and the gene(s) encoding ThiC, ThiD, ThiF, ThiS, ThiH, ThiG, ThiM, ThiE, TMP phosphatase, ThiK, ThiL, ThiI, IscR, TenI, IscS, tenA, Thi1, CARB, deCARB, ThiP, fur, cyaA, dxs or ThiO are heterologous to the host cell and optionally also to each other. In one embodiment the polynucleotide construct is an expression vector, comprising the gene(s) encoding the ThiC, ThiD, ThiF, ThiS, ThiH, ThiG, ThiM, ThiE, TMP phosphatase, ThiK, ThiL, ThiI, IscR, TenI, IscS, tenA, Thi1, CARB, deCARB, ThiP, fur, cyaA, dxs or ThiO operably linked to the one or more control sequences.

[0234] Polynucleotides may be manipulated in a variety of ways to modify the expression of the ThiC, ThiD, ThiF, ThiS, ThiH, ThiG, ThiM, ThiE, TMP phosphatase, ThiK, ThiL, ThiI, IscR, TenI, IscS, tenA, Thi1, CARB, deCARB, ThiP, fur, cyaA, dxs, sodA, PHOS or ThiO. Manipulation of the polynucleotide prior to its insertion into an expression vector may be desirable or necessary depending on the expression vector. The techniques for modifying polynucleotides utilizing recombinant DNA methods are well known in the art.

[0235] The control sequence may be a promoter, which is a polynucleotide that is recognized by a host cell for expression of a polynucleotide. The promoter contains transcriptional control sequences that mediate the expression of the polypeptide. The promoter may be any polynucleotide that shows transcriptional activity in the host cell including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell. The promoter may also be an inducible promoter. Selecting a suitable promoter for expression in yeast is well-known and is well understood by persons skilled in the art.

[0236] The control sequence may also be a transcription terminator, which is recognized by a host cellCase Ref. P180WO IPTector® to terminate transcription. The terminator is operably linked to the 3'-terminus of the polynucleotide encoding the polypeptide. Any terminator that is functional in the host cell may be used. The control sequence may also be an mRNA stabilizer region downstream of a promoter and upstream of the coding sequence of a gene which increases the expression of the gene.

[0237] The control sequence may also be a leader, a non-translated region of an mRNA that is important for translation by the host cell. The leader is operably linked to the 5'-terminus of the polynucleotide encoding the polypeptide. Any leader that is functional in the host cell may be used.

[0238] The control sequence may also be a polyadenylation sequence; a sequence operably linked to the 3'-terminus of the polynucleotide and, when transcribed, is recognized by the host cell as a signal to add multiple adenosine residues (poly(A) tail) to transcribed mRNA. Any polyadenylation sequence that is functional in the host cell may be used.

[0239] It may also be desirable to add regulatory sequences that regulate the expression of the ThiC, ThiD, ThiF, ThiS, ThiH, ThiG, ThiM, ThiE, TMP phosphatase, ThiK, ThiL, ThiI, IscR, TenI, IscS, tenA, Thi1, CARB, deCARB, ThiP, fur, cyaA, dxs, sodA, PHOS and / or ThiO genes relative to the growth of the host cell. Examples of regulatory systems are those that cause expression of the gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound.

[0240] Various nucleotide sequences in addition to the polynucleotide construct disclosed herein may be joined together to produce a recombinant expression vector, which may include one or more convenient restriction sites to allow for insertion or substitution of the polynucleotide sequence encoding the ThiC, ThiD, ThiF, ThiS, ThiH, ThiG, ThiM, ThiE, TMP phosphatase, ThiK, ThiL, ThiI, IscR, TenI, IscS, tenA, Thi1, CARB, deCARB, ThiP, fur, cyaA, dxs, sodA, PHOS, and / or ThiO genes at such sites. The recombinant expression vector may be any vector (e.g., a plasmid or virus or chromosomal) that can be conveniently subjected to recombinant DNA procedures and can bring about expression of the ThiC, ThiD, ThiF, ThiS, ThiH, ThiG, ThiM, ThiE, TMP phosphatase, ThiK, ThiL, ThiI, IscR, TenI, IscS, tenA, Thi1, CARB, deCARB, ThiP, fur, cyaA, dxs or ThiO genes. The choice of the vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced. The vector may be an autonomously replicating vector, i.e., a vector that exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid (linear or closed circular plasmid), an extrachromosomal element, a mini-chromosome, or an artificial chromosome. The vector may contain any means for assuring self-replication. Alternatively, the vector may, when introduced into the host cell, integrate into the genome, and replicate together with the chromosome(s) into which it has been integrated. Furthermore, a single vector or plasmid or two or more vectors or plasmids that together contain the total DNA to be introduced into the genome of the host cell, or a transposon, may be used.Case Ref. P180WO IPTector®

[0241] The vector may contain one or more selectable markers that permit easy selection of transformed, transfected, transduced, or the like cells. A selectable marker is a gene from which the product provides for biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, and the like. In some embodiments, antibiotic markers with surrounding FRT sequences are used.

[0242] The vector may further contain element(s) that permit integration of the vector into genome (being a vector in itself) of the host cell or permits autonomous replication of the vector in the cell independent of the genome. Alternatively, the vector may contain additional polynucleotides for directing integration by homologous recombination into the genome of the host cell at a precise location(s) in the chromosome(s). To increase the likelihood of integration at a precise location, the integrational elements should contain a sufficient number of nucleic acids, such as 100 to 10,000 base pairs or such as from 10 to 10,000 base pairs, for example from 50 to 10,000 base pairs, such as 400 to 10,000 base pairs, and such as 800 to 10,000 base pairs, which have a high degree of sequence identity to the corresponding target sequence to enhance the probability of homologous recombination. The integrational elements may be any sequence that is homologous with the target sequence in the genome of the host cell. Furthermore, the integrational elements may be non- encoding or encoding polynucleotides. On the other hand, the vector may be integrated into the genome of the host cell by non-homologous recombination.

[0243] For autonomous replication, the vector may further comprise an origin of replication enabling the vector to replicate autonomously in the host cell in question. The origin of replication may be any plasmid replicator mediating autonomous replication that functions in a cell. The term "origin of replication" or "plasmid replicator" refers to a polynucleotide that enables a plasmid or vector to replicate in vivo.

[0244] As mentioned, supra, more than one copy of a gene encoding pathway elements for sulfurol may be inserted into a host cell to increase the production of sulfurol. An increase in the gene copy number can be obtained by integrating one or more additional copies of a gene into the host cell genome or by including an amplifiable selectable marker gene with the gene so that cells containing amplified copies of the selectable marker gene - and thereby additional copies of the polynucleotide - can be selected by cultivating the cells in the presence of the appropriate selectable agent. The procedures used to ligate the elements described above to construct the recombinant expression vectors of the present disclosure are well known to one skilled in the art (see, e.g., Sambrook et al., 1989, supra).

[0245] In a separate embodiment, the host cell comprises the polynucleotide constructs and / or vectors as disclosed herein.Case Ref. P180WO IPTector® Cultures

[0246] Further provided for herein are cell cultures comprising the genetically modified host cells disclosed herein and a growth medium. Suitable growth mediums for relevant prokaryotic or eukaryotic host cells are widely known in the art. Methods of producing compounds.

[0247] This disclosure also describes a method for producing sulfurol, thiamine, 5-(2-hydroxyethyl)-4- methyl-1,3-thiazole-2-carboxylic phosphate acid (cTHZ-P) and / or 5-(2-hydroxyethyl)-4-methyl-1,3- thiazole-2-carboxylic acid (cTHZ) comprising a) culturing the cell culture disclosed herein at conditions allowing the host cells to produce the sulfurol; and b) optionally recovering and / or isolating the sulfurol.

[0248] This disclosure also describes a method for producing sulfurol, thiamine, 5-(2-hydroxyethyl)-4- methyl-1,3-thiazole-2-carboxylic phosphate acid (cTHZ-P) and / or 5-(2-hydroxyethyl)-4-methyl-1,3- thiazole-2-carboxylic acid (cTHZ) comprising a) culturing the cell culture disclosed herein at conditions allowing the host cells to produce the sulfurol, thiamine, 5-(2-hydroxyethyl)-4-methyl-1,3-thiazole-2-carboxylic phosphate acid (cTHZ-P) and / or 5-(2-hydroxyethyl)-4-methyl-1,3-thiazole-2-carboxylic acid (cTHZ); and b) optionally recovering and / or isolating the sulfurol, thiamine, 5-(2-hydroxyethyl)-4-methyl- 1,3-thiazole-2-carboxylic phosphate acid (cTHZ-P) and / or 5-(2-hydroxyethyl)-4-methyl-1,3- thiazole-2-carboxylic acid (cTHZ).

[0249] The cell culture can be cultivated in a nutrient medium and at conditions suitable for the production of the sulfurol disclosed herein and / or for propagating cell count using methods known in the art. For example, the culture may be cultivated by shake flask cultivation, or small-scale or large- scale fermentation (including continuous, batch, fed-batch, or solid state fermentations) in a laboratory or industrial fermenters in a suitable medium and under conditions allowing the host cells to grow and / or propagate, optionally to be recovered and / or isolated.

[0250] The cultivation can take place in a suitable nutrient medium comprising carbon and nitrogen sources and inorganic salts, using procedures known in the art. Suitable media are available from commercial suppliers or may be prepared according to published recipes (e.g., from catalogues of the American Type Culture Collection). The selection of the appropriate medium may be based on the choice of host cell and / or based on the regulatory requirements for the host cell. Such media are available in the art. The medium may, if desired, contain additional components favouring theCase Ref. P180WO IPTector® transformed expression hosts over other potentially contaminating microorganisms. Accordingly, in an embodiment a suitable nutrient medium can include one or more of (i) trace metals; (ii) vitamins; (iii) salts (such as salts of phosphate, magnesium, potassium, zinc, iron); (iv) nitrogen sources (such as YNB, ammonium sulfate, urea, yeast extracts, ammonium nitrate, ammonium chloride, malt extract, peptone and / or amino acids); (v) carbon source (such as dextrose, sucrose, glycerol, glucose, maltose, molasses, starch, cellulose, xylan, pectin, lignocellolytic biomass hydrolysate, and / or acetate); (vi) nucleobases; (vii) aminoglycosides; and / or (viii) antibiotics (such as G418 and hygromycin B, or such as spectinomycin and tetracycline).

[0251] The cultivation of the host cell may be performed over a period of time from about 0.5 to about 30 days. The cultivation process may be a batch process, continuous or fed-batch process, suitably performed at a temperature in the range of 0-100 °C or 0-80 °C, for example, from about 0 °C to about 50 °C and / or at a pH, for example, from about 2 to about 10. Preferred fermentation conditions are a temperature in the range of from about 25 °C to about 55 °C and at a pH of from about 3 to about 9. The appropriate conditions are usually selected based on the choice of host cell. Accordingly, in some embodiments the method disclosed herein comprises one or more elements selected from: a) culturing the cell culture under aerobic or anaerobic conditions b) cultivating the host cells under mixing; c) cultivating the host cells at a temperature of between 25°C to 50°C; d) cultivating the host cells at a pH of between 3-9; and e) cultivating the host cells for between 10 hours to 120 days.

[0252] The cell culture disclosed herein may be recovered and or isolated using methods known in the art. For example, the compound(s) may be recovered from the nutrient medium by conventional procedures including, but not limited to, centrifugation, filtration, spray-drying, or lyophilization. In a particular embodiment the method includes a recovery and / or isolation step comprising separating a liquid phase of the cell culture from a solid phase of the cell culture to obtain a supernatant comprising the sulfurol and subjecting the supernatant to one or more steps selected from: a) contacting the supernatant with one or more adsorbent resins to obtain at least a portion of the produced sulfurol, then optionally recovering the sulfurol from the resin in a concentrated solution prior to isolation of the sulfurol by crystallisation or solvent evaporation; b) contacting the supernatant with one or more ion exchange or reversed-phase chromatography columns to obtain at least a portion of the sulfurol, then optionally recovering the sulfurol from the resin in a concentrated solution prior to isolation of theCase Ref. P180WO IPTector® sulfurol by crystallisation or solvent evaporation; and c) extracting the sulfurol from the supernatant, such as by liquid-liquid extraction into an immiscible solvent, then optionally isolating the sulfurol by crystallisation or solvent evaporation; and thereby recovering and / or isolating the sulfurol.

[0253] The cell culture disclosed herein may be recovered and or isolated using methods known in the art. For example, the compound(s) may be recovered from the nutrient medium by conventional procedures including, but not limited to, centrifugation, filtration, spray-drying, or lyophilization. In a particular embodiment the method includes a recovery and / or isolation step comprising separating a liquid phase of the cell culture from a solid phase of the cell culture to obtain a supernatant comprising the sulfurol or a sulfurol precursor as defined herein, and subjecting the supernatant to one or more steps selected from: a) contacting the supernatant with one or more adsorbent resins to obtain at least a portion of the produced sulfurol or a sulfurol precursor as defined herein, then optionally recovering the sulfurol or the sulfurol precursor as defined herein from the resin in a concentrated solution prior to isolation of the sulfurol or the sulfurol precursor as defined herein by crystallisation or solvent evaporation; b) contacting the supernatant with one or more ion exchange or reversed-phase chromatography columns to obtain at least a portion of the sulfurol or the sulfurol precursor as defined herein, then optionally recovering the sulfurol or the sulfurol precursor as defined herein from the resin in a concentrated solution prior to isolation of the sulfurol or the sulfurol precursor as defined herein by crystallisation or solvent evaporation; and c) extracting the sulfurol or the sulfurol precursor as defined herein from the supernatant, such as by liquid-liquid extraction into an immiscible solvent, then optionally isolating the sulfurol or the sulfurol precursor as defined herein by crystallisation or solvent evaporation; and thereby recovering and / or isolating the sulfurol or the sulfurol precursor as defined herein.

[0254] The sulfurol yield provided by the method disclosed herein is typically higher than when producing sulfurol by methods employing host cells without reduced CRP-cAMP complex formation and / or increased degradation and / or decreased binding of cAMP, in some embodiments at least 10% higher such as at least 50%, such as at least 100%, such as least 150%, such as at least 200% higher.

[0255] In one embodiment the sulfurol yield and / or titer obtained by the method disclosed herein is from at least 50 mg / L to 800 mg / L sulfurol. In other embodiments sulfurol yield and / or titer obtained by the method is at least 400 mg / L, such as at least 800 mg / L, such as at least 1000 mg / L, such as at least 1500 mg / L, such as at least 2000 mg / L, such as at least 3000 mg / L, such as at least 5000 mg / L, such as at least 10000 mg / L, such as at least 15000 mg / L, such as at least 20000 mg / L.Case Ref. P180WO IPTector®

[0256] The method disclosed herein may further comprise one or more steps of mixing the sulfurol with one or more carriers, agents, adjuvants, additives and / or excipients, optionally pharmaceutical grade carriers, agents, adjuvants, additives and / or excipients.

[0257] The method disclosed herein may further comprise one or more in vitro steps in the process of producing the sulfurol. It may also comprise one or more in vivo steps performed in another cell than the host cell disclosed herein. For example, precursors for and / or intermediates in the pathway for the sulfurol may be produced in another cell and isolated therefrom and then fed to a cell culture disclosed herein for conversion into the sulfurol. Accordingly, in one embodiment the method disclosed herein further comprises feeding one or more exogenous sulfurol precursors to the host cell culture. In a preferred embodiment the feeding of one or more exogenous sulfurol precursors to the host cell culture comprises feeding HMP or THZ to the cell culture.

[0258] Accordingly, in one embodiment the method disclosed herein further comprises feeding one or more exogenous sulfurol precursors to the host cell culture. In a preferred embodiment the feeding of one or more exogenous sulfurol precursors to the host cell culture comprises feeding HMP, amino- HMP, or THZ to the cell culture. Fermentation composition

[0259] This disclosure also describes a fermentation composition comprising the cell culture disclosed herein and the sulfurol – either comprised in the cells or in the medium. In the fermentation composition the genetically modified host cells may be wholly or partially lysed and / or disintegrated. In some embodiments at least 50%, such as at least 75%, such as at least 95%, such as at least 99% of the genetically modified host cells in the fermentation composition are lysed and / or disintegrated. Further, in the fermentation composition disclosed herein at least 50%, such as at least 75%, such as at least 95%, such as at least 99% of solid cellular material may have been separated and / or removed from a liquid phase of the fermentation composition.

[0260] The fermentation composition may further comprise one or more compounds of a) precursor or products of the operative metabolic pathway producing the sulfurol; b) supplemental nutrients comprising; and wherein the concentration of the sulfurol is at least 1 mg / L composition. In particular the fermentation composition can comprise a concentration of sulfurol of at least 5 mg / kg, such as at least 10 mg / kg, such as at least 20 mg / kg, such as at least 50 mg / kg, such as at least 100 mg / kg, such as at least 500 mg / kg, such as at least 1000 mg / kg, such as at least 5000 mg / kg, such as at least 10000 mg / kg, such as at least 50000 mg / kg.

[0261] Suitable supplemental nutrients can include one or more of (i) trace metals; (ii) vitamins; (iii)Case Ref. P180WO IPTector® salts (such as salts of phosphate, magnesium, potassium, zinc, and iron); (iv) nitrogen sources (such as YNB, ammonium sulfate, urea, yeast extracts, ammonium nitrate, ammonium chloride, malt extract, peptone and / or amino acids); (v) carbon source (such as dextrose, sucrose, glycerol, glucose, maltose, molasses, starch, cellulose, xylan, pectin, lignocellolytic biomass hydrolysate, and / or acetate); (vi) nucleobases; (vii) aminoglycosides; and / or (viii) antibiotics (such as G418, hygromycin B, spectinomycin, tetracycline and / or Kanamycin, or any antibiotic as specified herein). Compositions and use

[0262] This disclosure also describes a composition comprising the fermentation composition disclosed herein and one or more carriers, agents, adjuvants, additives and / or excipients. Suitable carriers, agents, adjuvants, additives and / or excipients include formulation additives, stabilising agents, fillers, and the like. The composition and the one or more carriers, agents, adjuvants, additives and / or excipients can suitably be formulated into a dry solid form, e.g., by using methods known in the art, such as spray drying, spray cooling, lyophilization, flash freezing, granulation, microgranulation, encapsulation or microencapsulation. The composition and the one or more carriers, agents, adjuvants, additives and / or excipients can also be formulated into a liquid stabilized form using methods known in the art, such as adding to the fermentation composition one or more stabilizers such as sugars and / or polyols (e.g., sugar alcohols) and / or organic acids (e.g., lactic acid).

[0263] The composition disclosed herein may be further refined into a pharmaceutical preparation, a dietary supplement, a cosmetic, a food preparation, a feed preparation and / or an analytical or diagnostic reagent optionally using one or more steps of the methods described herein for producing the sulfurol including mixing the sulfurol with one or more pharmaceutical grade carriers, agents, adjuvants, additives and / or excipients. In one embodiment, the pharmaceutical composition is a pharmaceutical preparation obtainable from the method disclosed herein. The pharmaceutical preparation may be a dry preparation, optionally in the form of a powder, tablet, capsule, hard chewable and or soft lozenge or a gum. Alternatively, the pharmaceutical preparation may in form of a liquid pharmaceutical solution. Such pharmaceutical preparations may be used as a medicament in a method for treating and / or relieving a disease and / or medical condition, in particular in a mammal, in particular for use in the treatment of a nutritional deficiency. Accordingly, this disclosure further describes a method for preventing, treating and / or relieving a disease and / or medical condition comprising administering a therapeutically effective amount of the pharmaceutical composition disclosed herein to a mammal in need of treatment and / or relief. Diseases and / or medical conditions treatable or relievable by the pharmaceutical composition include but are not limited to diseases and / or medical conditions associated with lacking or insufficient bodily intake of sulfurol. TheCase Ref. P180WO IPTector® pharmaceutical preparation can be administered parenterally, such as topically, epicutaneously, sublingually, buccally, nasally, intradermally, intravenously, and / or intramuscularly. The pharmaceutical composition can also be administered enterally via the gastrointestinal tract.

[0264] This disclosure also describes a kit of parts comprising: a) the genetically modified host cell as described herein; b) instructions for use of the genetically modified host cell; c) the nucleic acid construct as described herein; d) instructions for use of the nucleic acid construct; and / or e) a host cell which can be genetically modified using the methods described herein.

[0265] In some embodiments, the kit comprises a genetically modified cell capable of producing sulfurol, wherein the genetically modified cell expresses pathway enzymes producing the sulfurol. In one embodiment the genetically modified cell expresses one or more sulfurol pathway enzymes or factors selected from ThiC, ThiD, ThiF, ThiS, ThiH, ThiG, ThiM, ThiE, TenI, TMP phosphatase, ThiL, ThiI, ThiO, IscR, IscS, tenA, CARB, deCARB, ThiP, fur, cyaA, dxs, sodA, PHOS, and / or Thi1. Sequence listing

[0266] The present application contains a Sequence Listing prepared in WIPO Sequence Suite included below but also submitted electronically in ST26 format which is hereby incorporated by reference in its entirety. SEQ ID NO: Host SEQ ID NO: 1 Escherichia coli SEQ ID NO: 2 Escherichia coli SEQ ID NO: 3 Escherichia coli SEQ ID NO: 4 Escherichia coli SEQ ID NO: 5 Escherichia coli SEQ ID NO: 6 Escherichia coli SEQ ID NO: 7 Escherichia coli SEQ ID NO: 8 Escherichia coli SEQ ID NO: 9 Escherichia coli SEQ ID NO: 10 Escherichia coli SEQ ID NO: 11 Escherichia coli SEQ ID NO: 12 Escherichia coli SEQ ID NO: 13 Escherichia coli SEQ ID NO: 14 Escherichia coli SEQ ID NO: 15 Escherichia coli SEQ ID NO: 16 Escherichia coliCase Ref. P180WO IPTector® SEQ ID NO: 17 Escherichia coli SEQ ID NO: 18 Escherichia coli SEQ ID NO: 19 Escherichia coli SEQ ID NO: 20 Escherichia coli SEQ ID NO: 21 Escherichia coliSEQ ID NO: 22Escherichia coliSEQ ID NO: 23Bacillus subtilisSEQ ID NO: 24Bacillus subtilisSEQ ID NO: 25 Arabidopsis thaliana SEQ ID NO: 26 Arabidopsis thaliana SEQ ID NO: 27 Pseudomonas putida SEQ ID NO: 28 Pseudomonas putidaSEQ ID NO: 29Gloeothece citriformis (Cyanothece)SEQ ID NO: 30Gloeothece citriformis (Cyanothece)SEQ ID NO: 31Stigmatella aurantiaca DW4SEQ ID NO: 32Stigmatella aurantiaca DW4SEQ ID NO: 33Acinetobacter nectaris DSM 29975SEQ ID NO: 34Acinetobacter nectaris DSM 29975SEQ ID NO: 35Megasphaera elsdenii DSM 20460SEQ ID NO: 36Megasphaera elsdenii DSM 20460SEQ ID NO: 37Naegleria gruberiSEQ ID NO: 38Naegleria gruberiSEQ ID NO: 39Burkholderia glumae BGR1SEQ ID NO: 40Burkholderia glumae BGR1SEQ ID NO: 41Haliangium ochraceum DSM14365SEQ ID NO: 42Haliangium ochraceum DSM14365SEQ ID NO: 43Oleidesulfovibrio alaskensis G20SEQ ID NO: 44Oleidesulfovibrio alaskensis G20SEQ ID NO: 45Bacillus subtilisSEQ ID NO: 46Bacillus subtilisSEQ ID NO: 47 Escherichia coli SEQ ID NO: 48 Escherichia coli SEQ ID NO: 49 Escherichia coli SEQ ID NO: 50 Escherichia coli SEQ ID NO: 51 Escherichia coli SEQ ID NO: 52 Escherichia coli SEQ ID NO: 53 Escherichia coli SEQ ID NO: 54 Escherichia coli SEQ ID NO: 55 Escherichia coliCase Ref. P180WO IPTector® SEQ ID NO: 56 Escherichia coli SEQ ID NO: 57 Staphylococcus aureus SEQ ID NO: 58 Staphylococcus aureus SEQ ID NO: 59 Pseudomonas aeruginosa SEQ ID NO: 60 Pseudomonas aeruginosa SEQ ID NO: 61 Pseudomonas Putida KT2440 SEQ ID NO: 62 Pseudomonas Putida KT2440 SEQ ID NO: 63 Pseudomonas Putida KT2440 SEQ ID NO: 64 Pseudomonas Putida KT2440 SEQ ID NO: 65 Pseudomonas Putida KT2440 SEQ ID NO: 66 Pseudomonas Putida KT2440 SEQ ID NO: 67 Pseudomonas Putida KT2440 SEQ ID NO: 68 Pseudomonas Putida KT2440 SEQ ID NO: 69 Pseudomonas Putida KT2440 SEQ ID NO: 70 Pseudomonas Putida KT2440 SEQ ID NO: 71 Escherichia coli SEQ ID NO: 72 Escherichia coli SEQ ID NO: 73 Shigella sonnei (strain Ss046) SEQ ID NO: 74 Shigella sonnei (strain Ss046) SEQ ID NO: 75 Pseudomonas asiatica SEQ ID NO: 76 Pseudomonas asiatica SEQ ID NO: 77 Hirschfeldia incana SEQ ID NO: 78 Hirschfeldia incana SEQ ID NO: 79 Citrus sinensis SEQ ID NO: 80 Citrus sinensis SEQ ID NO: 81 Escherichia coli SEQ ID NO: p82 Escherichia coli SEQ ID No: 83 Arabidopsis thaliana SEQ ID No: 84 Arabidopsis thaliana SEQ ID No: 85 Alternaria panax SEQ ID No: 86 Alternaria panaxCase Ref. P180WO IPTector® SEQ ID No: 87 Anaerotruncus colihominis SEQ ID No: 88 Anaerotruncus colihominis SEQ ID No: 89 Dorea longicatena SEQ ID No: 90 Dorea longicatena SEQ ID No: 91 Eubacterium ventriosum SEQ ID No: 92 Eubacterium ventriosum SEQ ID No: 93 Syntrophomonas wolfei SEQ ID No: 94 Syntrophomonas wolfei SEQ ID No: 95 Slackia equolifaciens SEQ ID No: 96 Slackia equolifaciens SEQ ID No: 97 Ruminococcus sp. SEQ ID No: 98 Ruminococcus sp. SEQ ID No: 99 Oscillospiraceae bacterium SEQ ID No: 100 Oscillospiraceae bacterium SEQ ID No: 101 Tepidanaerobacter sp. GT38 SEQ ID No: 102 Tepidanaerobacter sp. GT38 SEQ ID No: 103 Roseburia sp. SEQ ID No: 104 Roseburia sp. SEQ ID No: 105 Clostridiales bacterium SEQ ID No: 106 Clostridiales bacterium SEQ ID No: 107 Escherichia coli SEQ ID No: 108 Escherichia coli SEQ ID No: 109 Escherichia coli SEQ ID No: 110 Escherichia coli SEQ ID No: 111 Escherichia coli SEQ ID No: 112 Escherichia coli SEQ ID No: 113 Escherichia coli SEQ ID No: 114 Escherichia coli SEQ ID No: 115 Helicobacter pylori SEQ ID No: 116 Helicobacter pylori SEQ ID No: 117 Bacillus aneurinolyticus aka AneurinibacillusCase Ref. P180WO IPTector® aneurinilyticus SEQ ID No: 118 Bacillus aneurinolyticus aka Aneurinibacillus aneurinilyticus SEQ ID No: 119 Bacillus migulanus aka Aneurinibacillus migulanus SEQ ID No: 120 Bacillus migulanus aka Aneurinibacillus migulanus SEQ ID No: 121 Bacillus subtilis SEQ ID No: 122 Bacillus subtilis SEQ ID No: 123 Candida parapsilosis SEQ ID No: 124 Candida parapsilosis SEQ ID No: 125 Halalkalibacterium halodurans aka Bacillus halodurans SEQ ID No: 126 Halalkalibacterium halodurans aka Bacillus halodurans SEQ ID No: 127 Pyrobaculum aerophilum SEQ ID No: 128 Pyrobaculum aerophilum SEQ ID No: 129 Pyrococcus furiosus SEQ ID No: 130 Pyrococcus furiosus SEQ ID No: 131 Pyrococcus horikoshii SEQ ID No: 132 Pyrococcus horikoshii SEQ ID No: 133 Staphylococcus aureus SEQ ID No: 134 Staphylococcus aureus SEQ ID No: 135 Staphylococcus epidermidis SEQ ID No: 136 Staphylococcus epidermidis SEQ ID No: 137 Saccharolobus solfataricus aka Sulfolobus solfataricus SEQ ID No: 138 Saccharolobus solfataricus aka Sulfolobus solfataricus SEQ ID No: 139 Saccharomyces cerevisiae SEQ ID No: 140 Saccharomyces cerevisiae SEQ ID No: 141 Escherichia coli SEQ ID No: 142 Escherichia coli SEQ ID No: 143 Escherichia coli SEQ ID No: 144 Escherichia coli SEQ ID No: 145 Escherichia coli SEQ ID No: 146 Escherichia coliCase Ref. P180WO IPTector® SEQ ID No: 147 Escherichia coli SEQ ID No: 148 Escherichia coli Working Examples Materials and methods Materials

[0267] Chemicals used in the examples herein e.g. for buffers and substrates are commercial products of at least reagent grade. Solutions and media mMOPS medium

[0268] The minimal medium (mMOPS) used herein had the following composition in demineralized H2O (dH2O): Compound Concentration of compound in medium - [g / L] 3-(N-morpholino)propane sulfonic acid (MOPS) 8.3705 Tricine 0.7167 K2HPO4 0.2300 NH4Cl 0.5082 NaCl 2.9220 MgCl2 0.0050 ZnSO4^· 7H2O 0.00007 CuSO4^· 5H2O 0.00002 MnCl2 0.0002 FeSO43^· 7H2O 0.0029 CoCl2^· 6H2O 0.00007 CaCl2^· 2H2O 0.00007 K2SO4 0.0481 (NH4)6Mo7O24 ·4H2O 0.00004 H3BO3 0.0003 Calcium pantothenate 0.0190 P-aminobenzoic acid 0.0055 P-hydroxybenzoic acid 0.0055 2,3-dihydroxybenzoic acid 0.0062Case Ref. P180WO IPTector® D-biotin 0.000002 D-glucose 2.0000 LB agar medium

[0269] The LB agar medium used herein had the following composition in dH2O: Compound Concentration of compound in medium - [g / L] LB Broth (Lennox) 20.0000 Agar 20.0000 B medium

[0270] The fermentation batch medium (B medium 1) used herein had the following composition in dH2O: Compound Concentration of compound in medium - [g / L] (NH4)2SO4 10.000000 K2HPO4 4.000000 KH2PO4 4.000000 Na3Citrate · 2H2O 3.000000 Na2SO4 2.000000 NH4Cl 1.000000 MgSO4^· 7H2O 1.000000 ZnSO4^· 7H2O 0.000825 CuSO4^· 5H2O 0.000750 MnSO4^· H2O 0.000450 FeCl3^· 6H2O 0.062700 CoCl2^· 6H2O 0.000900 CaCl2^· 2H2O 0.003000 Boric acid 0.001125 Yeast extract 10.000000 Desthiobiotin (DTB) 0.400000 D-biotin 0.000020 D-glucose 10.000000

[0271] The optimized fermentation batch medium (UB medium) used herein had the followingCase Ref. P180WO IPTector® composition in dH2O: Compound Concentration of compound in medium - [g / L] (NH4)2SO4 10.000000 K2HPO4 4.000000 KH2PO4 4.000000 Na3Citrate · 2H2O 3.000000 Na2SO4 2.000000 NH4Cl 1.000000 MgSO4^· 7H2O 1.000000 ZnSO4^· 7H2O 0.000825 CuSO4^· 5H2O 0.000750 MnSO4^· H2O 0.000450 FeCl3^· 6H2O 0.062700 CoCl2^· 6H2O 0.000900 CaCl2^· 2H2O 0.003000 Boric acid 0.001125 Yeast extract 10.000000 Desthiobiotin (DTB) 0.400000 D-biotin 0.000020 D-glucose 10.000000

[0272] The optimized for upscaling fermentation batch medium (UB medium) used herein had the following composition in dH2O: Compound Concentration of compound in medium - [g / L] D-glucose 10.000000 Yeast extract 10.000000 MgSO4^· 7H2O 1.000000 (NH4)2S2O311.000000 K2HPO4 5.000000 KH2PO45.000000 Na3Citrate · 2H2O 3.000000 Na2SO42.000000 ZnSO4^· 7H2O 0.000825Case Ref. P180WO IPTector® CuSO4^· 5H2O 0.000750 MnSO4^· H2O 0.000450 FeSO4●7H2O 0.062700 F medium

[0273] The fermentation feed medium (F medium 1) used herein had the following composition in dH2O: Compound Concentration of compound in medium - [g / L] (NH4)2SO4 10.000000 K2HPO4 4.000000 KH2PO4 4.000000 Na3Citrate · 2H2O 3.000000 Na2SO42.000000 NH4Cl 1.000000 MgSO4^· 7H2O 1.000000 ZnSO4^· 7H2O 0.000825 CuSO4^· 5H2O 0.000750 MnSO4^· H2O 0.000450 FeCl3^· 6H2O 0.062700 CoCl2^· 6H2O 0.000900 CaCl2^· 2H2O 0.003000 Boric acid 0.001125 Yeast extract 10.000000 Desthiobiotin (DTB) 0.400000 D-biotin 0.000020 D-glucose 10.000000

[0274] The optimized fermentation feed medium (F medium 2) used herein had the following composition in dH2O: Compound Concentration of compound in medium - [g / L] D-glucose 10.000000 Yeast extract 10.000000 MgSO4^· 7H2O 1.000000Case Ref. P180WO IPTector® (NH4)2S2O3 11.000000 K2HPO4 5.000000 KH2PO4 5.000000 Na3Citrate · 2H2O 3.000000 Na2SO4 2.000000 ZnSO4^· 7H2O 0.000825 CuSO4^· 5H2O 0.000750 MnSO4^· H2O 0.000450 FeSO4●7H2O 0.062700

[0275] The optimized for upscaling fermentation feed medium (UF medium) used herein had the following composition in dH2O: Compounds MW Conc. (g / L) mM Feed Concentratio Glucose 180.16 550 n of glucose (concentration solution, base in feed) solution and MgSO4*7H2 246.47 10 trace metals O (concentration additions in feed) FeSO4●7H2O 278.01 0.278 (concentration in feed) NH4OH 35.04 175.20 5000 (concentration in base bottle) 3 extra H2SO4 98.08 additions of ZnSO4●7H2O 287.56 0.000825 0.011475866 This is the trace metals CuSO4●5H2O 285.72 0.000750 0.010499952 total trace during MnSO4●H2O 169.01 0.000450 0.01065032 metals fermentation FeSO4●7H2O 278.01 0.062700 0.902125823 concentratio n including batch and additions during fed batch Antifoam concentration

[0276] The antifoam used herein had the following concentrations: Antifoam 204 Batch0.005% v / v added Fed batch3 additions of 0.005% v / v (0.02% v / v total)Case Ref. P180WO IPTector® Antibiotics concentration

[0277] The antibiotics used herein had the following concentrations: Compound Concentration of compound in medium - [mg / L] Spectinomycin 50 Tetracycline 10 Kanamycin 50 Chloramphenicol 30 Ampicillin 100 Strains and plasmids

[0278] Strains of Escherichia coli used herein were the following: Strain name Background strain Plasmid Description BS1013 BS1013 - Publically available E. coli K-12 BW25113 parent strain1having genotype: rrnB3 ΔlacZ4787 hsdR514 Δ(araBAD)567 Δ(rhaBAD)568 rph-1 BS058012BS1013 - BS1013 having the following mutations and overexpressions of genes from the chromosome: ∆thiP Constitutive gene expression: apFAB70 / TH2_ATH (SEQ ID NO 26) iscR-H107Y ∆cyaA Constitutive gene expression: apFAB71 / thiE, thiM, thiD (SEQ ID NO 18, 14, 4) BS06893 BS05801 - BS05801 with the following mutation: fur-K14I3BS07063 BS06893 pBS3157 BS06893 carrying the hit from an RBS optimization experiment of rebalancing thiO_PPT and thiE yielding the thiamine production plasmid pBS3157 (p15A, SpecR) BS07191 BS05801 pBS3195 BS05801 carrying the hit from an RBS optimization experiment of rebalancing tenI_BSU yielding theCase Ref. P180WO IPTector® thiamine production plasmid pBS3195 (p15A, SpecR) BS07224 BS06893 - BS06893 with the following deletion: thiM translational KO (in apFAB synthetic operon) RBS optimization of thiE and thiD, thiM translational KO (all in synthetic operon apFAB71 / thiEMD) BS07228 BS07224 pBS3195 BS07224 carrying the thiamine production plasmid pBS3195 (p15A, SpecR) BS07288 BS07228 pBS3195 BS07228 with following mutation and deletion: RBS optimization of genomic thiD; carrying the thiamine production plasmid pBS3195 (p15A, SpecR) BS07309 BS07288 pBS3195, BS07288 carrying the thiamine production plasmid pBS3218 pBS3195 (p15A, SpecR) and IPTG inducible Thi1_CYA plasmid pBS3218 (pSC101, TetR) BS07310 BS07288 pBS3195, BS07288 carrying the thiamine production plasmid pBS3224 pBS3195 (p15A, SpecR) and IPTG inducible Thi1_STA plasmid pBS3224 (pSC101, TetR) BS07312 BS07288 pBS3195, BS07288 carrying the thiamine production plasmid pBS3048 pBS3195 (p15A, SpecR) and the empty control plasmid pBS3048 (pSC101, TetR) BS08569 BS07224 - BS07224 with following mutation and deletion: ∆thiM (native thiM, whole ORF deletion) BS08618 BS08569 pBS3195 BS08569 carrying the thiamine production plasmid pBS3195 (p15A, SpecR) BS07063 BS05801 pBS3157 BS05801 with following mutation and deletion: fur-K14I; carrying the thiamine production plasmid pBS3157 (p15A, SpecR) BS08711 BS07063 pBS3157, BS07063 carrying the thiamine production plasmid pBS3075 pBS3195 (p15A, SpecR) and constitutiveCase Ref. P180WO IPTector® expression of apFAB301 / sodA plasmid pBS3075 (pSC101, TetR) BS09106 BS04939 - BS05801, ∆TH2_ATH::[FRT,KanR,FRT]{ypjC,ileY} BS09234 BS09106 - BS09106, ∆TH2_ATH::[FRT]{ypjC,ileY} BS09143 BS04939 - BS04939, fur-K14I BS09160 BS09143 pBS3848 BS09143 carrying the cTHZ production plasmid pBS3848 (p15A, SpecR) Molecular biology techniques

[0279] Standard techniques were used for DNA isolation, amplification, purification and cloning (restriction digestion, ligation), transformation and the like. Such techniques are well known in the art and standard protocols can be found in: Maniatis et al., 1982 Molecular Cloning, Cold Spring Harbor Laboratory, Plainview NY. And M. Green, J. Sambrook (2012) Molecular Cloning: a laboratory manual. 4th Edition, Cold Spring Harbor Laboratory Press, CSH, NY., which are both hereby incorporated by reference in their entireties.

[0280] Genome Integrations of apFAB constitutive expression cassettes done according to Cui & Shearwin (2017)4. Deletion of genes according to Danesko & Wanner (2000)5. Point mutations introduced according to Gallagher et al. (2014)6.

[0281] Plasmids used herein were as follows: Plasmid ID Expression of genes / proteins Origin Antibiotic DNA marker sequence ID pBS31217p15A SpecRSEQ ID NO: apFAB071 / [thiF,thiS,thiG,thiI@TRC,thiO_PPT] 6, 8, 12, 22, 28 pBS3134 p15A SpecRSEQ ID NO: apFAB071 / [thiF,thiS,thiG,thiI@TRC,thiO_PPT] 6, 8, 12, 22, 28 pBS3157 p15A SpecRSEQ ID NO: apFAB071 / [thiF,thiS,thiG,thiI@TRC,thiO_PPT,t 6, 8, 12, 22, hiE] 28, 18 pBS3158 p15A SpecRSEQ ID NO: apFAB071 / [thiF,thiS,thiG,thiI@TRC,thiO_PPT,t 6, 8, 12, 22, hiE] 28, 18Case Ref. P180WO IPTector® pBS3183 p15A SpecRSEQ ID NO: apFAB071 / [thiF,thiS,thiG,thiI@TRC,thiO_PPT,t 6, 8, 12, 22, hiE, tenI_BSU] 28, 18, 24 pBS3195 p15A SpecRSEQ ID NO: apFAB071 / [thiF,thiS,thiG,thiI@TRC,thiO_PPT,t 6, 8, 12, 22, hiE, tenI_BSU] 28, 18, 24 pBS3739apFAB071 / [thiF,thiS,thiG,thiI@TRC,thiO_PPT,thiE] p15A SpecR SEQ ID NO: 6,8, 12, 22, 28, 18 pBS3808apFAB071 / [thiF,thiS,thiG,thiI@TRC,thiO_PPT,thiE],p15A SpecRSEQ ID NO: 6, apFAB310 / sodA8, 12, 22, 28, 18, 116 pBS3848 apFAB071 / [thiF,thiS,thiG,thiI@TRC,thiO_PPT , p15A SpecRSEQ ID NO: 6, tenI_BSU] 8, 12, 22, 28, 24pBS3048 NA (empty plasmid control) pSC101 TetRpSC101 TetRSEQ ID NO: pBS3203 T5lacO / TenA_BSU 46 pSC101 TetRSEQ ID NO: pBS3218 T5lacO / Thi1_CYA 30 pSC101 TetRSEQ ID NO: pBS3224 T5lacO / Thi1_SAU 32 pSC101 TetRSEQ ID NO: pBS3217 T5lacO / Thi1_ANE 34 pSC101 TetRSEQ ID NO: pBS3219 T5lacO / Thi1_MEL 36 pSC101 TetRSEQ ID NO: pBS3220 T5lacO / Thi1_NGR 38 pSC101 TetRSEQ ID NO: pBS3221 T5lacO / Thi1_BGL 40 pSC101 TetRSEQ ID NO: pBS3222 T5lacO / Thi1_HOC 42 pSC101 TetRSEQ ID NO: pBS3223 T5lacO / Thi1_OAL 44Case Ref. P180WO IPTector® pBS3224 pSC101 TetRSEQ ID NO: T5lacO / Thi1_SAU 32 pBS3199 T5LacO / phoA_ECO pSC101 TetRSEQ ID No: 48 pBS3200 T5LacO / nudJ_ECO pSC101 TetRSEQ ID No: 50 pBS3201 T5LacO / cof_ECO pSC101 TetRSEQ ID No: 52 pBS3215 T5LacO / TH2_ATH pSC101 TetRSEQ ID No: 26pBS3225 T5LacO / aphA_ECO pSC101 TetR SEQ ID No: 72pBS3863 T5LacO / kdsC_ECO pSC101 TetR SEQ ID No: 56pBS3864 T5LacO / ybhA_ECO pSC101 TetR SEQ ID No: 54ColE1, KanRNA pMB1, pBS3965 pET blank (empty plasmid control) pBR322, pUC ColE1, KanRSEQ ID No: pMB1, 100 pBS3850 apFAB70 / HAD_OBA@OPT,CHT*pBR322, pUC ColE1, KanRSEQ ID No: 88 pMB1, pBS3851 apFAB70 / HADPH_ACO@OPT,CHT pBR322, pUC ColE1, KanRSEQ ID No: 76 pMB1, pBS3852 apFAB70 / phoD_PAS@OPT,CHT pBR322, pUC ColE1, KanRSEQ ID No: 70 pMB1, pBS3853 apFAB70 / phoD_PPU@OPT,CHT pBR322, pUC ColE1, KanRSEQ ID No: 84 pMB1,pBS3855 T5LacO / At4g29530_ATH@OPT,CHTpBR322, pUC ColE1, KanRSEQ ID No: pMB1, 102 pBS3856 T5LacO / HADH_TSP@OPT,CHT pBR322, pUC ColE1, KanRSEQ ID No: pMB1, 104 pBS3857 T5LacO / HADPH_ROS@OPT,CHT pBR322, pUCCase Ref. P180WO IPTector® ColE1, KanRSEQ ID No: 94 pMB1, pBS3858 T5LacO / PSPH_SWO@OPT,CHT pBR322, pUC ColE1, KanRSEQ ID No: 86 pMB1, pBS3859 T5LacO / TMPPH_APA@OPT,CHT pBR322, pUC ColE1,Kan R SEQ ID No: 26pMB1, pBS3860 T5LacO / TH2_ATH_Z_WT@OPT,CHT pBR322, pUC ColE1, KanRSEQ ID No: pMB1,106pBS3955 T5LacO / HADPH_CBA@OPT,CHT pBR322, pUC ColE1, KanRSEQ ID No: pMB1, 100pBS3956 T5LacO / HAD_OBA@OPT,CHTpBR322, pUC ColE1,Kan R SEQ ID No: 88pMB1, pBS3957 T5LacO / HADPH_ACO@OPT,CHT pBR322, pUC ColE1, KanRSEQ ID No: 92 pMB1, pBS3958 T5LacO / HADPH_EVE@OPT,CHT pBR322, pUC ColE1, KanRSEQ ID No: 98 pMB1, pBS3959 T5LacO / HADPH_RUM@OPT,CHT pBR322, pUC ColE1, KanRSEQ ID No: 96 pMB1, pBS3960 T5LacO / HADPH_SEQ@OPT,CHT pBR322, pUC ColE1, KanRSEQ ID No: 76 pMB1, pBS3961 T5LacO / phoD_PAS@OPT,CHT pBR322, pUC ColE1, KanRSEQ ID No: 70 pMB1, pBS3962 T5LacO / phoD_PPU@OPT,CHT pBR322, pUC ColE1, KanRSEQ ID No: 60 pMB1, pBS3963 T5LacO / phoS_PAE@OPT,CHT pBR322, pUCCase Ref. P180WO IPTector® ColE1, KanRSEQ ID No: 90 pMB1, pBS3964 T5LacO / HADPH_DLO@OPT,CHT pBR322, pUC pBS3814 pSC101 TetRSEQ ID No: T5LacO / HP1287_HPY@OPT,CHT 116 pBS3815 pSC101 TetRSEQ ID No: T5LacO / tenA_AAN@OPT,CHT 118pBS3816 pSC101 TetRSEQ ID No: T5LacO / tenA_AMI@OPT,CHT 120 pBS3817 pSC101 TetRSEQ ID No: T5LacO / TenA_BSU@OPT,CHT 122 pBS3818 pSC101 TetRSEQ ID No: T5LacO / tenA_CPA@OPT,CHT 124 pBS3819 pSC101 TetRSEQ ID No: T5LacO / tenA_HHA@OPT,CHT 126 pBS3820 pSC101 TetRSEQ ID No: T5LacO / tenA_PAE@OPT,NHT 128 pBS3821 pSC101 TetRSEQ ID No: T5LacO / tenA_PFU@OPT,CHT 130 pBS3822 pSC101 TetRSEQ ID No: T5LacO / tenA_PHO@OPT,CHT 132 pBS3823 pSC101 TetRSEQ ID No: T5LacO / tenA_SAU@OPT,CHT 134pBS3824 pSC101 TetRSEQ ID No: T5LacO / tenA_SEP@OPT,CHT 136 pBS3825 pSC101 TetRSEQ ID No: T5LacO / tenA_SSO@OPT,CHT 138 pBS3826 pSC101 TetRSEQ ID No: T5LacO / THI20_SCE@OPT,CHT 140 pBS3802 pSC101 TetRSEQ ID No: T5LacO / THI20_SCE@TRC(1-302) 140 pBS3804 pSC101 TetRSEQ ID No: T5LacO / THI20_SCE@TRC(300-551),CHT 140 * @OPT = codon optimized using the Codon Optimization Tool algorithm from Twist Bioscience; CHT = C-terminal 6xHis tag; NHT = N-terminal 6xHis tag; TRC = truncated; Analytical ProceduresCase Ref. P180WO IPTector® Procedure I: Optical densities measurements

[0282] To measure optical densities (OD) of a cell culture as cuvette OD at 600 nm (cOD600), the culture was diluted 10-fold with dH2O to a final volume of 1 ml and transferred to a 1.5 ml transparent cuvette with 10 mm pathlength. The diluted culture was measured at 600 nm and 10 mm pathlength on a mySPEC (VWR). If the diluted culture was measured to cOD600>0.4, the culture was further diluted 10- fold and remeasured. Procedure 2: Thiochrome quantification of thiamine

[0283] For quantification of thiamine in supernatant samples from small-scale cultivations, the thiochrome assay was used following the assay described in section III of the methods in WO2017103221A1. Specifically, the supernatant from each culture was diluted alongside >20 thiamine standards in the concentration range of 0 µM (mg / L) to 60 µM (15.9 mg thiamine / L) prepared in Milli-Q water.50 µL of supernatant (either undiluted or a 1:4 dilution of the supernatant in water) and each of the thiamine standards were then added to a well of a 96 well microtiter plate. To each well, 100 μΙ of 4 M potassium acetate were added. Samples were then oxidized by the addition of 50 μΙ freshly prepared 3.8 mM potassium ferricyanide in 7 M NaOH. The solutions were mixed by pipetting and quenched byaddition of 50 μΙ fresh 0.06 % H202in saturated KH2P04. Samples were neutralized with 6M HCI and fluorescence was measured at 444 nm after excitation at 365 nm. The thiamine concentrations per well / sample were estimated based on comparison to standard curves included in the derivatization plate. Procedure 3: LCMS quantification of Sulfurol and its deriviatives

[0284] Commercial standards for the analytes of interest were purchased from suppliers listed in Table 1 below. Water (H2O) was purchased from Honeywell and methanol (MeOH), ascorbic acid and ammonium bicarbonate (NH4HCO3) from Sigma-Aldrich. Ammonium hydroxide was purchased from Carl Roth. Stock solutions of the analytes and internal standard were prepared in H2O:MeOH (50:50, v / v) + 0.1% ascorbic acid to a concentration of 1 mg mL-1. Working standard solutions of the stock solutions were then prepared in H2O:MeOH (50:50, v / v) + 0.1% ascorbic acid. Calibration curves in the concentrations of 0.1, 0.25, 0.5, 1, 2.5, 5, 10, 25, 50, 100, 250, 500 and 1000 ng mL-1 were prepared in H2O:MeOH (50:50, v / v) + 0.1% ascorbic acid and the internal standard 13C4-THI was added to the each calibration curve sample to correspond to a final concentration of 0.5 ng mL-1. A working standard solution of the internal standards was prepared in H2O:MeOH (50:50, v / v) + 0.1% ascorbic acid to contain 50 ng mL-1 of 13C4-THI. This internal standard solution (ISTD MIX) was added to each sample for quality control and normalization purposes. Sample preparation

[0122] Before analyses, the samples from the bioreactors were diluted and a mixture of internal standards (ISTD MIX) was added to correct for possible technical variation. To be able to quantify the analytes as accurately asCase Ref. P180WO IPTector® possible, two different dilution factors were applied. For analytes present in lower concentrations, a dilution factor of 1:50 was applied, while the analytes present in higher concentrations were quantified using a dilution factor of 1:1500. The 1:1500 dilution was prepared in two steps. First a 1:15 dilution was achieved by pipetting 980 L of H2O:MeOH (50:50, v / v) + 0.1% ascorbic acid into an Eppendorf tube and by adding 70 L of the supernatant. The tube was then vortex mixed thoroughly. The final 1:1500 dilution was achieved by pipetting 980 ^L of H2O:MeOH (50:50, v / v) + 0.1% ascorbic acid into a glass vial and by adding 10 L of the 1:15 diluted sample as well as 10 ^L of the ISTD MIX. The solution was again vortex mixed. The 1:50 dilution was achieved by pipetting 970 ^L of H2O:MeOH (50:50, v / v) + 0.1% ascorbic acid into a glass vial and by adding 20 ^L of the original sample as well as 10 L of the ISTD MIX. The solution was again vortex mixed. Liquid chromatography-tandem mass spectrometry

[0123] The samples were randomized after sample preparation and analyzed by ultra- high performance liquid chromatography (Infinity II, Agilent Technologies) coupled to tandem mass spectrometry (6470 Triple Quadrupole, Agilent Technologies) using electrospray ionization in positive ion mode. Selected reaction monitoring was used for quantifying the analytes and fragmentor voltages, collision energies and cell accelerator voltages were optimized for each ion transition.

[0124] The analytes were separated chromatographically before they enter the mass spectrometer. This was done using a XBridge Premier BEH C18 VanGuard FIT Column (4.6 mm X 100 mm, particle size 2.5 m, Waters Corporation) and H2O + 10 mM NH4HCO3 (pH 8.8, adjusted with NH4OH) as eluent A and MeOH + 10 mM NH4HCO3 (pH 8.8, adjusted with NH4OH) as eluent B with a flow rate of 0.6 mL min- 1. The elution gradient was as follows: 0-2.6 min 0% B to 20% B, 2.6-3 min 20% to 30% B, 3-3.5 min 30% B to 95% B, 3.5-5.5 min 95% B, 5.5-5.6 min 95% B to 0% B. After each run, the column was re- equilibrated at 0% B for 2.4 min. The injection volume for each sample was 2 microL. All data was acquired using the MassHunter Acquisition software (Version 10.0, Build 10.0.142) by Agilent Technologies and the retention times for each of the analytes are shown in Table 1. Table 1. Abbreviations and suppliers of commercial standards including retention times. Rt Analyte Abbreviation Supplier (min) (4-amino-2- Toronto methylpyrimidin-5- HMP-PP Research 1.3 yl)methyl Trihydrogen Chemicals Diphosphate 2-(2-carboxy-4- Analyticon methylthiazol-5-yl)ethyl cTHZ-P Biotechnologies 1.6 phosphate AG Toronto (4-amino-2- HMP-P Research 2 methylpyrimidin-5- ChemicalsCase Ref. P180WO IPTector® yl)methyl Dihydrogen Phosphate Toronto 4-Methyl-5-(2- THZ-P Research 3.1 phosphorylethyl)thiazole Chemicals Thiamine Pyrophosphate TPP Sigma Aldrich 3.5 Thiamine TMP Sigma Aldrich 3.7 Monophosphate 5-(2-hydroxyethyl)-4- methyl-1,3-thiazole-2- cTHZ Finetech 3.9 carboxylic acid Thiamine THI Sigma Aldrich 4 (4-amino-2-methyl-5- HMP Fluorochem 4.2 pyrimidinyl)methanol 13C4-thiamine13C4-THI Sigma Aldrich 4.5 Hydrocholoride Sulfurol / Thiazole THZ-P Sigma Aldrich 5.4

[0285] All data was processed using the MassHunter Quantitative Analysis software (Version B.09.00, Build 9.0.647.0) by Agilent Technologies. The peak areas for the analytes of interest were normalized against the peak areas of the internal standard C4 THI. Data quality was ensured by evaluating technical replicates of a specific fermentation sample as well as commercial standards. The quantified concentration of THI in these samples was required to remain within ±10% for the data set to be approved. Procedure 4: Improved LC-MS method

[0286] Commercial standards for the analytes of interest were purchased from suppliers listed in Table 1 below. Water (H2O) was purchased from Honeywell and acetonitrile (ACN) and ammonium bicarbonate (NH4HCO3)from Sigma-Aldrich. Ammonium hydroxide was purchased from Carl Roth. Stock solutions of the analytes and internal standard were prepared in H2O:ACN (50:50, v / v) to a concentration of 1 mg mL-1. Working standard solutions of the stock solutions were then prepared in H2O:ACN (50:50, v / v). Calibration curves in the concentrations of 25, 50, 100, 200, 400, 600, 800 and 1000 ng mL-1 were prepared in H2O:ACN (50:50, v / v) and the internal standard 13C4-THI was added to the each calibration curve sample to correspond to a final concentration of 0.5 ng mL-1. A working standard solution of the internal standards was prepared in H2O:ACN (50:50, v / v) to contain 50 ng mL- 1 of 13C4-THI. This internal standard solution (ISTD MIX) was added to each sample for quality control and normalization purposes. Sample preparation

[0122] Before analyses, the samples from the bioreactors were diluted and a mixture of internal standards (ISTD MIX) was added to correct for possible technical variation. The samples were prepared with a dilution factor of 1:2500. The 1:2500Case Ref. P180WO IPTector® dilution was prepared in two steps. First a 1:25 dilution was achieved by pipetting 960 µL of H2O:ACN (50:50, v / v) into a vial and by adding 40 L of the supernatant. The tube was then vortex mixed thoroughly. The final 1:2500 dilution was achieved by pipetting 980 µL of H2O:ACN (50:50, v / v) into a glass vial and by adding 10 L of the 1:25 diluted sample as well as 10 µL of the ISTD MIX. Liquid chromatography-tandem mass spectrometry. The samples were randomized after sample preparation and analyzed by ultra-high performance liquid chromatography (Infinity II, Agilent Technologies) coupled to tandem mass spectrometry (6470 Triple Quadrupole, Agilent Technologies) using electrospray ionization in positive ion mode. Selected reaction monitoring was used for quantifying the analytes and fragmentor voltages, collision energies and cell accelerator voltages were optimized for each ion transition.

[0124] The analytes were separated chromatographically before they enter the mass spectrometer. This was done using a XBridge Premier BEH C18 VanGuard FIT Column (4.6 mm X 100 mm, particle size 2.5 m, Waters Corporation) and H2O + 10 mM NH4HCO3 (pH 8.8, adjusted with NH4OH) as eluent A and ACN as eluent B with a flow rate of 0.6 mL min- 1. The elution gradient was as follows: 0-2.6 min 0% B to 20% B, 2.6-3 min 20% to 30% B, 3-3.5 min 30% B to 95% B, 3.5-5.5 min 95% B, 5.5-5.6 min 95% B to 0% B. After each run, the column was re-equilibrated at 0% B for 2.4 min. The injection volume for each sample was 2 µL. All data was acquired using the MassHunter Acquisition software (Version 10.0, Build 10.0.142) by Agilent Technologies and the retention times for each of the analytes are shown in Table 2. Table 2. Abbreviations and suppliers of commercial standards including retention times. Analyte Abbreviation Supplier Rt (min) (4-amino-2-methylpyrimidin-5- Toronto Research HMP-PPyl)methyl Trihydrogen Diphosphate Chemicals 2-(2-carboxy-4-methylthiazol-5- Analyticon cTHZ-Pyl)ethyl phosphate Biotechnologies AG (4-amino-2-methylpyrimidin-5- Toronto Research HMP-Pyl)methyl Dihydrogen Phosphate Chemicals 4-Methyl-5-(2- Toronto Research THZ-Pphosphorylethyl)thiazole Chemicals Thiamine Pyrophosphate TPP Sigma AldrichThiamine Monophosphate TMP Sigma Aldrich5-(2-hydroxyethyl)-4-methyl-1,3- CTHZ Finetechthiazole-2-carboxylic acidCase Ref. P180WO IPTector® Thiamine THI Sigma Aldrich 4 (4-amino-2-methyl-5- HMP Fluorochem 4.2 pyrimidinyl)methanol Sulfurol / Thiazole THZ-P Sigma Aldrich 5.4

[0287] All data was processed using the MassHunter Quantitative Analysis software (Version B.09.00, Build 9.0.647.0) by Agilent Technologies. The peak areas for the analytes of interest were normalized against the peak areas of the internal standard C4 THI. Data quality was ensured by evaluating technical replicates of a specific fermentation sample as well as commercial standards. The quantified concentration of THI in these samples was required to remain within ±10% for the data set to be approved. Procedure 5: HPLC quantification of Sulfurol and its derivatives

[0288] A 10 mM stock solution of carboxysulfurol (MW: 187.22 g / mol, CAS 75113-60-9) was prepared using a 1:1 methanol: water solvent. This stock was used to prepare the working standard solutions to create the six-point calibration curve that contains the following concentrations: 0, 50, 100, 200, 500, 1000, 3000 µM. Broth samples were centrifuged at 4000 x g for 15 minutes at 4 °C to separate cells from the supernatant. Fifty microliter aliquots of the supernatants were diluted in 450 µL solvent (1:1 MeOH: water) and passed through a 0.22 µm filter to create working samples. Carboxysulfurol was analyzed by HPLC using an Agilent HPLC system equipped with a photodiode array detector and autoinjector. Aliquots (2 uL) of the working samples and calibration standards were injected onto a reversed-phase C18 analytical column (Kinetex® 2.6µm EVO C18100 Å) with a gradient method flowing at 0.4 mL / min starting at 1% mobile phase B (H2O + 0.1% TFA: Methanol + 0.1% TFA) for 1.5 minutes and increased until 35 % mobile phase B for 5.5 minutes. From there, mobile phase B was further increased to 95% for 1.5 minutes before bringing the column back to the initial mobile phase B state of 1% for 2 minutes. Detection of 5-(2-hydroxyethyl)-4-methyl-1,3-thiazole-2-carboxylic acid (cTHZ) was done by monitoring absorbance at 290 nm. Example 1 – Conversion of a Thiamine producer to a Sulfurol and Sulfurol intermediates producing strain Introduction

[0289] The thiamine production strain (BS05801) and thiamine plasmid (pBS3121) were constructed as described in WO2024013212, Example 28. From this starting-point, various genetic modifications were made to convert the thiamine strain into a strain suitable for producing Sulfurol instead. ThisCase Ref. P180WO IPTector® was done by increasing the 5-(2-hydroxyethyl)-4-methyl-1,3-thiazole-2-carboxylic phosphate acid (cTHZ-P) production capacity of the strain while removing the ability of the strain to re-phosphorylate THZ / 5-(2-hydroxyethyl)-4-methyl-1,3-thiazole-2-carboxylic acid (cTHZ), allowing these to be secreted into the supernatant. Specifically, ThiM overexpression was removed and changes in the balancing of the expression of the ThiF, ThiS, ThiG, TenI pathway were made to increase production of the bottleneck molecule, 5-(2-hydroxyethyl)-4-methyl-1,3-thiazole-2-carboxylic phosphate acid (cTHZ-P) and thereby also capacity for 5-(2-hydroxyethyl)-4-methyl-1,3-thiazole-2-carboxylic acid (cTHZ), THZ- P and THZ (sulfurol) either directly, or indirectly via thiamine. Protocol

[0290] pBS3134 was cloned by amplification of the ThiE (SEQ ID NO: 18) gene from E. coli chromosomal DNA and a library of random ribosome binding sites (RBS) using Phusion U polymerase (Thermo Fischer Scientific) following manufacturer’s protocol and using primers containing Uracils for recognition by USER enzyme (New England Biolabs). Similarly, a plasmid backbone from pBS3124, containing thiF, thiS, thiG, thiI_TRC, thiO_PPT (SEQ ID NO: 6, 8, 12, 22, 28) expressed from a constitutive promoter apFAB071 and carrying origin p15A and a spectinomycin resistance cassette was amplified. During the backbone amplification a library of random RBSs was introduced in front of ThiO_PPT (SEQ ID NO: 28). DNA fragments were digested and ligated using USER enzyme (New England Biolabs) and T4 ligase (Thermo Fischer Scientific) following the manufacturer's protocols. These mixtures were introduced by electroporation into BS05801, and transformed cells were grown on selective LB agar supplemented with spectinomycin overnight at 37°C. The strains were grown in deep well plates in mMOPS media, with addition of 50 uM HMP, for 24 hours at 37°C and 275 rpm, after which thiamine production was evaluated using the thiochrome assay as described above in procedure 2. The highest producing strain was isolated, giving rise to a plasmid with balanced expression of ThiO_PPT and ThiE expression. This plasmid hit, pBS3158, was further cloned with a RBS library of TenI_BSU expression with USER cloning as described above, giving rise to pBS3183, and likewise screening for the highest thiamine production, yielding plasmid hit pBS3195 encoding balanced constitutive expression of thiF, thiS, thiG, thiI_TRC, thiO_PPT, thiE, and TenI_BSU (SEQ ID NO: 6, 8, 12, 22, 28, 18).

[0291] To effectively eliminate phosphorylation of 5-(2-hydroxyethyl)-4-methyl-1,3-thiazole-2- carboxylic acid (cTHZ) and THZ, allowing export of both to the supernatant without cell disruption, the gene ThiM (SEQ ID NO: 14) was completely removed from the chromosome of strain BS07224 using methods known in the art and replaced with only a short FRT sequence. One method to achieve this removal of the thiM gene is by generating a DNA fragment carrying a Kanamycin resistance gene flanked by homologous regions of ThiM and transforming strain BS07224 (carrying the λRedCase Ref. P180WO IPTector® recombinase genes expressed from an inducible promoter) with this DNA cassette. One protocol for such a method can be found in Datsenko, K. A. and Wanner, B. L., One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products, PNAS, 2000, 97(12):6640-5, DOI:10.1073 / pnas.120163297. The knockout of ThiM resulted in the E. coli strain BS08569 which was combined with the pBS3195 plasmid by electroporation resulting in BS08618. Production of thiamine (THI) and 5-(2-hydroxyethyl)-4-methyl-1,3-thiazole-2-carboxylic acid (cTHZ) in Figure 3 was analyzed in a Biolector®Pro experiment, as fed batch, using the B media as the batch and F media as the feed. Thiamine and 5-(2-hydroxyethyl)-4-methyl-1,3-thiazole-2-carboxylic acid (cTHZ) were quantified in the supernatant by LCMS as described in the analytical procedures. Results

[0292] The Knock-out of genomic ThiM over-expression in BS07312 led to accumulation of 5-(2- hydroxyethyl)-4-methyl-1,3-thiazole-2-carboxylic acid (cTHZ) in the supernatant, without any further modifications made. The parent strain, over-expressing ThiM, BS07191, accumulated no 5-(2- hydroxyethyl)-4-methyl-1,3-thiazole-2-carboxylic acid (cTHZ) under the same production conditions. Both strains contain the pBS3195 production plasmid and should have similar capacities for 5-(2- hydroxyethyl)-4-methyl-1,3-thiazole-2-carboxylic phosphate acid (cTHZ-P) production. 5-(2- hydroxyethyl)-4-methyl-1,3-thiazole-2-carboxylic acid (cTHZ) is likely produced via 5-(2-hydroxyethyl)- 4-methyl-1,3-thiazole-2-carboxylic phosphate acid (cTHZ-P) dephosphorylation, either via TH2_ATH, the overexpressed TMP phosphatase, or other native phosphatase activities present in E. coli. Without the over-expression of ThiM, the 5-(2-hydroxyethyl)-4-methyl-1,3-thiazole-2-carboxylic acid (cTHZ) is not re-phosphorylated to 5-(2-hydroxyethyl)-4-methyl-1,3-thiazole-2-carboxylic phosphate acid (cTHZ-P) and is therefore unable to be converted to Thiamine. This phenomenon also decreases the thiamine production capacity of the strain. Example 2 – Biological conversion of Thiamine to Sulfurol with Thiaminase I Introduction

[0293] Thiaminases are a class of enzymes that break down thiamine into Sulfurol and other byproducts. Two classes exist. Type I thiaminase, Thiamine pyridinylase (represented here by Thi1_CYA, SEQ ID NO: 30) and Type II thiaminases, Aminopyrimidine aminohydrolase (represented here by TenA_BSU, SEQ ID NO: 46). Using the expression of these thiaminases, biologically produced thiamine can be converted to Sulfurol in a 1:1 molar stoichiometry in vivo. Thiaminases can additionally act on Thiamine-phosphate (TMP) and Thiamine-pyrophosphate (TPP) to produce Sulfurol-phosphate (THZ-P) or sulfurol-pyrophosphate (THZ-PP). ProtocolCase Ref. P180WO IPTector®

[0294] Thiaminase sequences were amplified from E. coli codon optimized DNA sequences ordered as gBlocks from IDT, or from native DNA. They were cloned behind an IPTG-inducible promoter as described in Example 1, yielding plasmids pBS3203 to pBS3223. This plasmid were transformed into the thiamine production strain BS07288. The resulting strains were assayed for thiamine production as described in Example 1, but with the addition of IPTG to induce expression of thiaminases. The strain with the highest reduction in thiamine production, BS07309 and therefore highest presumed generation of Sulfurol as a product of the thiaminase reaction, was chosen for fed-batch fermentation in Biolector®Pro as described in Example 1. Results

[0295] The IPTG induced over-expression of Thi_CYA in BS07309 led to successful biological conversion of a fraction of the Thiamine produced, converting the thiamine production strain to a 5- (2-hydroxyethyl)-4-methyl-1,3-thiazole-2-carboxylic acid (cTHZ) and sulfurol (THZ) producer, as seen in comparison to the control strain BS07312 in Figure 3. Example 3 – Observed formation of the sulfurol intermediates 5-(2-hydroxyethyl)-4-methyl-1,3- thiazole-2-carboxylic acid (cTHZ) and THZ-P Introduction

[0296] In a sulfurol production strain, e.g. BS07309, we could observe detectable amounts of the sulfurol intermediates 5-(2-hydroxyethyl)-4-methyl-1,3-thiazole-2-carboxylic acid (cTHZ) and THZ-P, which derive from enzymatic activity in the host cell. We therefore deemed it important to establish a possible chemical conversion route for these intermediates (see Example 4). Protocol

[0297] A set of sulfurol production strains such as BS07309 and BS07310 were grown in a BioLector® Pro (m2p.labs) experiment under the following conditions: Experiments BLP-22-011 and BLP-22-012 were run with either 0 or 20 mM IPTG induction for 24 hours according to the manufacturer’s standard protocol. Results

[0298] We could observe up to 15.5 mg / L 5-(2-hydroxyethyl)-4-methyl-1,3-thiazole-2-carboxylic acid (cTHZ) in BS07309, and up to 16.7 mg / L THZ-P in BS7310, measured by LCMS as described above. The fact that we were producing these intermediates opened up the possibility for further chemical conversion to sulfurol as described in Example 4. Example 4 – Chemical conversion of biological 5-(2-hydroxyethyl)-4-methyl-1,3-thiazole-2- carboxylic phosphate acid (cTHZ-P), 5-(2-hydroxyethyl)-4-methyl-1,3-thiazole-2-carboxylic acidCase Ref. P180WO IPTector® (cTHZ) and THI / TMP to Sulfurol Introduction

[0299] 5-(2-hydroxyethyl)-4-methyl-1,3-thiazole-2-carboxylic acid (cTHZ) (1), 5-(2-hydroxyethyl)-4- methyl-1,3-thiazole-2-carboxylic phosphate acid (cTHZ-P) (2), THI (4), and THZ-P (5), the sulfurol precursors, were produced in certain abundance in the sulfurol production strain (Example 1) depending on combinatorial overexpression of thiazole phosphatases, decarboxylases (Example 3), and thiaminases (Example 2). These precursors can be chosen to be produced as the main fermentation product, as they are water-soluble and have downstream recoverability characteristics. These sulfurol precursors can then be chemically converted to sulfurol by chemical reactions (a, b and c). Also, THI / TMP produced in a thiamine fermentation can be converted to sulfurol by chemistry (d). (a) Chemical decarboxylation of fermented 5-(2-hydroxyethyl)-4-methyl-1,3-thiazole-2-carboxylic acid (cTHZ) 1

[0300] Chemical reaction of the conversion of 5-(2-hydroxyethyl)-4-methyl-1,3-thiazole-2-carboxylic acid (cTHZ) to sulfurol (a) as shown in Figure 4. Acid-catalyzed decarboxylation of 5-(2-hydroxyethyl)- 4-methyl-1,3-thiazole-2-carboxylic acid (cTHZ) can be performed over a range of conditions. The choice of acid, temperature and time conditions for the decarboxylation reaction is based on a reaction optimization process shown in table 3. Table 3 Choice of acid, temperature and time conditions for the decarboxylation reaction solvent acid temp (°C) reaction time (h) neat none 80 19 neat none 150 10 water none 100 10 ethanol none 80 19 acetic acid none 100 29 water 0.2M acetic acid 100 2 water 0.025 acetic acid 100 1 water 0.5M HCl 100 1 Protocol

[0301] Acetic acid or hydrochloric acid was added to a supernatant containing 2 g / L of 5-(2- hydroxyethyl)-4-methyl-1,3-thiazole-2-carboxylic acid (cTHZ). This mixture was then stirred for 2 hours at 100°C to generate THZ. Sulfurol was extracted and purified using DSP protocols.Case Ref. P180WO IPTector® (b) Chemical dephosphorylation of fermented 4-methyl-5-hydroxyethylthiazole phosphate (THZ-P) 6.

[0302] Chemical dephosphorylation of 6 is done by base hydrolysis as shown in Figure 5. The atoms that make up the hydroxyl group of 1 are all derived from fermentation making it natural. This subtle feature is due to an intermolecular rearrangement that happens during the course of hydrolysis. Protocol

[0303] NaOH is dissolved in fermentation broth to make a 100 mM solution of NaOH containing 2g / L of 6. The mixture is then stirred for 2 hours at 100 °C. Sulfurol will then be extracted and purified using DSP protocols. (c) Chemical conversion of fermented thiamine 4 to sulfurol.

[0304] Biologically produced thiamine 4 produced via fermentation is used as a chemical precursor to sulfurol as shown in Figure 6. These can be converted to sulfurol by chemistry using reactions based on the known chemical degradation and enzymatic conversion pathways of 4. In general, this reaction happens when 4 or 5 undergoes a nucleophilic releasing sulfurol along with a sulfonated pyrimidine derivative. The nucleophile for the release mechanism (c) is based on sulfite chemistry (e.g. sodium sulfite, bisulfite). Protocol

[0305] Solid NaHSO3 was dissolved in fermentation broth to make a 2.0 % solution of NaHSO3 w per volume of containing 2g per L of 4. The mixture had a pH of 5.5 upon dissolution and was then stirred for 2 hours at room temperature. Sulfurol was then extracted and purified using DSP protocols. Results

[0306] Sulfurol 2 formation through degradation of fermented THI 4. A supernatant containing THI 4 was converted to THI by addition of 2.0 % NaHSO3. Product formation was analyzed by HPLC at 254 nM by taking aliquots of the reaction mixture at 30, 60 and 120 minutes starting from t = 0. Complete conversion was achieved in 120 minutes. (d) Combined chemical dephosphorylation and decarboxylation of fermented 5-(2-hydroxyethyl)-4- methyl-1,3-thiazole-2-carboxylic phosphate acid (cTHZ-P) 3. cTHZ-P is a water-soluble precursor that can be recovered and converted to sulfurol during the down stream process. Except for the metal divalent cation requirement, the process of converting cTHZ-P to sulfurol is similar to the decarboxylation reaction. Metal cations are required to coordinate the phosphate group and facilitate the nucleophilic attack of water. When combined with the decarboxylation process, this acid-catalyzed dephosphorylation reaction to the monophosphate esterCase Ref. P180WO IPTector® yields sulfurol. Table 4. Choice of acid, metal cation and reaction duration for the combined decarboxylation and dephosphrylation reaction solvent acid Cation (0.1 equiv) Temp (°C) Reaction time (h) Water none Zn 100 2 Water HCl, 1M None 100 2 Water HCl, 0.1M Zn 100 2 water HCl, 1M Mg 100 2 Water H2SO41M None 100 2 Water H2SO40.1M Zn 100 2 water H2SO41M Mg 100 2 Protocol Hydrochloric acid or sulfuric acid as well as zinc sulfate or magnesium sulfate are added to the supernatant containing 2 g / L of carboxy sulfurol phosphate. The mixture was then stirred for 2 hours at 100 °C to generate THZ. Sulfurol will then be extracted and purified using DSP protocols. Example 5 – Thiamine fermentation route Introduction

[0307] The thiamine production strain(s) can produce high titers of thiamine when the substrate HMP is fed. The produced thiamine can then be processed in DSP with chemistry to yield sulfurol with high purity. Protocol

[0308] The thiamine fermentations were performed as described previously in WO2024013212, Example 2., with the following changes: Fermentation under different conditions was carried out for strain BS07063. This strain is based on strain BS06893 carrying the pBS3157 plasmid that is optimized for expression of thiE and thiO_PPU. In initial experiments, different temperature and dissolved oxygen (DO) conditions were tested. In FERM-24-012, the fermentation of strain BS07063 was carried out with fermentation batch media B medium and feed media F medium (which were not optimized for scale up). This fermentation was carried out at 31 °C, 30% DO, and a feed rate of 9 g glucose solution per liter per hour.15 mM HMP (hydroxymethylpyrimidine phosphate) was fed as substrate. The fermentation was buffered with NH4OH to maintain a stable pH of 7.

[0309] Fermentation conditions were optimized for strain BS07063. The batch and feed media were optimized to be compatible with upscaling conditions, such as the elimination of chlorides and boricCase Ref. P180WO IPTector® acid that corrode metal tanks. The optimized media upscaling batch media UB medium and optimized upscaling feed media UF medium were used in fermentation FERM-24-035. The optimized feeding profile (OFP) used was a slow linear increase from 4 g of glucose solution per liter per hour to 12 g of glucose solution per liter per hour for 63h, following a constant feeding profile of 11 g of glucose solution per liter per hour until the end of fermentation. The feeding profile started at the end of the batch phase and was triggered by an increase in the pH. 15 mM HMP (hydroxymethylpyrimidine phosphate) was fed as substrate, and the fermentation was carried out at 31 °C with 15% DO.

[0310] Strain BS08711 was constructed by transforming strain BS07063 with the additional plasmid pBS3075 carrying sodA expressed constitutively. Strain BS08711 was fermented in FERM-24-037 with media UB and UF, 15 mM HMP concentration in the reactor, with the OFP feeding profile, at 31 °C, with 15% DO, at pH 7.ResultsIn FERM-24-012, the fermentation of strain BS07063 yielded a maximum titer of 1452 mg / L thiamine (Figure 7, left bar) and Table 5. In FERM-24-035, the fermentation of strain BS07063 yielded a maximum titer of 2246 mg / L thiamine (Figure 7, middle bar) and Table 5 In FERM- 24-037, the fermentation of strain BS08711 yielded a maximum titer of 2575 mg / L thiamine (Figure 7, right bar) and Table 5. Table 5: Thiamine production in fermentation by different strains. Fermentation FERM-24012 FERM-24013 FERM-24035 FERM-24037 FERM-24058 ID Strain BS07063 BS07063 BS07063 BS08711 BS08809 (SodA) (parDE stabilized) Fermentation 31 ° C, 15% 31 ° C, 15% 31 ° C, 15% 31 ° C, 15% 31 ° C, 15% conditions DO, pH=7 DO, pH=7 DO, pH=7 DO, pH=7 DO, pH=7 Feeding Profile 9g glucose 9g glucose 4g glucose 4g glucose 4g glucose solution / L / h solution / L / h - solution / L / h - solution / L / h - solution / L / h - -> 22 for 18h, -> 12 for 63h, -> 12 for 63h, -> 12 for 63h, then 9 then 11 then 11 then 11 Media Media with Media with Media Media Media chlorides chlorides without without without chlorides chlorides chlorides HMP (mM) 6 (enough for 6 (enough for 15 (enough 15 (enough 15 (enough 1592 mg / L 1592 mg / L for 3980 mg / L for 3980 mg / L for 3980 mg / L THI) THI) THI) THI) THI) Overflow? No Yes No No NoCase Ref. P180WO IPTector® Thiamine 1452 1500 2246 2575 2418 (mg / L) %Fold - - 54.7 14.7 12.1 improvement Example 6 – c-sulfurol fermentation route Introduction

[0311] The 5-(2-hydroxyethyl)-4-methyl-1,3-thiazole-2-carboxylic acid (cTHZ) production strain(s) can produce relevant titers of 5-(2-hydroxyethyl)-4-methyl-1,3-thiazole-2-carboxylic acid (cTHZ) in the supernatant. The produced 5-(2-hydroxyethyl)-4-methyl-1,3-thiazole-2-carboxylic acid (cTHZ) can then be processed in DSP with chemistry to yield sulfurol with high purity.

[0312] ProtocolStrains BS07288 and BS08618 were analyzed for 5-(2-hydroxyethyl)-4-methyl-1,3- thiazole-2-carboxylic acid (cTHZ) production in fermentation. Strain BS07288 is based on the thiamine production strain BS06893 with optimized RBS sequences for thiE and thiD and a translational KO of thiM, all located in the synthetic operon apFAB71 / thiEMD. This strain still has a functional native copy of thiM. Strain BS08618 is based on BS07224 and has the same genotype as BS07288 with following addition that the native thiM copy is deleted as a whole ORF deletion, yielding ∆thiM. This strain is a true thiM negative strain with no sulfurol-kinase activity. Fermentations:

[0313] Strain BS07288 was run in FERM-24-044. The optimized media upscaling batch media UB medium and optimized upscaling feed media UF medium were used in fermentation FERM-24-044. The optimized feeding profile (OFP) used was a slow linear increase from 4 g of glucose solution per liter per hour to 12 g of glucose solution per liter per hour for 63h, following a constant feeding profile of 11 g of glucose solution per liter per hour until the end of fermentation. The feeding profile started at the end of the batch phase and was triggered by an increase in the pH. The fermentation was carried out at 31 °C with 15% DO.Strain BS08618 was run in FERM-24-022. The non-optimized batch media (containing all chlorides and boric acid) and the non-optimized feeding solution were used here. The feeding profile was also not optimized. It was a constant rate of 9 grams of glucose solution per liter per hour. The fermentation was carried out at 31 °C with 15% DO. Results

[0314] Strain BS07288 was run again in FERM-25-018 with the exact same conditions as FERM-24- 044Strain BS07288 run in FERM-24-044 yielded a titer of 819 mg / L 5-(2-hydroxyethyl)-4-methyl-1,3- thiazole-2-carboxylic acid (cTHZ) measured by HPLC. Strain BS08618 run in FERM-24-022 yielded a titer of 386 mg / L 5-(2-hydroxyethyl)-4-methyl-1,3-thiazole-2-carboxylic acid (cTHZ) measured byCase Ref. P180WO IPTector® HPLC.

[0315] The concentrations of both 5-(2-hydroxyethyl)-4-methyl-1,3-thiazole-2-carboxylic acid (cTHZ) and 5-(2-hydroxyethyl)-4-methyl-1,3-thiazole-2-carboxylic phosphate acid (cTHZ-P) were quantified in the supernatant as shown in table 6. A significant accumulation of 1151 mg / L 5-(2-hydroxyethyl)-4- methyl-1,3-thiazole-2-carboxylic phosphate acid (cTHZ-P) was detected extracellularly as early as 24 hours into the fermentation. However, its concentration decreased substantially towards the end of the fermentation to 8 mg / L, while 5-(2-hydroxyethyl)-4-methyl-1,3-thiazole-2-carboxylic acid (cTHZ) levels increased continuously throughout the process, reaching a final titer of 724 mg / L. Quantification of 5-(2-hydroxyethyl)-4-methyl-1,3-thiazole-2-carboxylic phosphate acid (cTHZ-P) was not performed in fermentations prior to FERM-25-018 since there had been no commercially available standard. The 5-(2-hydroxyethyl)-4-methyl-1,3-thiazole-2-carboxylic phosphate acid (cTHZ-P) standard was obtained by ordered synthesis at Analyticon® Biotechnologies GmbH (Germany). Table 6 Fermentation ID Strain Time (h) c-sulfurol c-sulfurol (mg / L) phosphate (mg / L) 18 42 929 24 63 1151 FERM-25-018 BS07288 42 211 767 65 472 318 72 503 198 90 724 8 Example 7 – Sulfurol fermentation route Introduction

[0316] The sulfurol production strain(s) can produce sulfurol in the supernatant. The produced sulfurol can then be processed in DSP with chemistry to yield sulfurol with high purity. Protocol

[0317] Strain BS07309 was analyzed for sulfurol production in bench fermentation FERM-24-008. This strain is based on BS07288, a strain where the overexpression of thiM was abolished by a translational KO of the thiM gene in the overexpression operon apFAB71 / thiEMD. BS07309 carries the thiamine production plasmid pBS3195 (p15A, SpecR) and the IPTG-inducible Thi1_CYA plasmid pBS3218 (pSC101, TetR).

[0318] Strain BS07309 was fermented in FERM-24-008. The process was run at 31°C, 15% DOCase Ref. P180WO IPTector® (controlled by stirring), pH=7 using the initial fermentation medium before removing the chlorides, B and F medium. Results

[0319] Strain BS07309 run in FERM-24-008 yielded a titer of 81 mg / L sulfurol (THZ), 124 mg / L THI, and 218 mg / L 5-(2-hydroxyethyl)-4-methyl-1,3-thiazole-2-carboxylic acid (cTHZ) measured by LCMS. Example 8 – Chemical route from thiamine to sulfurolIntroduction

[0320] Purification of sulfurol from thiamine-producing strains is a multistep process that begins with the selective capture of thiamine followed by a decomposition reaction using chemistry to generate sulfurol. Central to the design of this sulfurol purification process is the different chemical natures of thiamine and sulfurol. This difference allows orthogonal capture strategies based on charge (for thiamine) and polarity (for sulfurol) to be arranged in series to recover high purity sulfurol from a cell- free supernatant. The general overview of this purification process is shown in Figure 8. Protocol

[0321] Step 1. Harvested broth was centrifuged at 4000 g for 20 minutes at 4 °C to separate the cells from the supernatant.

[0322] Step 2. Supernatant was passed through a membrane microfiltration system to generate a cell- free supernatant.

[0323] Step 3. To capture thiamine, cell-free supernatant was passed through a column containing PuroLite C104Plus, a weak anion exchange resin charged to pH 5.5 using deionized water. The load ratio is maintained at 300 mL of supernatant for every 20 mL of dry resin. After the supernatant has passed through the resin column, the resin was washed with 4 column volumes of deionized water. Finally, resin-bound thiamine is recovered by elution using 2 column volumes of water acidified with 0.1 M aqueous HCl.

[0324] Step 4. To convert thiamine to sulfurol, the highly acidic thiamine-containing eluant is first neutralized with 1M aqueous NaOH to at least pH 6 by titration. Sodium bisulfite was then added to the solution at 2.0 % w / v at pH and the mixture was stirred for two hours at room temperature. The pH of this reaction goes to 5.5 upon addition of sodium bisulfite.

[0325] Step 5. Sulfurol produced from the breakdown of thiamine in solution was extracted twice using ethyl acetate. This was done by adding 400 mL ethyl acetate for every 1000 mL of reaction mixture.

[0326] Step 6. The organic phases were combined and dried using sodium sulphate. Ethyl acetate is then evaporated to yield sulfurolCase Ref. P180WO IPTector® Results

[0327] Sulfurol recovered from thiamine producing strains was analyzed for purity using GCMS and NMR. Conversion of thiamine to sulfurol using bisulfite works optimally from pH 4.5-6. Purity of sulfurol resulting from the downstream process was estimated to be at 93.0% using GCMS (Figure 9). One dimensional NMR spectrum shows identical signals when compared to a reference standard (Figure 10). Example 9 – Chemical route from c-sulfurol to sulfurol Introduction

[0328] Purification of sulfurol from carboxysulfurol producing strains is a multistep process wherein the key step is an acid-catalyzed decarboxylation of caboxysulfurol to generate sulfurol in aqueous solution. Prior to decarboxylation, the supernatant is stripped off nonpolar compounds and other impurities that interfere with the reaction using solvent extraction. Sulfurol generated from the decarboxylation process is then captured by another round of solvent extraction and is followed by distillation to recover high purity sulfurol. The general overview of this purification process is shown in Figure 11. Protocol

[0329] Step 1. Harvested broth is centrifuged at 4000 g for 20 minutes at 4 °C to separate the cells from the supernatant.

[0330] Step 2. The supernatant is then passed through a membrane filtration system to generate a cell-free supernatant.

[0331] Step 3. Supernatant containing carboxy sulfurol was extracted three times with ethyl acetate to remove non-polar impurities.

[0332] Step 4. To the aqueous phase, 6M aqueous HCl solution was added to set the pH at 2.0. The mixture was then heated at 100°C for 2.5 h to allow decarboxylation of carboxysulfurol as shown in figure 4. The choice of acid, temperature and time conditions for the decarboxylation reaction is based on a reaction optimization process shown in table 7. Table 7 Choice of acid, temperature and time conditions for the decarboxylation reaction solvent acid temp (°C) reaction time (h) neat none 80 19 neat none 150 10 water none 100 10 ethanol none 80 19 acetic acid none 100 29Case Ref. P180WO IPTector® water 0.2M acetic acid 100 2 water 0.025 acetic acid 100 1 water 0.5M HCl 100 1

[0333] Step 5. After heating, the reaction mixture was neutralized to pH 7 with 10M aqueous sodium hydroxide solution at room temperature and the mixture was extracted eight times with ethyl acetate to recover sulfurol.

[0334] Step 6. The combined organic phases were dried over sodium sulphate and the solvent was evaporated. The crude product was resolved in ethyl acetate and the formed salt was filtered off. The solvent was evaporated to yield a crude brown oil that contained sulfurol.

[0335] Step 7. Crude sulfurol was then distilled to purify sulfurol.

[0336] Overview of the sulfurol purification process on the carboxysulfurol producing strains (Figure 11) Results Sulfurol generated from carboxysulfurol producing strains from this downstream process was analyzed for purity and identity using GCMS (Figure 12) and NMR (Figure 13). The decarboxylation reaction works optimally between pH 1.0-3.0. Other than the major product sulfurol, traces of pantolactone and another unknown compound were detected in the distillate. Example 10 – Chemical route from c-sulfurol to sulfurol using resins Introduction Purification of sulfurol from carboxysulfurol producing strains using resins is similar to the direct carboxysulfrol to sulfurol conversion process except for a capturing step. Resins of different affinity mechanisms were used to directly capture carboxysulfurol in the supernatant prior to decarboxylation. This step allows carboxysulfurol to be transferred to an aqueous environment different from the supernatant and allows for a more favorable reaction environment. The general overview of this purification process is shown in Figure 14. Protocol

[0337] Step 1. Harvested broth is centrifuged at 4000 g for 20 minutes at 4 °C to separate the cells from the supernatant.

[0338] Step 2. The supernatant is then passed through a membrane filtration system to generate a cell-free supernatant.

[0339] Step 3. From a list of various ion-exchange resins charged over a range of pH (table 6), a weak anion exchange resin was added to a carboxysulfurol solution and the reaction mixture was shakenCase Ref. P180WO IPTector® overnight at room temperature. Following overnight incubation, more resin was added, and the mixture was shaken for another 4h at room temperature. After full binding, the resin was filtered off and the resin was washed with water several times. To strip the resin of carboxysulfurol, 0.5M aqueous HCl solution was added, and the mixture was shaken for 1h at room temperature. The resin was filtered off and the solution was collected. Table 8 List of various ion-exchange resins charged over a range of pH. Resin initial pH end pH AmberLite FPA66 OH 4 6.5 AmberLite FPA66 OH 7 6.5 AmberLite FPA66 OH 9 6.5 AmberLite IRN78 OH 4 10 AmberLite IRN78 OH 7 10 AmberLite IRN78 OH 9 10 AmberLyst A26 OH 4 10 AmberLyst A26 OH 7 10 AmberLyst A26 OH 9 10 Ambersep 900 4 10 Ambersep 900 7 10 Ambersep 900 9 10 AmberLite IRA67 4 8 AmberLite IRA67 7 8 AmberLite IRA67 9 9

[0340] Step 4. The combined HCl phases was heated at 100°C for 2h to facilitate decarboxylation.

[0341] Step 5. After heating, the reaction mixture was neutralized with 2M aqueous sodium hydroxide solution at room temperature and the mixture was extracted with ethyl acetate.

[0342] Step 6. The organic phase was dried over sodium sulphate and the solvent was evaporated to yield crude sulfurol.

[0343] Step 7. Crude sulfurol was then distilled to purify sulfurol. Results

[0344] Sulfurol generated from carboxysulfurol producing strains from this downstream process was analyzed for purity and identity using NMR (Figure 15). The decarboxylation reaction in this process works optimally between pH 1.0-3.0 Other than the major product sulfurol, traces of pantolactone and other unknown compounds were detected in the distillate.Case Ref. P180WO IPTector® Example 11 – Overexpression of phosphatases for production of 5-(2-hydroxyethyl)-4-methyl-1,3- thiazole-2-carboxylic acid (cTHZ) Introduction

[0345] In order to achieve a higher conversion rate of 5-(2-hydroxyethyl)-4-methyl-1,3-thiazole-2- carboxylic phosphate acid (cTHZ-P) to 5-(2-hydroxyethyl)-4-methyl-1,3-thiazole-2-carboxylic acid (cTHZ), we are testing a panel of different alkaline phosphatases on their ability to produce 5-(2- hydroxyethyl)-4-methyl-1,3-thiazole-2-carboxylic acid (cTHZ). Protocol

[0346] We have constructed auxiliary plasmids containing a panel of alkaline phosphatases sourced from E. coli or other organisms in order to find a candidate for the conversion of 5-(2-hydroxyethyl)- 4-methyl-1,3-thiazole-2-carboxylic phosphate acid (cTHZ-P) to 5-(2-hydroxyethyl)-4-methyl-1,3- thiazole-2-carboxylic acid (cTHZ). The phosphatases are expressed under the IPTG-inducible pT5LacO promoter and are analyzed in a strain carrying the 5-(2-hydroxyethyl)-4-methyl-1,3-thiazole-2- carboxylic acid (cTHZ) production plasmid pBS3848. Results

[0347] The overexpression of additional phosphatases is increasing the overall production of 5-(2- hydroxyethyl)-4-methyl-1,3-thiazole-2-carboxylic acid (cTHZ).

[0348] We sourced a panel of phosphatases from E. coli or other organisms (archae, bacteria, fungi or plant) in order to find an efficient phosphatase for the conversion of cTHZ-P to cTHZ. The phosphatase sequences were analyzed and the respective ESP (Enzyme-Substrate Pair Prediction) score on the DeepMolecules server calculated (https: / / esp.cs.hhu.de / )(Kroll et al., 2023, Kroll et al., 2024). The substrates analyzed were cTHZ-P (KEGG ID C20247), cTHZ-P* (tautomer, KEGG ID C20246), TMP (KEGG ID C01081) and THI (KEGG ID C00378). The results are shown in Table 9Case Ref. P180WO IPTector® Table 9 ESP score of sourced phosphatases

[0349] Due to low ESP scores, we did not continue to work with PHOS_nudJ_PPU, PHOS_alkP_PPU, PHOS_ygiF_PPU, PHOS_upxB_PPU, PHOS_nudJ_SSO, PHOS_AH2_HIN and PHOS_AH2_CSI. PHOS_rsgA_SAU had been sourced previously but failed to yield correct clones in the cloning, and was also not pursued further.

[0350] We constructed auxiliary plasmids containing the remaining phosphatases of the panel. E. coli genes were cloned from wild type K-12 genomic DNA, heterologous genes were ordered from Twist Bioscience. The first batch of phosphatases was expressed under the IPTG-inducible T5LacO promoter, on a backbone with pSC101 origin and tetracyclin resistance, and was analyzed in the production strain BS09106 which is ΔTH2_ATH and carrying the cTHZ production plasmid pBS3848. The strains used in this experiment are listed in Table 10.Case Ref. P180WO IPTector® Table 10 Strains tested in Example 11 - batch 1 STRAIN Parent Plasmid Plasmid Genomic Phosphatase ID strain 1 2 background 1 Phosphatase 2 ∆cyaA, BS07288 BS08542 pBS3195 - thiM@STOP gTH2_ATH - ∆cyaA, BS08618 BS08569 pBS3195 - thiM@STOP / ΔthiM gTH2_ATH - BS09097 BS04939 pBS3848 - ∆cyaA, thiM gTH2_ATH - BS09211 BS09106 pBS3848 pBS3199 ∆cyaA, thiM ΔTH2_ATH T5LacO / phoA_ECO BS09212 BS09106 pBS3848 pBS3200 ∆cyaA, thiM ΔTH2_ATH T5LacO / nudJ_ECO BS09213 BS09106 pBS3848 pBS3201 ∆cyaA, thiM ΔTH2_ATH T5LacO / cof_ECO BS09214 BS09106 pBS3848 pBS3215 ∆cyaA, thiM ΔTH2_ATH T5LacO / TH2_ATH BS09215 BS09106 pBS3848 pBS3225 ∆cyaA, thiM ΔTH2_ATH T5LacO / aphA_ECO BS09216 BS09106 pBS3848 pBS3863 ∆cyaA, thiM ΔTH2_ATH T5LacO / kdsC_ECO BS09217 BS09106 pBS3848 pBS3864 ∆cyaA, thiM ΔTH2_ATH T5LacO / ybhA_ECO None / empty BS09218 BS09106 pBS3848 pBS3048 ∆cyaA, thiM ΔTH2_ATH plasmid

[0351] The second batch of phosphatases are expressed under the IPTG-inducible T5LacO promoter, on a backbone with ColE1, pMB1, pBR322, pUC (rop) origin and kanamycin resistance, and are analyzed in the production strain BS09234 which is ΔTH2_ATH (no kan marker) and carrying the cTHZ production plasmid pBS3848. Results

[0352] The overexpression of the phosphatases in batch 1 PHOS_phoA_ECO (BS09211, 14-15% increase compared to control BS09218), PHOS_TH2_ATH (BS09214, 17-22% increase), PHOS_aphA_ECO (BS09215, 7-8% increase) and PHOS_kdsC_ECO (BS09216, 44-60% increase) increased the overall titer of cTHZ compared to the control strain BS09218 (Table 11). These are promising candidates to explore further in fermentation in future experiments. Table 11. cTHZ titer, OD600 and yield in strains overexpressing additional phosphatases of batch 1. No induction Median % Median % Increase titer Median increase yield increase Strain %RSD over %RSD %RSD (mg / L OD over (mg / L over Ctrl (%) cTHZ) Ctrl pr. OD) Ctrl BS072882,45 2,82 -11,3 2,7 4,38 -20,6 0,92 5,829,92BS086183,13 1,73 13,5 3,5 2,86 2,94 0,89 2,385,87BS092113,18 1,07 15,3 3,6 5,89 5,88 0,88 7,095,11BS092120,68 60,4 -75,3 3,0 3,77 -11,8 0,23 62,4-72,9Case Ref. P180WO IPTector® BS092132,76 2,99 0,04 3,5 4,41 2,94 0,77 5,50-7,60BS092143,21 2,39 16,5 3,5 7,26 2,94 0,92 5,769,45BS092152,95 2,14 7,11 3,2 15,6 -5,88 0,92 18,6110,2BS092163,98 4,79 44,5 3,5 5,84 2,94 1,14 10,335,9BS092172,44 8,78 -11,4 2,6 40,8 -23,5 1,07 45,627,6BS092182,76 5,47 0,00 3,4 7,48 0,00 0,84 4,320,0015 µM IPTG induction Median % Median % Increase titer Median increase yield increase Strain %RSD over %RSD %RSD (mg / L OD over (mg / L over Ctrl (%) cTHZ) Ctrl pr. OD) Ctrl BS072882,39 3,11 -14,7 2,7 7,52 -22,9 0,87 5,259,20BS086183,02 1,41 7,92 3,8 9,25 8,57 0,81 8,981,47BS092113,21 1,33 14,4 2,4 2,44 -31,4 1,36 1,7569,9BS092120,43 26,7 -84,8 1,0 49,9 -71,4 0,47 51,5-40,9BS092132,73 3,60 -2,43 3,6 1,62 2,86 0,77 4,44-4,10BS092143,42 2,53 22,2 3,6 5,68 2,86 0,96 5,9020,2BS092153,04 1,47 8,49 2,7 10,1 -22,9 1,12 10,039,7BS092164,49 2,73 60,1 3,0 3,94 -14,3 1,56 3,2795,3BS092172,39 24,4 -14,7 2,9 55,1 -17,1 0,99 53,123,8BS092182,80 1,57 0,00 3,5 1,67 0,00 0,80 2,990,00Example 12 – Overexpression of auxiliary genes supporting THI production Introduction

[0353] In order to boost the flux to 5-(2-hydroxyethyl)-4-methyl-1,3-thiazole-2-carboxylic phosphate acid (cTHZ-P) and thus more downstream thiamine, we overexpressed auxiliary genes involved in ROS damage repair, chaperones or DXP synthesis. Protocol

[0354] We constructed strains overexpressing sodA, sodB, sodC, yndG, dnaK, grxA, trxC, msrA, msrB, xylB, dxs, yajO either IPTG-inducible (pT5 / lacO promoter) or constitutively expressed (apFAB promoter) on a second plasmid with the S101 origin and tetracyclin resistance marker. Results

[0355] We made strains overexpressing sodA, sodB, sodC, yndG, dnaK, grxA, trxC, msrA, msrB, xylB, dxs, yajO that had improved production of thiamine in small scale.Case Ref. P180WO IPTector® Example 13 – Feeding of amino-HMP as alternative substrate to HMP for THI production (I) Introduction

[0356] The feeding of amino-HMP in the presence of thiaminase II enzymes that can convert amino- HMP to HMP is an alternative feeding strategy that might become commercially relevant. Results

[0357] We introduced different tenA thiaminase II enzymes into the thiamine production strain, and a subset could utilize amino-HMP as substrate for producing good titers of thiamine in small scale. Example 14 – Deletion of native thiamine operons in THI production strain Introduction

[0358] The native thiamine operons are regulated by intrinsic mechanism such as feedback inhibition. In order to avoid any effect of inhibition (which might also occur at the translational level), we deleted the native thiamine operons thiCEFSGH, thiEFSGH (the same operon but not disturbing thiC), and thiMD and analyzed the ability to produce thiamine with HMP-feeding. Protocol

[0359] The strains used in this experiment are listed in Table 12. Table 12. Strains tested in Example 15 Parent STRAIN ID Plasmid 1 Operon deletion strain BS08809 BS05801 pBS3739 BS08983 BS08879 - thiCEFSGH::[FRT,KanR,FRT]{rsd,yjaZ} BS08984 BS08880 - thiEFSGH::[FRT,KanR,FRT]{thiC,yjaZ} BS08985 BS08881 - thiMD::[FRT,KanR,FRT]{rcnR,yegX} BS09003 BS08983 pBS3739 thiCEFSGH::[FRT,KanR,FRT]{rsd,yjaZ} BS09004 BS08983 pBS3808 thiCEFSGH::[FRT,KanR,FRT]{rsd,yjaZ} BS09005 BS08984 pBS3739 thiEFSGH::[FRT,KanR,FRT]{thiC,yjaZ} BS09006 BS08984 pBS3808 thiEFSGH::[FRT,KanR,FRT]{thiC,yjaZ} BS09007 BS08985 pBS3739 thiMD::[FRT,KanR,FRT]{rcnR,yegX} BS09008 BS08985 pBS3808 thiMD::[FRT,KanR,FRT]{rcnR,yegX} Results

[0360] We deleted several native thiamine operons in the thiamine production strain and deleting the thiCEFSGH, the thiEFSGH and the thiMD operon had a positive effect and increased thiamin titers in small scale, see Table 13. Table 13. Thiochrome titer, OD600 and yield in strains with deleted native thiamine operonsCase Ref. P180WO IPTector® Median Median Increase Increase Increase titer Median yield Strain %RSD over %RSD over %RSD over (mg / L OD (mg / L Ctrl (%) Ctrl (%) Ctrl (%) THIeq) pr. OD) BS08809 8,81 1,2 3,59 4,9 2,45 6,0BS08983 0,19 8,2 3,35 4,9 0,06 10,2 BS08984 0,19 13,2 3,23 7,9 0,06 16,7 BS08985 0,19 8,7 3,35 9,8 0,06 24,1 BS09003 11,32 0,4 28,5 3,08 4,0 -14,2 3,67 4,4 49,8 BS09004 12,12 16,7 37,6 3,12 2,0 -13,1 3,88 18,3 58,4 BS09005 12,64 2,9 43,5 3,39 5,8 -5,6 3,73 8,3 52,2 BS09006 11,70 4,9 32,8 3,55 0,7 -1,1 3,30 4,3 34,7 BS09007 12,30 0,7 39,6 3,39 10,6 -5,6 3,63 10,7 48,2 BS09008 13,74 4,5 56,0 3,62 7,6 0,8 3,80 3,1 55,1 Example 15 – Feeding of amino-HMP as alternative substrate to HMP for THI production (II) Introduction

[0361] The feeding of amino-HMP in the presence of thiaminase II enzymes that can convert amino- HMP to HMP is an alternative feeding strategy that might become commercially relevant. Protocol

[0362] We sourced thiaminase II enzymes base on literature research describing thiaminase II activity. We constructed strains overexpressing these newly sourced thiaminase II genes under the control of the IPTG-inducible T5LacO promoter on a second plasmid with the pSC101 origin and tetracyclin resistance marker. The plasmids were transformed into the strain BS08809 carrying the cTHZ production plasmid pBS3739, and a small scale screening was carried out and titers measured by Thiochrome assay. The strains used in this experiment are listed in Table 14. Table 14. Strains tested in Example 14 STRAIN ID Parent Plasmid Plasmid 2 Thiaminase II strain 1 BS08809 BS05801 pBS3739 - - BS08905 BS08809 pBS3739pBS3048empty plasmid controlBS09046 BS08809 pBS3739pBS3814T5LacO / HP1287_HPY@OPT,CHTBS09047 BS08809 pBS3739pBS3815T5LacO / tenA_AAN@OPT,CHTBS09048 BS08809 pBS3739pBS3816T5LacO / tenA_AMI@OPT,CHTBS09049 BS08809 pBS3739pBS3817T5LacO / TenA_BSU@OPT,CHT BS09050 BS08809 pBS3739pBS3818T5LacO / tenA_CPA@OPT,CHTBS09051 BS08809 pBS3739pBS3819T5LacO / tenA_HHA@OPT,CHTBS09052 BS08809 pBS3739pBS3820T5LacO / tenA_PAE@OPT,NHTCase Ref. P180WO IPTector® BS09053 BS08809 pBS3739pBS3821T5LacO / tenA_PFU@OPT,CHTBS09054 BS08809 pBS3739pBS3822T5LacO / tenA_PHO@OPT,CHTBS09055 BS08809 pBS3739pBS3823T5LacO / tenA_SAU@OPT,CHTBS09056 BS08809 pBS3739pBS3824T5LacO / tenA_SEP@OPT,CHTBS09057 BS08809 pBS3739pBS3825T5LacO / tenA_SSO@OPT,CHTBS09058 BS08809 pBS3739pBS3826T5LacO / THI20_SCE@OPT,CHTBS09059 BS08809 pBS3739pBS3802T5LacO / THI20_SCE@TRC(1-302)BS09060 BS08809 pBS3739pBS3804T5LacO / THI20_SCE@TRC(300-551),CHTNote: @OPT = codon optimized; CHT = C-terminal 6xHis tag; NHT = N-terminal 6xHis tag Results

[0363] We introduced a panel of tenA thiaminase II enzymes into the thiamine production strain, and a subset could utilize amino-HMP as substrate for producing good titers of thiamine in small scale, see Table 15. Importantly, thiamine production is completely dependent on the presence of HMP, as we observed no thiochrome titers in the “no HMP” condition. Regarding the “HMP-fed” panel, the proper control BS08905 underperformed, so % increase was calculated compared to the parent strain BS08809. Strains BS09051 (tenA_HHA@OPT,CHT , 17% increase compared to control), BS09054 (tenA_PHO@OPT,CHT, 10% increase), BS09055 (tenA_SAU@OPT,CHT, 18% increase), BS09057 (tenA_SSO@OPT,CHT, 23% increase) and BS09059 (THI20_SCE@TRC(1-302), the N-terminal HMP kinase and HMP-P kinase activity domain, 26% increase) showed increased ability to produce thiamine when HMP was fed as substrate.

[0364] When feeding the alternative substrate amino-HMP, strains BS09046 (HP1287_HPY@OPT,CHT, 943% increase compared to control BS08905), BS09047 (tenA_AAN@OPT,CHT, 1764% increase), BS09050 (tenA_CPA@OPT,CHT, 2045% increase), BS09052 (tenA_PAE@OPT,NHT, 2045% increase), BS09053 (tenA_PFU@OPT,CHT, 1136% increase), BS09054 (tenA_PHO@OPT,CHT, 1500% increase), BS09055 (tenA_SAU@OPT,CHT, 489% increase), BS09056 (tenA_SEP@OPT,CHT, 2081% increase), BS09057 (tenA_SSO@OPT,CHT, 2400% increase) and BS09059 (THI20_SCE@TRC(1-302), 172% increase) were able to utilize this substrate for thiamine production. These candidates are interesting when exploring an alternative feeding strategy for commercial thiamine production. Further testing and optimization is necessary since total titers were not increased compared to HMP-fed candidates (max titer of 11.75 mg / L thiochrome in BS09057-amino-HMP-fed vs to max titer of 13.98 mg / L thiochrome in BS09059-HMP-fed). Table 15. Thiochrome titer, OD600 and yield in strains with overexpressed thiaminase II genes and different fed substrates.Case Ref. P180WO IPTector® No HMP Median Median Increase Increase yield Increase titer Median Strain %RSD over %RSD over (mg / L %RSD over (mg / L OD Ctrl (%) Ctrl (%) THIeq Ctrl (%) THIeq) pr. OD) BS08809 0,00 - - 3,51 2,3 -8,2 0,00 - - BS08905 0,00 - - 3,82 3,1 0,0 0,00 - - BS09046 0,00 - - 3,66 1,6 -4,1 0,00 - - BS09047 0,00 - - 3,74 3,7 -2,0 0,00 - - BS09048 0,00 - - 3,55 1,1 -7,1 0,00 - - BS09049 0,00 - - 3,51 2,9 -8,2 0,00 - - BS09050 0,00 - - 3,47 4,3 -9,2 0,00 - - BS09051 0,00 - - 3,47 12,0 -9,2 0,00 - - BS09052 0,00 - - 3,43 3,5 -10,2 0,00 - - BS09053 0,00 - - 3,47 3,7 -9,2 0,00 - - BS09054 0,00 - - 3,47 4,2 -9,2 0,00 - - BS09055 0,00 - - 3,39 1,3 -11,2 0,00 - - BS09056 0,00 - - 3,51 4,0 -8,2 0,00 - - BS09057 0,00 - - 3,55 4,0 -7,1 0,00 - - BS09058 0,00 - - 3,78 12,0 -1,0 0,00 - - BS09059 0,00 - - 2,92 11,2 -23,4 0,00 - - BS09060 0,00 - - 3,35 8,9 -12,2 0,00 - - HMP fed Median Median Increase Increase yield Increase titer Median Strain %RSD over %RSD over (mg / L %RSD over (mg / L OD Ctrl (%) Ctrl (%) THIeq Ctrl (%) THIeq) pr. OD) BS08809 11,06 0,6 461,4 3,08 9,0 -15,9 3,30 86,9 500,0 BS08905 1,97 6,9 0,0 3,66 1,6 0,0 0,55 8,2 0,0 BS09046 9,49 2,0 381,7 3,20 4,3 -12,8 3,05 5,0 454,5 BS09047 10,23 6,5 419,3 3,35 3,8 -8,5 3,06 9,4 456,4 BS09048 11,48 14,7 482,7 3,35 1,4 -8,5 3,43 15,8 523,6 BS09049 1,55 4,4 -21,3 3,16 3,9 -13,8 0,50 1,2 -9,1 BS09050 11,03 3,5 459,9 3,00 7,9 -18,1 3,60 6,9 554,5 BS09051 12,97 5,2 558,4 2,88 12,4 -21,3 4,70 14,9 754,5 BS09052 11,47 4,6 482,2 3,35 7,0 -8,5 3,42 10,9 521,8 BS09053 11,29 73,4 473,1 3,47 9,2 -5,3 3,22 110,6 485,5Case Ref. P180WO IPTector® BS09054 12,18 3,4 518,3 3,16 2,5 -13,8 3,77 4,6 585,5 BS09055 13,10 6,6 565,0 2,96 2,6 -19,1 4,39 98,9 698,2 BS09056 11,06 6,1 461,4 2,96 2,0 -19,1 3,67 6,0 567,3 BS09057 13,64 25,4 592,4 3,00 7,0 -18,1 5,07 24,3 821,8 BS09058 10,65 35,9 440,6 2,96 18,4 -19,1 3,74 19,2 580,0 BS09059 13,98 9,9 609,6 3,04 1,3 -17,0 4,61 10,9 738,2 BS09060 0,63 160,6 -68,0 2,92 3,3 -20,2 0,22 160,4 -60,0 amino- HMP fed Median Median Increase Increase yield Increase titer Median Strain %RSD over %RSD over (mg / L %RSD over (mg / L OD Ctrl (%) Ctrl (%) THIeq Ctrl (%) THIeq) pr. OD) BS08809 0,11 113,4 -76,6 3,66 4,4 -1,1 0,03 105,8 -75,0 BS08905 0,47 22,6 0,0 3,70 2,6 0,0 0,12 21,1 0,0 BS09046 4,90 5,4 942,6 3,43 1,7 -7,4 1,43 7,3 1091,7 BS09047 8,76 8,4 1763,8 3,27 3,0 -11,6 2,74 10,4 2183,3 BS09048 0,33 29,9 -29,8 3,55 3,1 -4,2 0,09 26,5 -25,0 BS09049 0,73 7,0 55,3 3,35 1,8 -9,5 0,22 9,1 83,3 BS09050 10,08 2,7 2044,7 2,92 3,3 -21,0 3,54 5,3 2850,0 BS09051 0,49 14,6 4,3 3,35 8,2 -9,5 0,14 18,4 16,7 BS09052 10,08 5,5 2044,7 3,12 4,4 -15,8 3,30 2,6 2650,0 BS09053 5,81 89,0 1136,2 3,08 9,5 -16,8 1,99 83,1 1558,3 BS09054 7,52 25,0 1500,0 2,73 9,1 -26,3 2,75 15,5 2191,7 BS09055 2,77 8,4 489,4 3,43 2,4 -7,4 0,84 61,2 600,0 BS09056 10,25 6,2 2080,9 2,96 2,8 -20,0 3,50 8,1 2816,7 BS09057 11,75 9,0 2400,0 3,12 4,2 -15,8 3,77 12,1 3041,7 BS09058 0,00 - -100,0 3,43 2,8 -7,4 0,00 - -100,0 BS09059 1,28 24,9 172,3 3,39 4,1 -8,4 0,39 21,6 225,0 BS09060 0,00 173,2 -100,0 3,62 4,8 -2,1 0,00 173,2 -100,0 We also analyzed the cTHZ titer of these thiaminase II strains, and we observed significant amounts of up to 4 mg / L in the panel that was not fed HMP nor amino-HMP, see Table Example 16 Table 16 cTHZ titer, OD600 and yield in strains with overexpressed thiaminase II genes and different fed substrates.Case Ref. P180WO IPTector® No HMP Median Median Increase Increase Increase titer Median yield Strain %RSD over %RSD over %RSD over (mg / L OD (mg / L Ctrl (%) Ctrl (%) Ctrl (%) cTHZ) pr. OD) BS08809 0,07 165 -97,9 3,51 2,33 -8,2 0,02 165 -97,7 BS08905 3,16 81,4 0,0 3,82 3,09 0,0 0,83 81,7 0 BS09046 3,47 86,6 10,0 3,66 1,63 -4,1 0,95 86,6 14 BS09047 3,17 85,3 0,44 3,74 3,71 -2,0 0,85 85,1 1,8 BS09048 0,00 173 -100 3,55 1,10 -7,1 0,00 173 -100 BS09049 0,00 173 -100 3,51 2,90 -8,2 0,00 173 -100 BS09050 4,09 82,8 29,4 3,47 4,33 -9,2 1,18 82,5 41,4 BS09051 2,23 91,9 -29,5 3,47 12,0 -9,2 0,64 90,6 -23,0 BS09052 3,70 87,9 17,3 3,43 3,49 -10,2 1,08 88,5 29,6 BS09053 0,11 164 -96,5 3,47 3,66 -9,2 0,03 164 -96,2 BS09054 0,00 173 -100 3,47 4,17 -9,2 0,00 173 -100 BS09055 3,65 85,5 15,5 3,39 1,34 -11,2 1,08 85,5 29,1 BS09056 0,07 169 -97,9 3,51 3,96 -8,2 0,02 169 -97,7 BS09057 3,28 18,9 3,8 3,55 4,00 -7,1 0,00 - -100 BS09058 0,00 173 -100 3,78 12,0 -1,0 0,00 - -100 BS09059 0,00 173 -100 2,92 11,2 -23,4 0,00 - -100 BS09060 0,38 86,8 -88,0 3,35 8,91 -12,2 0,11 86,7 -87,0 HMP fed Median Median Increase Increase Increase titer Median yield Strain %RSD over %RSD over %RSD over (mg / L OD (mg / L Ctrl (%) Ctrl (%) Ctrl (%) cTHZ) pr. OD) BS08809 0,00 - - 3,08 86,8 -15,9 0 - - BS08905 0,00 173 - 3,66 1,63 0,00 0 173 - BS09046 0,06 15,6 - 3,20 4,26 -12,8 0,0194 18,8 - BS09047 0,00 173 - 3,35 3,79 -8,50 0 173 - BS09048 0,00 - - 3,35 1,35 -8,50 0 - - BS09049 0,35 87 - 3,16 3,90 -13,8 0,112 86,8 - BS09050 0,04 87 - 3,00 7,89 -18,1 0,0117 87,6 - BS09051 0,00 - - 2,88 12,4 -21,3 0 - - BS09052 0,00 - - 3,35 6,98 -8,50 0 - - BS09053 0,00 - - 3,47 9,18 -5,31 0 - - BS09054 0,00 - - 3,16 2,47 -13,8 0 - -Case Ref. P180WO IPTector® BS09055 0,00 - - 2,96 2,63 -19,1 0 - - BS09056 0,00 - - 2,96 2,00 -19,1 0 - - BS09057 0,00 - - 3,00 7,05 -18,1 0 - - BS09058 0,00 - - 2,96 18,4 -19,1 0 - - BS09059 0,00 - - 3,04 1,28 -17,0 0 - - BS09060 0,00 - - 2,92 3,34 -20,2 0 - - amino- HMP fed Median Median Increase Increase Increase titer Median yield Strain %RSD over %RSD over %RSD over (mg / L OD (mg / L Ctrl (%) Ctrl (%) Ctrl (%) cTHZ) pr. OD) BS08809 0,00 173 -100 3,66 4,40 -1,05 0,00 173 -100 BS08905 0,68 19,1 0,00 3,70 2,64 0,00 0,19 21,0 0,00 BS09046 0,00 173 -100 3,43 1,74 -7,36 0,00 173 -100 BS09047 0,00 173 -100 3,27 2,98 -11,6 0,00 173 -100 BS09048 0,00 173 -100 3,55 3,11 -4,21 0,00 173 -100 BS09049 0,53 88,0 -21,9 3,35 1,78 -9,46 0,16 88,5 -16,5 BS09050 0,17 86,7 -75,4 2,92 3,34 -21,0 0,06 86,6 -68,7 BS09051 0,77 86,7 13,8 3,35 8,22 -9,46 0,23 86,6 21,7 BS09052 0,00 173 -100 3,12 4,44 -15,8 0,00 173 -100 BS09053 0,05 86,6 -93,3 3,08 9,54 -16,8 0,01 87,9 -93,1 BS09054 0,00 - -100 2,73 9,07 -26,3 0,00 - -100 BS09055 0,00 - -100 3,43 2,38 -7,36 0,00 - -100 BS09056 0,00 173 -100 2,96 2,76 -20,0 0,00 173 -100 BS09057 0,00 173 -100 3,12 4,22 -15,8 0,00 - -100 BS09058 0,60 12,1 -11,5 3,43 2,85 -7,36 0,00 - -100 BS09059 0,63 88,0 -6,80 3,39 4,09 -8,41 0,00 - -100 BS09060 0,04 159 -94,2 3,62 4,78 -2,10 0,01 161 -94,3 References Jenkins, A. H., Schyns, G., Potot, S., Sun, G., & Begley, T. P. (2007). A new thiamine salvage pathway. Nat Chem Biol, 3(8), 492–497.Case Ref. P180WO IPTector® Kreinbring CA, Remillard SP, Hubbard P, Brodkin HR, Leeper FJ, Hawksley D, Lai EY, Fulton C, Petsko GA, Ringe D. Structure of a eukaryotic thiaminase I. Proc Natl Acad Sci U S A. 2014; 111(1):137-42. Abe M, Ito S, Kimoto M, Hayashi R, Nishimune T. Molecular studies on thiaminase I. Biochim Biophys Acta.1987; 909(3):213-21. Agee CC, Airth RL. Reversible inactivation of thiaminase I of Bacillus thiamineolyticus by its primary substrate, thiamine. J Bacteriol.1973; 115(3):957-65. Costello CA, Kelleher NL, Abe M, McLafferty FW, Begley TP. Mechanistic studies on thiaminase I. Overexpression and identification of the active site nucleophile. J Biol Chem. 1996; 271(7):3445-52. Hazra A, Chatterjee A, Begley TP. Biosynthesis of the thiamine thiazole in Bacillus subtilis: identification of the product of the thiazole synthase-catalyzed reaction. J Am Chem Soc.2009; 131(9):3225-9. Hazra AB, Han Y, Chatterjee A, Zhang Y, Lai RY, Ealick SE, Begley TP. A missing enzyme in thiamin thiazole biosynthesis: identification of TenI as a thiazole tautomerase. J Am Chem Soc. 2011 Jun 22;133(24):9311-9. Aleku GA, Roberts GW, Titchiner GR, Leys D. Synthetic Enzyme-Catalyzed CO2Fixation Reactions. ChemSusChem.2021; 14(8):1781-1804. Wieser M, Fujii N, Yoshida T, Nagasawa T. Carbon dioxide fixation by reversible pyrrole-2- carboxylate decarboxylase from Bacillus megaterium PYR2910. Eur J Biochem. 1998; 257(2):495-9. Kroll, A., Ranjan, S., Engqvist, M. K., & Lercher, M. J. (2023). A general model to predict small molecule substrates of enzymes based on machine and deep learning. Nature Communications, 14(1), 2787. Kroll, A., Ranjan, S., & Lercher, M. J. (2024). A multimodal Transformer Network for protein-Case Ref. P180WO IPTector® small molecule interactions enhances predictions of kinase inhibition and enzyme-substrate relationships. PLOS Computational Biology, 20(5), e1012100. * * *

Claims

Case Ref. P180WO IPTector® Claims 1. A genetically modified host cell having improved production of thiamine (THI), carboxysulfurol phosphate (cTHZ-P), sulfurol phosphate (THZ-P), carboxysulfurol (cTHZ), and / or sulfurol (THZ), wherein the host cell expresses one or more heterologous and / or genetically modified native genes encoding enzymes selected from a thiaminase I, a thiaminase II and / or a phosphatase and / or a decarboxylase whereby the production of the THI, THZ-P, cTHZ, cTHZ-P and / or sulfurol in the genetically modified host cell is improved compared to an unmodified parent host cell.

2. The host cell of claim 1, wherein the: a) thiaminase I or thiaminase II converts thiamine to sulfurol; and / or b) a phosphatase which dephosphorylates THZ-P to sulfurol and / or cTHZ-P to cTHZ.

3. The host cell of claim 1, wherein the thiaminase I, thiaminase II and / or phosphatase are bacterial enzymes.

4. The host cell of claim 1 or 3, wherein the thiaminase I and / or thiaminase II is from a genus selected from Gloeothece (Cyanothece), Megasphaera, Naegleria, Burkholderia, Haliangium, Oleidesulfovibrio, Stigmatella, Acinetobacter, Helicobacter, Bacillus, Candida, Halaalkalibacterium, Pyrobaculum, Pyrococcus Staphylococcus and / or Saccharomyces 5. The host cell of claim 1 or 3, wherein the thiaminase I and / or thiaminase II is from a species selected from Gloeothece citriformis, Megasphaera elsdenii DSM 20460, Naegleria gruberi, Burkholderia glumae BGR1, Haliangium ochraceum DSM14365, Oleidesulfovibrio alaskensis G20, Stigmatella aurantiaca DW4 and / or Acinetobacter nectaris, Helicobacter pylori, Bacillus anerurinolyticus, Bacilluc migulanus, Bacillus subtilis, Candida parapsilosis, Halaalkalibacterium halodurans, Pyrobaculum aerophilum, Pyrococcus furiosus, Pyrococcus horikoshii, Staphylococcus aureus, Staphylococcus epidermidis, Saccharomyces solfataricus and / or Saccharomyces cerevisiae.

6. The host cell of claim 1 or 3, wherein the phosphatase is from a species selected from Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Pseudomonas Putida KT2440, Shigella sonnei (strain Ss046), Pseudomonas asiatica, Hirschfeldia incana, and / or Citrus sinensis.Case Ref. P180WO IPTector® 7. The host cell of any preceding claim wherein the: a) thiaminase I has an amino acid sequence which is at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the thiaminase I comprised in SEQ ID NO: 29, 31, 33, 35, 37, 39, 41 and / or 43; b) thiaminase II has an amino acid sequence which is at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the thiaminase II comprised in SEQ ID NO: 45, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137 and / or 139 and / or c) phosphatase has an amino acid sequence which is at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the phosphatases comprised in SEQ ID NO: 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103 and / or 105.

8. The host cell of claims 4 or 5, wherein the thiaminase I has an amino acid sequence as set forth in SEQ ID NO: 29, 31, 33, 35, 37, 39, 41 and / or 43 and / or the thiaminase II has an amino acid sequence as set forth in SEQ ID NO: 45, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137 and / or 139.

9. The host cell of claim 3, wherein the phosphatase has an amino acid sequence as set forth in SEQ ID NO: 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103 and / or 105.

10. The host cell of claims 4 or 5, wherein the thiaminase I is from a genus or species selected from Cyanothece, Megasphaera elsdenii DSM 20460, Naegleria gruberi, Burkholderia glumae BGR1, Haliangium ochraceum DSM14365, Oleidesulfovibrio alaskensis G20, Stigmatella aurantiaca DW4 and / or Acinetobacter nectaris having SEQ ID NO: 29, 31, 33, 35, 37, 39, 41 and / or 43 and / or the thiaminase II is from a genus or species selected from Helicobacter pylori, Bacillus anerurinolyticus, Bacilluc migulanus, Bacillus subtilis, Candida parapsilosis, Halaalkalibacterium halodurans, Pyrobaculum aerophilum, Pyrococcus furiosus, Pyrococcus horikoshii, Staphylococcus aureus, Staphylococcus epidermidis, Saccharomyces solfataricus and / or Saccharomyces cerevisiae.Case Ref. P180WO IPTector® 11. The host cell of any preceding claim, wherein the gene encoding: a) the thiaminase I is least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 30, 32, 34, 36, 38, 40, 42 and / or 44 or genomic DNA thereof encoding the thiaminase I enzyme comprised in SEQ ID NO: 29, 31, 33, 35, 37, 39, 41 and / or 43; b) the thiaminase II (TenA) is least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 46, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138 and / or 140 or genomic DNA thereof encoding the thiaminase II enzyme comprised in SEQ ID NO: 45, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137 and / or 139; and / or c) the phosphatase is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68,70, 72, 74, 76, 78 and / or 80, or genomic DNA thereof encoding the phosphatase enzyme comprised in SEQ ID NO: 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103 and / or 105.

12. The host cell of any preceding claim, wherein the a) thiaminase I gene is at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the thiaminase I gene comprised in SEQ ID NO: 30, 32, 34, 36, 38, 40, 42 and / or 44; b) thiaminase II gene is at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the thiaminase II gene comprised in SEQ ID NO: 46, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138 and / or 140; and / or c) phosphatase gene is at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the phosphatase gene comprised in SEQ ID NO: 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68,70, 72, 74, 76, 78, 80, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104 and / or 106.

13. The host cell of any preceding claims, wherein the thiaminase I gene is a truncated thiaminase ICase Ref. P180WO IPTector® gene, and / or the thiaminase II gene is a truncated thiaminase II gene.

14. The host cell of any preceding claims, wherein the phosphatase gene is a truncated phosphatase gene.

15. The host cell of any preceding claim, further comprising an operative metabolic pathway comprising one or more native or heterologous pathway elements producing cTHZ, THZ-P, cTHZ- P and / or sulfurol.

16. The host cell of any preceding claim, wherein the one or more native or heterologous pathway elements are selected from: a) a phosphomethylpyrimidine synthase (ThiC); that catalyzes the synthesis of the hydroxymethylpyrimidine phosphate (HMP-P) moiety of thiamine from aminoimidazole ribotide (AIR) in a radical S-adenosyl-L-methionine (SAM)-dependent reaction; b) a hydroxymethylpyrimidine / phosphomethylpyrimidine kinase (ThiD) that catalyzes the phosphorylation of hydroxymethylpyrimidine phosphate (HMP-P) to HMP-PP, and of HMP to HMP-P; c) an adenylyltransferase (ThiF) that catalyzes the adenylation of the carboxy terminus of the sulfur carrier protein ThiS and the subsequent displacement of AMP catalyzed by ThiI-persulfide to give a ThiS-ThiI acyl disulfide ThiS; d) a sulfur carrier protein (ThiS) in which its C-terminal thiocarboxylation occurs in 2 steps: first, it is acyl-adenylated and then thiocarboxylated by ThiL; e) a 2-iminoacetate synthase (ThiH) that catalyzes the radical-mediated cleavage of tyrosine to 2- iminoacetate and 4-cresol; f) a thiazole synthase (ThiG) that catalyzes the rearrangement of 1-deoxy-D-xylulose 5-phosphate (DXP) to produce the thiazole phosphate moiety of thiamine (cTHZ-P); g) a hydroxyethylthiazole kinase (ThiM) that catalyzes the phosphorylation of the hydroxyl group of 4-methyl-5-beta-hydroxyethylthiazole (THZ); h) a thiamine mono-phosphate (TMP) phosphatase (TH2) that dephosphorylate thiamine monophosphate to thiamine; i) a thiamine kinase (ThiK) that catalyzes the phosphorylation of thiamine to thiamine- monophosphate (TMP); j) a thiamine-monophosphate kinase (ThiL) which catalyzes the ATP-dependent phosphorylation of thiamine-monophosphate (TMP) to form thiamine-pyrophosphate (TPP);Case Ref. P180WO IPTector® k) a thiamine-phosphate synthase (ThiE) that condenses 4-methyl-5-(beta-hydroxyethyl)thiazole monophosphate (THZ-P) and 2-methyl-4-amino-5-hydroxymethyl pyrimidine pyrophosphate (HMP-PP) to form thiamine monophosphate (TMP); l) an HTH-type transcriptional regulator (IscR) that regulates the transcription of several operons and genes involved in the biogenesis of Fe-S clusters and Fe-S-containing proteins; m) a tRNA sulfotranferase (ThiI) which catalyzes the transfer of sulfur to the sulfur carrier protein ThiS, forming ThiS-thiocarboxylate; n) a glycine oxidase (ThiO) converting glycine into dehydroglycine (DHG); o) a cysteine desulfurase (IscS) which provides sulfur for Fe-S cluster synthesis in vitro; p) a thiazole tautomerase (TenI) which catalyzes the aromatization of cThz*-P to cThz-P; q) a thiaminase I (Thi1) that degrades thiamine by catalyzing the base-exchange substitution of thiazole with a nucleophile; r) a 2-(2-Carboxy-4-methylthiazol-5-yl)ethyl phosphate (cTHZ-P) phosphatase (PHOS) that dephosphorylates cTHZ-P to cTHZ; s) a thiaminase II (tenA) which either degrades thiamine by catalyzing the base-exchange substitution of thiazole with a nucleophile, or which can carry out the hydrolysis of aminomethylpyrimidine to HMP; t) a deCARB decarboxylase that can decarboxylate the carboxy group on cTHZ to yield THZ; u) a thiamine transporter (ThiP) that is a thiamine ABC transporter membrane subunit facilitating thiamine import; v) a DNA-binding transcriptional dual regulator (fur) that can lead to higher Fe2+ availability when mutated to fur-K14I; w) an adenylate cyclase (cyaA) that catalyzes the synthesis of cyclic AMP (cAMP) by an intramolecular transfer of the adenylyl group of ATP to the 3'-hydroxy group, releasing pyrophosphate; and / or x) a superoxide dismutase (sodA) that catalyzes conversion of superoxide radicals, optionally into oxygen and hydrogen peroxide.

17. The host cell of claim 16, wherein the one or more native or heterologous pathway elements are selected from: aa) ThiC has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the ThiC comprised in SEQ ID NO: 1; bb) ThiD has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%,Case Ref. P180WO IPTector® such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the ThiD comprised in SEQ ID NO: 3; cc) ThiE has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the ThiE comprised in SEQ ID NO: 17; dd) ThiF has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the ThiF comprised in SEQ ID NO: 5; ee) ThiS has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the ThiS comprised in SEQ ID NO: 7; ff) ThiO has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the ThiO comprised in SEQ ID NO: 27; gg) ThiH has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the ThiH comprised in SEQ ID NO: 9; hh) ThiG has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the ThiG comprised in SEQ ID NO: 11; ii) ThiM has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the ThiM comprised in SEQ ID NO: 13; jj) ThiL has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the ThiL comprised in SEQ ID NO: 15; kk) ThiI has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the ThiI comprised in SEQ ID NO: 19; ll) TenI has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the Ten I comprised in SEQ ID NO: 23; mm) Thiaminase I (Thi1) has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100%Case Ref. P180WO IPTector® identity to the Thi1 comprised in SEQ ID NO: 29, 31, 33, 35, 37, 39, 41 and / or 43; nn) Thiaminase K (ThiK) has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the ThiK comprised in SEQ ID NO: 141; oo) IscR has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the IScR comprised in SEQ ID NO: 109; pp) IscS has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the IscS comprised in SEQ ID NO: 145; qq) TenA has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the TenA comprised in SEQ ID NO: 45, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137 and / or 139; rr) deCARB has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the deCARB comprised in SEQ ID NO: 81; ss) ThiP has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the ThiP comprised in SEQ ID NO: 107; tt) fur-k14I has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the fur-k14I comprised in SEQ ID NO: 111; uu) CyaA has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the CyaA comprised in SEQ ID NO: 113; vv) SodA has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the SodA comprised in SEQ ID NO: 143; ww) TH2 has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the TH2 comprised in SEQ ID NO: 25; xx) PHOS has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to theCase Ref. P180WO IPTector® PHOS comprised in SEQ ID NO: 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 83, 85, 87, 89,91, 93, 95, 97, 99, 101, 103 and / or 105; and / or yy) dxs has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the dxs comprised in SEQ ID NO:

147.

18. The host cell of the preceding claims, wherein the host cell comprises a mutation which is a deletion, disruption, and / or an attenuation of the gene.

19. The host cell of claim 18, wherein the mutation is a deletion through complete removal of the gene or a translational knockout by introducing one more stop codons or frameshift mutations preventing expression of an active peptide.

20. The host cell of any of the claims 18 and 19, wherein the deletion, disruption and / or attenuation comprise a translational knockout of ThiM, CyaA, FurK14I, iscR-H107Y and / or thiP.

21. The host cell of any of the claims 18 and 19, wherein the gene is disrupted or deleted, wherein the gene is: a. thiC, thiE, thiF, thiS, thiG, and / or thiH; b. thiE, thiF, thiS, thiG, and / or thiH; or c. thiM, and / or thiD.

22. The host cell of any of the claims 18-21, wherein the deletion, disruption and / or attenuation comprise a deletion, such as an open reading frame (ORF) deletion, for example of ThiM, CyaA, and / or thiP.

23. The host cell of claim 18 to 22, wherein the one or more native or heterologous pathway elements are encoded by one or more genes selected from the group consisting of: a) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 2 or genomic DNA thereof encoding the ThiC comprised in SEQ ID NO: 1; b) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at leastCase Ref. P180WO IPTector® 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 4 or genomic DNA thereof encoding the ThiD comprised in SEQ ID NO: 3; c) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 18 or genomic DNA thereof encoding the ThiE comprised in SEQ ID NO: 17; d) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 6 or genomic DNA thereof encoding the ThiF comprised in SEQ ID NO: 5; e) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 8 or genomic DNA thereof encoding the ThiS comprised in SEQ ID NO: 7; f) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 28 or genomic DNA thereof encoding the ThiO comprised in SEQ ID NO: 27; g) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 12 or genomic DNA thereof encoding the ThiG comprised in SEQ ID NO: 11; h) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 14 or genomic DNA thereof encoding the ThiM comprised in SEQ ID NO: 13; i) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 16 or genomic DNA thereof encoding the ThiL comprised in SEQ ID NO: 15; j) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 20 or genomic DNA thereof encoding the ThiI comprised in SEQ ID NO: 19;Case Ref. P180WO IPTector® k) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 10 or genomic DNA thereof encoding the ThiH comprised in SEQ ID NO: 9; l) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 24 or genomic DNA thereof encoding the TenI comprised in SEQ ID NO: 23; m) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 30, 32, 34, 36, 38, 40, 42 and / or 44 or genomic DNA thereof encoding the Thiaminase I comprised in SEQ ID NO: 29, 31, 33, 35, 37, 39, 41 and / or 43; n) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 142 or genomic DNA thereof encoding the ThiK comprised in SEQ ID NO: 141; o) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 110 or genomic DNA thereof encoding the IscR comprised in SEQ ID NO: 109; p) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 146 or genomic DNA thereof encoding the IscS comprised in SEQ ID NO: 145; q) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 82 or genomic DNA thereof encoding the deCARB comprised in SEQ ID NO: 81; r) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 108 or genomic DNA thereof encoding the ThiP comprised in SEQ ID NO: 107; s) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, suchCase Ref. P180WO IPTector® as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 112 or genomic DNA thereof encoding the fur-k14I comprised in SEQ ID NO: 111; t) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 46, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138 and / or 140 or genomic DNA thereof encoding the TenA Thiaminase II comprised in SEQ ID NO: 45, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137 and / or 139; u) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 114 or genomic DNA thereof encoding the CyaA comprised in SEQ ID NO: 113; v) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68,70, 72, 74, 76, 7880, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104 and / or 106 or genomic DNA thereof encoding the PHOS comprised in SEQ ID NO: 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 83, 85, 87, 89,91, 93, 95, 97, 99, 101, 103 and / or 105; w) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 144 or genomic DNA thereof encoding the SodA comprised in SEQ ID NO: 143; x) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 26 or genomic DNA thereof encoding the TH2 TMP phosphatase comprised in SEQ ID NO: 25; and y) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 148 or genomic DNA thereof encoding the dxs comprised in SEQ ID NO:

147.

24. The host cell of any preceding claim, further comprising an operative metabolic pathwayCase Ref. P180WO IPTector® comprising one or more native or heterologous pathway elements producing the sulfurol compound and / or its precursors.

25. The host cell of any preceding claim, further comprising an operative metabolic pathway comprising one or more native or heterologous pathway elements producing the sulfurol, cTHZ- P, THZ-P, cTHZ and / or thiamine.

26. The host cell of any of claims 18-25 wherein the one or more pathway elements comprise one or more FeS cluster dependent enzymes.

27. The host cell of any of claims 18-26, wherein the one or more pathway elements comprise one or more radical SAM enzymes.

28. The host cell of any of claims 18-27, wherein one or more pathway enzymes carry N-terminal signal peptides directing newly synthesized proteins toward the secretory pathway.

29. The host cell of any preceding claim, wherein one or more pathway elements are selected from: a) ThiC has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the ThiC comprised in SEQ ID NO: 1; b) ThiD has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the ThiD comprised in SEQ ID NO: 3; c) ThiE has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the ThiE comprised in SEQ ID NO: 17; d) ThiF has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the ThiF comprised in SEQ ID NO: 5; e) ThiS has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the ThiS comprised in SEQ ID NO: 7; f) ThiO_PPT has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity toCase Ref. P180WO IPTector® the ThiO_PPT comprised in SEQ ID NO: 27; g) ThiH has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the ThiH comprised in SEQ ID NO: 9; h) ThiG has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the ThiG comprised in SEQ ID NO: 11; i) TenI_BSU has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the TenI_BSU comprised in SEQ ID NO: 23; j) TMP phosphatase (TH2_ATH) has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the TH2_ATH comprised in SEQ ID NO: 25; k) Thiaminase I (Thi1_CYA) has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the Thi1_CYA comprised in SEQ ID NO: 29; l) Thiaminase I (Thi1_SAU) has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the Thi1_SAU comprised in SEQ ID NO: 31; m) Thiaminase I (Thi1_ANE) has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the Thi1_ANE comprised in SEQ ID NO: 33; n) Thiaminase I (Thi1_MEL) has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the Thi1_MEL comprised in SEQ ID NO: 35; o) Thiaminase I (Thi1_NGR) has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the Thi1_NGR comprised in SEQ ID NO: 37; p) Thiaminase I (Thi1_BGL) has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the Thi1_BGL comprised in SEQ ID NO: 39; q) Thiaminase I (Thi1_HOC) has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the Thi1_HOC comprised in SEQ ID NO: 41;Case Ref. P180WO IPTector® r) Thiaminase I (Thi1_OAL) has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the Thi1_OAL comprised in SEQ ID NO: 43; and / or s) Thiaminase II (TenA_BSU) has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the TenA_BSU comprised in SEQ ID NO:

45.

30. The host cell of any preceding claim, wherein one or more pathway elements are selected from: a) ThiC has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the ThiC comprised in SEQ ID NO: 1; b) ThiD has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the ThiD comprised in SEQ ID NO: 3; c) ThiE has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the ThiE comprised in SEQ ID NO: 17; d) ThiF has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the ThiF comprised in SEQ ID NO: 5; e) ThiS has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the ThiS comprised in SEQ ID NO: 7; f) ThiO_PPT has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the ThiO_PPT comprised in SEQ ID NO: 27; g) ThiH has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the ThiH comprised in SEQ ID NO: 9; h) ThiG has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the ThiG comprised in SEQ ID NO: 11; i) TMP phosphatase (TH2_ATH) has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, suchCase Ref. P180WO IPTector® as 100% identity to the TH2_ATH comprised in SEQ ID NO: 25; and / or j) TenI_BSU has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the TenI_BSU comprised in SEQ ID NO:

23.

31. The host cell of any preceding claim, wherein one or more pathway elements are selected from: a) ThiF has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the ThiF comprised in SEQ ID NO: 5; b) ThiS has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the ThiS comprised in SEQ ID NO: 7; c) ThiO_PPT has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the ThiO_PPT comprised in SEQ ID NO: 28; d) ThiH has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the ThiH comprised in SEQ ID NO: 9; e) ThiG has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the ThiG comprised in SEQ ID NO: 11; f) TMP phosphatase (TH2_ATH) has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the TH2_ATH comprised in SEQ ID NO: 25; g) phosphatase PHOS_PhoA has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the phosphatase PHOS_PhoA comprised in SEQ ID NO: 47; h) Phosphatase PHOS_NudJ has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the PHOS_NudJ comprised in SEQ ID NO: 49; i) Phosphatase PHOS_Cof has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the PHOS_Cof comprised in SEQ ID NO: 51;Case Ref. P180WO IPTector® j) Phosphatase PHOS_YbhA has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the PHOS_YbhA comprised in SEQ ID NO: 53; k) Phosphatase PHOS_KdsC has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the PHOS_KdsC comprised in SEQ ID NO: 55; l) Phosphatase PHOS_RsgA_SAE has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the PHOS_RsgA_SAE comprised in SEQ ID NO: 57; m) Phosphatase PHOS_PhoS_PAE has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the PHOS_PhoS_PAE comprised in SEQ ID NO: 59; n) Phosphatase PHOS_NudJ_PPU has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the PHOS_NudJ_PPU comprised in SEQ ID NO: 61; o) Phosphatase PHOS_AlkP_PPU has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the PHOS_AlkP_PPU comprised in SEQ ID NO: 63; p) Phosphatase PHOS_ygiF_PPU has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the PHOS_ygiF_PPU comprised in SEQ ID NO: 65; q) Phosphatase PHOS_upxB_PPU has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the PHOS_upxB_PPU comprised in SEQ ID NO: 67; r) Phosphatase PHOS_PhoD_PPU has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the PHOS_PhoD_PPU comprised in SEQ ID NO: 69; s) Phosphatase PHOS_AphA has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the PHOS_AphA comprised in SEQ ID NO: 71; t) Phosphatase PHOS_NudJ_SSO has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the PHOS_NudJ_SSO comprised in SEQ ID NO: 73; u) Phosphatase PHOS_PhoD_PAS has an amino acid sequence which is at least 70%, such at leastCase Ref. P180WO IPTector® 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the PHOS_PhoD_PAS comprised in SEQ ID NO: 75; v) Phosphatase PHOS_AH2_HIN has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the PHOS_AH2_HIN comprised in SEQ ID NO: 77; and / or w) Phosphatase PHOS_AH2_CSI has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the PHOS_AH2_CSI comprised in SEQ ID NO:

79.

32. The host cell of any preceding claim, wherein the one or more pathway elements are encoded by one or more genes selected from the group of: a) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 2 or genomic DNA thereof encoding the ThiC comprised in SEQ ID NO: 1; b) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 4 or genomic DNA thereof encoding the ThiD comprised in SEQ ID NO: 3; c) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 18 or genomic DNA thereof encoding the ThiE comprised in SEQ ID NO: 17; d) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 6 or genomic DNA thereof encoding the ThiF comprised in SEQ ID NO: 5; e) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 8 or genomic DNA thereof encoding the ThiS comprised in SEQ ID NO: 7; f) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 28 or genomic DNA thereof encoding the ThiO_PPT comprised in SEQ ID NO: 27;Case Ref. P180WO IPTector® g) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 12 or genomic DNA thereof encoding the ThiG comprised in SEQ ID NO: 11; h) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 10 or genomic DNA thereof encoding the ThiH comprised in SEQ ID NO: 9; i) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 24 or genomic DNA thereof encoding the TenI_BSU comprised in SEQ ID NO: 23; j) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: : 26 or genomic DNA thereof encoding the TH2_ATH comprised in SEQ ID NO: 25; k) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 30 or genomic DNA thereof encoding the Thiaminase I Thi1_CYA comprised in SEQ ID NO: 29; l) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 32 or genomic DNA thereof encoding the Thiaminase I Thi1_SAU comprised in SEQ ID NO: 31; m) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 34 or genomic DNA thereof encoding the Thiaminase I Thi1_ANE comprised in SEQ ID NO: 33; n) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 36 or genomic DNA thereof encoding the Thiaminase I Thi1_MEL comprised in SEQ ID NO: 35; o) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at leastCase Ref. P180WO IPTector® 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 38 or genomic DNA thereof encoding the Thiaminase I Thi1_NGR comprised in SEQ ID NO: 37; p) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 40 or genomic DNA thereof encoding the Thiaminase I Thi1_BGL comprised in SEQ ID NO: 39; q) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 42 or genomic DNA thereof encoding the Thiaminase I Thi1_HOC comprised in SEQ ID NO: 41; r) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 44 or genomic DNA thereof encoding the Thiaminase I Thi1_OAL comprised in SEQ ID NO: 43; and / or s) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 46 or genomic DNA thereof encoding the Thiaminase II TenA_BSU comprised in SEQ ID NO:

45.

33. The host cell of any preceding claim, wherein the one or more pathway elements are encoding one or more genes selected from the group of: a) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 2 or genomic DNA thereof encoding the ThiC comprised in SEQ ID NO: 1; b) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 4 or genomic DNA thereof encoding the ThiD comprised in SEQ ID NO: 3; c) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 18 or genomic DNA thereof encoding the ThiE comprised in SEQ ID NO: 17; d) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, suchCase Ref. P180WO IPTector® as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 6 or genomic DNA thereof encoding the ThiF comprised in SEQ ID NO: 5; e) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 8 or genomic DNA thereof encoding the ThiS comprised in SEQ ID NO: 7; f) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 28 or genomic DNA thereof encoding the ThiO_PPT comprised in SEQ ID NO: 27; g) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 12 or genomic DNA thereof encoding the ThiG comprised in SEQ ID NO: 11; h) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 10 or genomic DNA thereof encoding the ThiH comprised in SEQ ID NO: 9; i) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 26 or genomic DNA thereof encoding the TH2_ATH comprised in SEQ ID NO: 25; and / or j) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 24 or genomic DNA thereof encoding the TenI_BSU comprised in SEQ ID NO:

23.

34. The host cell of any preceding claim, wherein the one or more pathway elements are encoded by one or more genes selected from the group of: a) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 116 or genomic DNA thereof encoding the tenA_HP1287_HPY comprised in SEQ ID NO: 115;Case Ref. P180WO IPTector® b) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 118 or genomic DNA thereof encoding the tenA_AAN comprised in SEQ ID NO: 117; c) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 120 or genomic DNA thereof encoding the tenA_AMI comprised in SEQ ID NO: 119; d) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 122 or genomic DNA thereof encoding the TenA_BSU comprised in SEQ ID NO: 121; e) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 124 or genomic DNA thereof encoding the tenA_CPA comprised in SEQ ID NO: 123; f) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 126 or genomic DNA thereof encoding the tenA_HHA comprised in SEQ ID NO: 125; g) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 128 or genomic DNA thereof encoding the tenA_PAE comprised in SEQ ID NO: 127; h) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 130 or genomic DNA thereof encoding the tenA_PFU comprised in SEQ ID NO: 129; i) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 132 or genomic DNA thereof encoding the tenA_PHO comprised in SEQ ID NO: 131; j) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at leastCase Ref. P180WO IPTector® 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 134 or genomic DNA thereof encoding the tenA_SAU comprised in SEQ ID NO: 133; k) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 136 or genomic DNA thereof encoding the tenA_SEP comprised in SEQ ID NO: 135; l) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 138 or genomic DNA thereof encoding the tenA_SSO comprised in SEQ ID NO: 137; m) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 140 or genomic DNA thereof encoding the tenA_THI20_SCE comprised in SEQ ID NO: 139; n) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 142 or genomic DNA thereof encoding the thiK comprised in SEQ ID NO: 141; o) gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 144 or genomic DNA thereof encoding the SodA comprised in SEQ ID NO: 143; p) gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 146 or genomic DNA thereof encoding the iscS comprised in SEQ ID NO: 145; and / or q) gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 148 or genomic DNA thereof encoding the dxs comprised in SEQ ID NO:

147.

35. The host cell of any preceding claim, wherein the one or more pathway elements are encoded by one or more genes selected from the group of: a) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, suchCase Ref. P180WO IPTector® as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 6 or genomic DNA thereof encoding the ThiF comprised in SEQ ID NO: 5; b) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 8 or genomic DNA thereof encoding the ThiS comprised in SEQ ID NO: 7; c) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 28 or genomic DNA thereof encoding the ThiO_PPT comprised in SEQ ID NO: 27; d) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 12 or genomic DNA thereof encoding the ThiG comprised in SEQ ID NO: 11; e) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 10 or genomic DNA thereof encoding the ThiH comprised in SEQ ID NO: 9; f) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 26 or genomic DNA thereof encoding the phosphatase TH2_ATH comprised in SEQ ID NO: 25; g) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 48 or genomic DNA thereof encoding the phosphatase PHOS_PhoA comprised in SEQ ID NO: 47; h) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 50 or genomic DNA thereof encoding the phosphatase PHOS_NudJ comprised in SEQ ID NO: 49; i) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 52 orCase Ref. P180WO IPTector® genomic DNA thereof encoding the phosphatase PHOS_Cof comprised in SEQ ID NO: 51; j) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 54 or genomic DNA thereof encoding the phosphatase PHOS_YbhA comprised in SEQ ID NO: 53; k) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 56 or genomic DNA thereof encoding the phosphatase PHOS_KdsC comprised in SEQ ID NO: 55; l) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 58 or genomic DNA thereof encoding the phosphatase PHOS_RsgA_SAE comprised in SEQ ID NO: 57; m) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 60 or genomic DNA thereof encoding the phosphatase PHOS_PhoS_PAE comprised in SEQ ID NO: 59; n) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 62 or genomic DNA thereof encoding the phosphatase PHOS_NudJ_PPU comprised in SEQ ID NO: 61; o) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 64 or genomic DNA thereof encoding the phosphatase PHOS_AlkP_PPU comprised in SEQ ID NO: 63; p) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 66 or genomic DNA thereof encoding the phosphatase PHOS_ygiF_PPU comprised in SEQ ID NO: 65; q) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 68 or genomic DNA thereof encoding the phosphatase PHOS_upxB_PPU comprised in SEQ ID NO: 67; r) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, suchCase Ref. P180WO IPTector® as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 70 or genomic DNA thereof encoding the phosphatase PHOS_PhoD_PPU comprised in SEQ ID NO: 69; s) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 72 or genomic DNA thereof encoding the phosphatase PHOS_AphA comprised in SEQ ID NO: 71; t) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 74 or genomic DNA thereof encoding the phosphatase PHOS_NudJ_SSO comprised in SEQ ID NO: 73; u) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 76 or genomic DNA thereof encoding the phosphatase PHOS_PhoD_PAS comprised in SEQ ID NO: 75; v) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 78 or genomic DNA thereof encoding the phosphatase PHOS_AH2_HIN comprised in SEQ ID NO: 77; w) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 80 or genomic DNA thereof encoding the phosphatase PHOS_AH2_CSI comprised in SEQ ID NO: 79; and / or x) a gene which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 82 or genomic DNA thereof encoding the deCARB_CadA comprised in SEQ ID NO:

81.

36. The host cell of any preceding claim, wherein the host cell is configured for producing cTHZ-P and comprises or expresses one or more or all genes encoding: a. thiF, such as the ThiF having an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the ThiF comprised in SEQ ID NO: 5;Case Ref. P180WO IPTector® b. thiS, such as the ThiS having an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the ThiS comprised in SEQ ID NO: 7; c. thiG, such as the ThiG having an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the ThiG comprised in SEQ ID NO: 11; d. thiI, such as the ThiI having an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the ThiI comprised in SEQ ID NO: 19; e. thiO and / or ThiH, such as the ThiO having an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the ThiO comprised in SEQ ID NO: 27 and / or such as ThiH having an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the ThiH comprised in SEQ ID NO: 9; f. iscS, such as the IscS having an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the IscS comprised in SEQ ID NO: 145; g. tenI, such as the tenI having an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the Ten I comprised in SEQ ID NO: 23, and h. dxs, such as the dxs having an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the dxs comprised in SEQ ID NO:

147.

37. The host cell of any preceding claim, wherein the host cell is configured for producing thiamine and comprises or expresses genes encoding: a. optionally ThiC, such as ThiC having an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the ThiC comprised in SEQ ID NO: 1; b. ThiD, such as ThiD having an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the ThiD comprised in SEQ ID NO: 3; c. ThiE, such as ThiE having an amino acid sequence which is at least 70%, such at least 75%, suchCase Ref. P180WO IPTector® as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the ThiE comprised in SEQ ID NO: 17;ThiF has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the ThiF comprised in SEQ ID NO: 5; and d. TH2, such as the TH2 having an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the TH2 comprised in SEQ ID NO: 25; and e. optionally a thiaminase, such as Thi1 or TenA, for example wherein the Thi1 has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the Thi1 comprised in SEQ ID NO: 29, 31, 33, 35, 37, 39, 41 and / or 43, or wherein the TenA has an amino acid sequence which is at least 70%, such at least 75%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 99%, such as 100% identity to the TenA comprised in SEQ ID NO: 45, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137 and / or 139.

38. The host cell of any preceding claim, wherein the host cell is configured for producing carboxy sulfurol (cTHZ) and comprises or expresses genes encoding a phosphatase capable of dephosphorylating cTHZ-P to cTHZ, such as wherein the phosphatase has an amino acid sequence which is at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the phosphatase comprised in SEQ ID NO: 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 83, 85, 87, 89,91, 93, 95, 97, 99, 101, 103 and / or 105.

39. The host cell of any preceding claim, wherein one or more genes and / or polypeptides of the pathway for the sulfurol are heterologous to the host cell.

40. The host cell of any preceding claim, further comprising at least 2 copies of one or more genes and / or polypeptides of the pathway for the sulfurol compound.

41. The host cell of any preceding claim, further comprising a transporter molecule facilitating transport of a precursor for or a product of the pathway for the sulfurol compound.

42. The host cell of any preceding claim, wherein the host cell is further genetically modified to provide an increased amount of a substrate in the pathway for the sulfurol compound.Case Ref. P180WO IPTector® 43. The host cell of any preceding claim, wherein the host cell is further genetically modified to exhibit increased tolerance towards one or more substrates, intermediates, or products in the pathway for the sulfurol compound.

44. The host cell of any preceding claim, wherein one or more additional native or endogenous genes of the host cell are deleted, disrupted and / or attenuated.

45. The host cell of any preceding claim, wherein one or more genes in the pathway for the sulfurol compound is overexpressed.

46. The host cell of any preceding claim, wherein the substrate in the pathway for the sulfurol compound is selected from the group consisting of: cTHZ-P, THZ-P, cTHZ and thiamine.

47. The host cell of any preceding claim, wherein the host cell is prokaryotic or eukaryotic.

48. The host cell of claim 47, wherein the host cell is prokaryotic and of a genus selected from the group consisting of Escherichia, Bacillus, Brevibacterium, Burkholderia, Campylobacter, Corynebacterium, Serratia, Lactobacillus, Lactococcus, Acinetobacter, Acetobacter and Pseudomonas.

49. The host cell of claim 48, wherein the host cell is of the genus Escherichia, Corynebacterium, Bacillus, Serratia, Pseudomonas.

50. The host cell of claim 48, wherein the host cell is selected from the species Escherichia coli, Corynebacterium glutamicum, Bacillus subtilis, Serratia marcescens, pseudomonas putida and / or Pseudomonas mutabilis.

51. The host cell of claim 47, wherein the host cell is selected from the group consisting of mammalian, insect, plant, fungal or archaeal cells.

52. The host cell of claim 47, wherein the host cell is eukaryotic and selected from the group consisting of mammalian, insect, plant, fungal or archaeal cells.

53. The host cell of claim 51, wherein the host cell is a fungal cell of a genus selected fromCase Ref. P180WO IPTector® Saccharomyces, Pichia and / or Ashbya.

54. The host cell of claim 53, wherein the host cell is selected from the species Saccharomyces cerevisiae, Pichia pastoris and / or Ashbya gossypii.

55. A mutated polypeptide selected from the group consisting of a polypeptide which is at least 90% identical to: a. the mutant phosphatase having a sequence comprised in SEQ ID NO: 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103 and / or 105; b. the mutant thiaminase I having a sequence comprised in SEQ ID NO: 29, 31, 33, 35, 37, 39, 41 and / or 43; and / or c. the mutant thiaminase II having a sequence comprised in SEQ ID NO: 45, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137 and / or 139.

56. A polynucleotide construct comprising a polynucleotide sequence encoding a phosphatase, thiaminase I, and / or thiaminase II operably linked to one or more control sequences, wherein the polynucleotide construct comprises one or more mutations deleting, disrupting and / or attenuating the phosphatase, thiaminase I, and / or thiaminase II.

57. The polynucleotide construct of claim 56, wherein the construct is an expression vector.

58. The host cell of any preceding claim comprising the polynucleotide construct of claim 57.

59. A cell culture, comprising the host cell of any preceding claim, and a growth medium.

60. A method for producing sulfurol comprising: a. culturing the cell culture of claim 59 at conditions allowing the host cells to produce the sulfurol compound; b. optionally recovering and / or isolating the sulfurol compound.

61. A method for producing sulfurol comprising: a. culturing the cell culture of claim 59 at conditions allowing the host cells to produce the thiamine compound; b. optionally recovering and / or isolating the thiamine compound broth;Case Ref. P180WO IPTector® c. Subjecting the thiamine broth to thiaminase I; and / or d. optionally recovering and / or isolating the sulfurol compound.

62. A method for producing sulfurol comprising: a. culturing the cell culture of claim 59 at conditions allowing the host cells to produce the cTHZ compound; b. optionally recovering and / or isolating the cTHZ compound; c. chemical decarboxylation of cTHZ (1)and / or d. optionally recovering and / or isolating the sulfurol compound.

63. A method of producing sulfurol comprising a. culturing the cell culture of claim 59 at conditions allowing the host cells to produce the cTHZ- P compound; b. optionally recovering and / or isolating the cTHZ-P compound; c. chemical dephosphorylation of cTHZ-P to sulfurol by hydrolysis of cTHZ-Pand / or d. optionally recovering and / or isolating the sulfurol compound.

64. A method of producing sulfurol comprising a. culturing the cell culture of claim 59 at conditions allowing the host cells to produce the thiamine compound; b. optionally recovering and / or isolating the thiamine compound; c. chemical conversion of thiamine to sulfurol byCase Ref. P180WO IPTector®and / or d. optionally recovering and / or isolating the sulfurol compound.

65. A method for producing a sulfurol compound comprising: a. culturing the cell culture of claim 59 at conditions allowing the host cells to produce the thiamine compound; b. chemically converting the thiamine compound to sulfurol; c. optionally recovering and / or isolating the sulfurol compound.

66. The method of claim 65, wherein the method comprises a step of recovering thiamine from the cell culture prior to step b.

67. The method for producing a sulfurol compound of any of claims 60-66, comprising a chemical conversion of thiamine with a bisulphite in a concentration from 0,2 to 4,0 w / v such as 1,0, 1,5, 2,0, 2,5, 3,0, 3,5 or 4,0% w / v.

68. The method of any of claims 60-67, wherein the bisulphite is selected from Potassium bisulphite, Sodium metabisulphite and / or Sodium bisulphite (NaHSO3).

69. The method of any of claims 60-68, wherein the bisulphite is Sodium bisulphite (NaHSO3).

70. The method of any of claims 60-69, wherein the Sodium bisulphite (NaHSO3) is in a concentration of 2,0 w / v.

71. The method of any of claims 60-70, comprising a chemical conversion of thiamine with a bisulphite at a pH from 3.0 to 8.0, such as 3,0, 3,5, 4,0, 4,5, 5,0, 5,5, 6,0, 6,5, 7,0 and / or 8,0.

72. The method of any of claims 60-70, comprising a chemical conversion of thiamine with a bisulphiteCase Ref. P180WO IPTector® at a pH of 4.5 to 6.0 such as a pH of 5,5.

73. The method of any of claims 60-72, comprising a chemical conversion of thiamine with a bisulphite at a temperature from 5 to 30 degrees Celsius, such as 5, 10, 15, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 to 30 degrees Celsius or at room temperature such as 15 to 25 degree Celsius.

74. The method of any of claims 60-73, comprising a chemical conversion of thiamine with a bisulphite at room temperature such as from 15 to 25 degrees Celsius such as 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 degrees Celcius.

75. The method for producing sulfurol of claim 60 to 74 at conditions allowing the host cells to produce a thiamine compound; and a. chemically converting the thiamine compound to sulfurol by adding 2,0, % w / v of NaHSO3at pH 5,5 at room temperature; b. optionally recovering and / or isolating the sulfurol compound.

76. A method for producing a sulfurol compound comprising: a. culturing the cell culture of claim 59 at conditions allowing the host cells to produce the carboxy-sulfurol compound; b. recovering and / or isolating the carboxy-sulfurol compound by a resin and / or an organic solvent; c. chemically converting the carboxy-sulfurol compound to sulfurol; d. optionally recovering and / or isolating the sulfurol compound.

77. The method for producing a sulfurol compound of claim 76, wherein recovering and / or isolating the carboxy-sulfurol compound comprises a strong anionic or weak anionic resin such as AmberLite FPA66 OH, AmberLite IRN78 OH, AmberLyst A26 OH, Ambersep 900 and / or AmberLite IRA67.

78. The method of any of claims 76 to 77, wherein recovering and / or isolating the carboxy-sulfurol compound broth comprises an organic solvent such as Ethyl acetate (EtOAc / CH3COOC2H5), Ethanol (C2H6O), n-Butanol (C2H6O), and / or methylethyl ketone for extraction.

79. The method of any of claims 76 to 78, wherein recovering and / or isolating the carboxy-sulfurolCase Ref. P180WO IPTector® compound broth comprises a resin, such as a strong anionic or a weak anionic resin, and Ethyl acetate (EtOAc / CH3COOC2H5) for extraction.

80. The method of any of claims 76 to 79, comprising a chemical conversion of carboxy-sulfurol with an acid.

81. The method of any of claims 76 to 80, comprising a chemical conversion of carboxy-sulfurol with an acid such as HCl, Acetic acid (HC2H3O2), Phosphoric acid (H3PO4), nitric acid (HNO3) and / or sulfuric acid (H2SO4).

82. The method of any of claims 76 to 81, comprising a chemical conversion of carboxy-sulfurol with an acid at an acidic pH from 0.1 to 6.0, such as 1.0, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0 or 6.0 and / or an acid in the molarity from 0.05 to 2 such as 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 2.

0.

83. The method of any of claims 76 to 82, comprising a chemical conversion of carboxy-sulfurol with an acid at an acidic pH such as 1.0 to 4.0 and / or a molarity of 0.1 to 1.

0.

84. The method of any of claims 76 to 83, comprising a chemical conversion of carboxy-sulfurol with an acid at a temperature from 50 to 150 degrees Celsius, such as a temperature from 50, 60, 70, 80, 90, 100, 110, 120, 130, 140 and / or 150 degrees Celsius.

85. The method of any of claims 76 to 84, comprising a chemical conversion of carboxy-sulfurol with an acid at 100 degrees Celsius.

86. The method of producing a sulfurol compound of claim 76 to 85 at conditions allowing the host cells to produce a carboxy-sulfurol compound comprising: a. recovering and / or isolating the carboxy-sulfurol compound broth by a resin, such as a strong anionic or a weak anionic resin, such as AmberLite FPA66 OH, AmberLite IRN78 OH, AmberLyst A26 OH, Ambersep 900 and / or AmberLite IRA67, and / or EtOAc extraction; b. chemically converting the carboxy-sulfurol compound to sulfurol with CH3COOH and / or HCl at an acidic pH at 100 degree Celsius; c. optionally recovering and / or isolating the sulfurol compound.Case Ref. P180WO IPTector® 87. The method of any preceding claims, further comprising feeding one or more exogenous sulfurol precursor therefore to the host cell culture, optionally selected from the group of O- methylpimeloyl-acyl carrier protein, pimeloyl-acyl carrier protein, KAPA, DAPA, DTB, amino-HMP, and pimelate.

88. The method of any of claims 60 to 87, further comprising one or more elements selected from: a. culturing the cell culture under aerobic or anaerobic conditions b. cultivating the host cells under mixing; c. cultivating the host cells at a temperature of between 25°C to 50°C; d. cultivating the host cells at a pH of between 3-9; and / or e. cultivating the host cells for between 10 hours to 120 days.

89. The method of any of claims 60 to 88, wherein the recovering and / or isolation step comprises separating a liquid phase of the cell culture from a solid phase of the cell culture to obtain a supernatant comprising the sulfurol compound and subjecting the supernatant to one or more steps selected from: a) contacting the supernatant with one or more adsorbent resins in order to obtain at least a portion of the produced sulfurol compound, then optionally recovering the sulfurol compound from the resin in a concentrated solution prior to isolation of the sulfurol compound by crystallisation or solvent evaporation; b) contacting the supernatant with one or more ion exchange or reversed-phase chromatography columns to obtain at least a portion of the sulfurol compound, then optionally recovering the sulfurol compound from the resin in a concentrated solution prior to isolation of the sulfurol compound by crystallisation or solvent evaporation; and / or c) extracting the thiamine compound from the supernatant, such as by liquid-liquid extraction into an immiscible solvent, then optionally isolating the sulfurol compound by crystallisation or solvent evaporation; and thereby recovering and / or isolating the sulfurol compound.

90. The method of any of claims 60 to 89, wherein the sulfurol compound yield is at least 10% higher such as at least 50%, such as at least 100%, such as least 150%, such as at least 200% higher than production by a host cell by a. expressing one or more heterologous phosphatase and / or thiaminase genes capable of converting carboxysulfurol-phosphate (cTHZ-P) or thiamine into sulfurol, sulfurol-phosphate (THZ-P) and / or carboxysulfurol (cTHZ) in the host cell; and / orCase Ref. P180WO IPTector® b. over-expressing one or more ThiO genes capable of catalysing the glycine to DHG in the host cell.

91. The method of any of claims 60-90, wherein the sulfurol yield and / or titer is at least 4 mg / L, optionally at least 8 mg / L, optionally at least 10mg / L, optionally at least 15 mg / L, optionally at least 20 mg / L, optionally at least 30mg / L, optionally at least 50 mg / L, optionally at least 100 mg / L, optionally at least 150mg / L, optionally at least 200 mg / L, 300mg / L, optionally at least 500 mg / L, optionally at least 1 g / L, optionally at least 1.5 g / L, optionally at least 2 g / L, for example at least 10 g / L, such as at least 15 g / L.

92. The method of any of claims 60-91, wherein the carboxy-sulfurol (cTHZ) yield and / or titer is at least 4 mg / L, optionally at least 8 mg / L, optionally at least 10mg / L, optionally at least 15 mg / L, optionally at least 20 mg / L, optionally at least 30mg / L, optionally at least 50 mg / L, optionally at least 100 mg / L, optionally at least 150mg / L, optionally at least 200 mg / L, 300mg / L, optionally at least 500 mg / L, optionally at least 1 g / L, optionally at least 1.5 g / L, optionally at least 2 g / L, for example at least 10 g / L, such as at least 15 g / L.

93. The method of any of claims 60-92, wherein the thiamine yield and / or titer is at least 4 mg / L, optionally at least 8 mg / L, optionally at least 10mg / L, optionally at least 15 mg / L, optionally at least 20 mg / L, optionally at least 30mg / L, optionally at least 50 mg / L, optionally at least 100 mg / L, optionally at least 150mg / L, optionally at least 200 mg / L, 300mg / L, optionally at least 500 mg / L, optionally at least 1 g / L, optionally at least 1.5 g / L, optionally at least 2 g / L, for example at least 10 g / L, such as at least 15 g / L.

94. The method of claims 60-93, wherein one or more steps of producing the sulfurol compound is performed in vitro, such as by chemical synthesis.

95. A method of preparing sulfurol from a sulfurol precursor comprising: a. Obtaining the sulfurol precursor by culturing the host cell of any preceding claims in a growth medium to provide the sulfurol precursor; b. Recovering the sulfurol precursor from the growth medium, and c. Converting the sulfurol precursor to sulfurol in vitro, optionally by chemical synthesis.

96. The method of claim 95, wherein the sulfurol precursor is selected from the group consisting of:Case Ref. P180WO IPTector® thiamine (THI), carboxy-sulfurol (cTHZ), 4-methyl-5-beta-hydroxyethylthiazole phosphate (THZ-P), and 5-(2-hydroxyethyl)-4-methyl-1,3-thiazole-2-carboxylic phosphate acid (cTHZ-P).

97. The method of any of claims 95-96, wherein the sulfurol precursor is thiamine and step c) is conducted using a bisulfite, such as NaHSO3.

98. The method of any of claims 95-97, wherein the sulfurol precursor is cTHZ and step c) is conducted using a base.

99. The method of any of claims 95-98, wherein the sulfurol precursor is THZ-P and step c) is conducted using a base and optionally a metal cation.

100. The method of any of claims 95-99, wherein the sulfurol precursor is cTHZ-P and step c) is conducted using a base, and optionally a metal cation, such as Zn2+.

101. The method according to any of claims 95-100, wherein the method further comprises the method of any one of claims 60-100.

102. The method according to any of the preceding claims, wherein a substrate in the pathway for producing sulfurol, THI, cTHZ, THZ-P, and / or cTHZ-P is added to the host cell.

103. The method of claim 102, wherein the substrate is amino-HMP and / or HMP.

104. The method of any of claims 102-103, wherein the host cell does not comprise or express ThiC.

105. The method of any of claims 102-104, wherein the host cell comprises or expresses a gene encoding: a thiaminase, such as a thiaminase defined in any one of the preceding claims, for example Thi1 or TenA.

106. The method of any of claims 102-105, wherein the host cell comprises one or more pathway elements as defined in any one of the preceding claims, for example all the pathway elements of the operative metabolic pathway for producing thiamine.

107. The method of any of claims 60 to 105, further comprising mixing the sulfurol compound withCase Ref. P180WO IPTector® one or more carriers, agents, adjuvants, additives and / or excipients, optionally pharmaceutical grade carriers, agents, adjuvants, additives and / or excipients.

108. A fermentation composition comprising the cell culture of claim 59 or resulting from the method of any claims 60 to 107, and the sulfurol compound.

109. The fermentation composition of claim 108, wherein at least 50%, such as at least 75%, such as at least 95%, such as at least 99% of the genetically modified host cells are lysed and / or disintegrated.

110. The fermentation composition of any of claims 108 to 109, wherein at least 50%, such as at least 75%, such as at least 95%, such as at least 99% of solid cellular material has been separated from the composition.

111. The fermentation composition of any of claims 108 to 110, further comprising one or more compounds selected from: a. precursor or products of the operative metabolic pathway producing the sulfurol compound; b. supplemental nutrients; and wherein the concentration of the sulfurol compound is at least 1 mg / L composition.

112. A composition comprising the fermentation composition of claim 108 to 111, and one or more carriers, agents, adjuvants, additives and / or excipients and at least trace amounts of one or more metabolites of the cell culture, optionally signature metabolites for the genetically modified host cell.

113. The composition of claim 112, wherein the fermentation composition and the one or more carriers, agents, adjuvants, additives and / or excipients are in a dry solid form.

114. The composition of any of claims 112-113, wherein the fermentation composition and the one or more carriers, agents, adjuvants, additives and / or excipients are in a liquid stabilized form.

115. The composition of any of claims 112-114, refined into a pharmaceutical preparation, a dietary supplement, a cosmetic, a food preparation, a flavor preparation, a feed preparation and / or an analytical or diagnostic reagent.Case Ref. P180WO IPTector® 116. The composition of any of claims 112-115, wherein the preparation is in form of a powder, tablet, capsule, hard chewable and / or soft lozenge, liposomes or a gum.

117. The composition of any of claims 112-116, wherein the pharmaceutical preparation is in form of a liquid pharmaceutical solution. * * *

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