Microbial cell factory for producing thiamine

Abstract

The present disclosure relates to a genetically modified host cell with improved thiamine production wherein the host cell expresses: one or more heterologous ThiO enzymes, converting glycine in the host cell to dehydroglycine (DHG), and / or one or more heterologous ThiI enzymes, a sulfur carrier protein ThiS that catalyzes sulfur transfer from IscS to the host cell, and / or one or more heterologous ThiI enzymes that catalyze sulfur transfer from IscS to the host cell. Thus, the yield of thiamine in the genetically modified host cell is improved as compared to an unmodified parent host cell.

Description

Technical Field

[0001] The present invention relates to genetically modified host cells having increased production of thiamine by providing increased production of dehydroglycine (DHG) and ThiS thiocarboxylation protein, to genetic constructs for expressing such mutants; to cultures of genetically modified host cells and their use for producing said products; to fermentation broths comprising said products resulting from such production; to compositions comprising the fermentation broths; to dietary, seasoning or pharmaceutical preparations made from such compositions and the use of such compositions and preparations. Background Art

[0002] In nature, thiamine is produced by some microorganisms and plants. The biosynthetic pathway of thiamine in natural organisms is generally well described in the art. Figure 1 shown.

[0003] Today, thiamine is produced industrially by using chemical synthesis processes based on non-renewable fossil fuels, and there is no fermentation-based production available. However, there is a need to develop more sustainable bio-based processes using renewable sources such as sugars. Since microorganisms only need small amounts of thiamine, the biggest challenge is to engineer them to produce large amounts of this vitamin.

[0004] The use of microbial-based cell factories is a potential route for the biosynthetic production of B vitamins (Acevedo-Rocha et al., 2019). The advantages of recombinant microorganisms such as Escherichia coli as microbial cell factories for the production of bioproducts are widely recognized because: (i) E. coli has unparalleled rapid growth kinetics; its doubling time is approximately 20 minutes when grown in glucose-salt medium and under optimal environmental conditions, and (ii) it can easily reach high cell densities; the theoretical density limit of E. coli liquid culture is estimated to be approximately 200 g dry cell weight / L or approximately 1X10 13 In addition, there are many molecular tools and protocols available for genetic optimization of E. coli; and it is a suitable organism for expressing heterologous proteins; both of which are essential for obtaining high levels of production of desired bioproducts.

[0005] Thiamine is produced in nature via a multistep enzymatic pathway that can be divided into two branches ( Figure 1 ) : pyrimidine (PYR) and thiazole (THZ). In the PYR branch, the radical SAM phosphomethylpyrimidine synthase ThiC converts the substrate 5-aminoimidazole nucleotide (AIR) into the pathway intermediate 4-amino-5-hydroxymethyl-2-methylpyrimidine-phosphate (HMP-P), which is phosphorylated to HMP-PP by the kinase ThiD.

[0006] In the THZ branch, there are three sub-branches that form the thiazole ring. The first, second, and third sub-branches contain sugar backbones formed by pyruvate and 3-phosphoglyceraldehyde catalyzed by Dxs, sugar backbones from the sulfur transfer step formed by cysteine ​​catalyzed by IscS, ThiI, ThiF, and ThiS, and sugar backbones formed by dehydroglycine (DHG) that can be formed by tyrosine catalyzed by ThiH or glycine catalyzed by ThiO, respectively. These three components are combined by the ThiG enzyme to generate carboxythiazole phosphate (cTHZ-P). Finally, ThiE combines with the pyrimidine (HMP-PP) and thiazole (cTHZ-P) moieties to generate thiamine monophosphate (TMP), which can be dephosphorylated by Arabidopsis phosphatase to form thiamine ( Figure 1 ).

[0007] In E. coli and anaerobic organisms, DHG is produced from tyrosine by the action of the enzyme ThiH through a complex free radical-SAM mediated mechanism (D1 Broderick et al., 2014). The ThiH gene product of E. coli is oxygen sensitive and has a turnover value of 3, which is very low for the production of DHG (D1 Broderick et al., 2014). Other organisms that are usually aerobic use glycine instead of tyrosine as a substrate to produce DHG by glycine oxidase (ThiO). In such organisms, ThiH is replaced by ThiO.

[0008] In this regard, the turnover values ​​of the enzymes in Bacillus subtilis (BsThiO) (D2 Boselli et al., 2007) and Pseudomonas putida (PpThiO) (Equar et al., 2015) are several orders of magnitude higher than those of ThiH in Escherichia coli (Broderick et al., 2014). Interestingly, it has been suggested that ThiH can form a complex with ThiG to deliver DHG, since it is an active compound due to the aldehyde group (Leonardi et al., 2003), while ThiF also interacts with ThiS (Lehmann et al., 2006) and then transfers sulfur from the inactive thiocarboxylated form to ThiG to generate cTHZ-P in the presence of DXP and DHG.

[0009] In the case of the sulfur transferor branch, IscS transfers the sulfur atom from cysteine ​​to ThiI and then from ThiI to ThiS (Taylor et al. 1998; Martinez-Gomez et al. 2011), while ThiF catalyzes the sulfur carboxylation of ThiS ( Figure 2). Interestingly, E. coli contains all of the following thiamine genes ThiF, ThiS, ThiI, IscS, ThiG, and ThiH, but lacks ThiO. It is unclear what the rate-limiting step is in the THZ subbranch. However, while sulfur transfer and sugar formation are likely rate-limiting, DHG formation could also be improved by replacing ThiH with ThiO, as the ThiO enzyme has a less complex mechanism and a higher turnover rate. Summary of the invention

[0010] Against this background, an improvement has been provided that the ThiO enzymes from Bacillus subtilis and Pseudomonas putida can replace the function of the E. coli ThiH enzyme, and if the native ThiI gene is also co-expressed at an appropriate level, the level of thiamine is greatly improved. Therefore, the combination of replacing the ThiH enzyme from E. coli with the ThiO enzyme from Bacillus subtilis or Pseudomonas putida and overexpressing ThiI at an appropriate level is crucial for improving the formation of cTHZ-P, and can significantly improve the flux of thiamine compared to overexpressing ThiH or ThiH and ThiI genes alone. This is in Figure 2 3, showing that ThiI co-expression increased the titer by >300% to about 13 mg / L in strains BS06285 or BS06287 containing the P. putida ThiO or B. subtilis ThiO enzymes, respectively.

[0011] Thus, overexpression of ThiO and ThiI increased the flux of thiamine in small-scale cultures by approximately 30% compared to controls using ThiH and ThiI. It is hypothesized that not only is ThiO a better enzyme than ThiH, but ThiI can also synergize well with ThiO from different organisms to increase sulfur mobilization and DHG formation, thereby increasing thiamine production. It was also found that a truncated form of the ThiI enzyme, containing the rhodanese domain involved in sulfur mobilization to ThiS, could effectively replace the full-length native ThiI enzyme, further increasing thiamine production. Thus, the results obtained herein suggest that by replacing the radical SAM enzyme ThiH with the appropriate combination and expression of ThiI and ThiO, the thiamine pathway can be significantly improved, enabling efficient thiamine production from glucose or added precursors.

[0012] Thus, in a first aspect, provided herein is a genetically modified host cell with improved thiamine production, wherein the host cell expresses one or more heterologous ThiO enzymes that convert glycine in the host cell to dehydroglycine (DHG), thereby improving thiamine production in the genetically modified host cell compared to an unmodified parent host cell.

[0013] In a second aspect, the present invention provides a genetically modified host cell with improved thiamine production, wherein the host cell expresses one or more heterologous ThiI enzymes, which catalyze the transfer of sulfur from IscS in the host cell to the sulfur carrier protein ThiS, thereby improving thiamine production in the genetically modified host cell compared to an unmodified parent host cell.

[0014] In a third aspect, the present invention provides a method for producing thiamine, comprising:

[0015] a) cultivating the cell culture under conditions that allow the host cells to produce thiamine; and

[0016] b) optionally recovering and / or isolating the thiamine.

[0017] In a fourth aspect, provided herein is a fermentation composition comprising a cell culture and thiamine.

[0018] In a fifth aspect, provided herein is a composition comprising a fermentation composition and one or more carriers, agents, adjuvants, additives and / or excipients.

[0019] In a final aspect, provided herein is a method of treating a disease in a mammal, comprising administering to the mammal a therapeutically effective amount of the composition. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The drawings contained herein are schematic and simplified for clarity, and they merely show details that are essential to the understanding of the invention, while other details may have been omitted.

[0021] Figure 1 The modules of the pathway for microbial thiamine production in Escherichia coli are shown. In the pyrimidine branch ( Figure 1 , module 1, left), ThiC (phosphomethylpyrimidine synthase) converts its substrate AIR (aminoimidazole riboside) obtained from the purine pathway into HMP-P (hydroxymethylpyrimidine phosphate), which is then phosphorylated to HMP-PP (hydroxymethylpyrimidine diphosphate) by ThiD (hydroxymethylpyrimidine / phosphomethylpyrimidine kinase). The production of high levels of HMP-P is limited by the low turnover rate of ThiC, which can be overcome by co-expressing ThiD and externally feeding HMP, which is phosphorylated twice in a 2-step reaction to form HMP-PP (dashed arrows). In the thiazole (THZ) branch ( Figure 1, module 2, right), the major product is carboxythiazole phosphate (cTHZ-P), which is formed by the action of ThiG, combining 3 molecules including sulfur from cysteine ​​(through the action of IscS, ThiI, ThiS and ThiF), dehydroglycine (DHG) from tyrosine (ThiH) in E. coli or glycine (ThiO) in various organisms, and the sugar DXP (1-deoxy-D-xylulose 5-phosphate). THZ can also be supplied to the cell factory by co-expression with ThiM (hydroxyethylthiazole kinase) to catalyze the phosphorylation of THZ to THZ-P (dashed arrow). ThiE (thiamine phosphate synthase) condenses HMP-PP and cTHZ-P to form thiamine monophosphate (TMP), which is dephosphorylated by Arabidopsis phosphatase to produce thiamine ( Figure 1 , module 3 (bottom). The genes can be overexpressed from plasmids (e.g., particularly ThiF, ThiS, ThiG, ThiH or ThiO, ThiI) or chromosomes (e.g., particularly ThiD, ThiE ThiM and phosphatases). According to LC-MS data (not shown), a single copy of phosphatases is sufficient to produce about 98% of thiamine, with the remainder being a mixture of TMP and thiamine diphosphate (TPP).

[0022] Figure 2 Bar graphs showing thiamine titer (mg / L), growth (OD) and OD normalized titer (mg / L / OD) of E. coli strains expressing the native thiamine genes ThiF, ThiS, ThiG and ThiH after 24 h of batch culture in mMOPS (supplemented with 500 μM HMP) with either ThiI not co-expressed (BS05850) or ThiI co-expressed (BS06232). The other strains contained heterologous ThiO from Pseudomonas putida (BS06278) or Bacillus subtilis (BS06280) instead of native ThiH. These strains were transformed with plasmids encoding ThiI from E. coli to generate strains BS06285 (Pseudomonas putida ThiO) and BS06287 (Bacillus subtilis ThiO) containing ThiI. The bar graphs show the median of 4 replicates, with each point representing one replicate.

[0023] Figure 3The four domains of E. coli ThiI are shown. The first domain (residues 1-63) contains the NFLD (N-terminal ferredoxin-like domain). The second domain (residues 61-165) contains the THUMP (thiouridine synthase, methylase, and pseudouridine synthase) domains, which all interact with tRNA. The third domain (residues 189-360) belongs to the adenine nucleotide α-hydrolase (AANH) domain, which is responsible for uridine adenylation. The fourth domain (residues 404-482) has a rhodanese domain, which is involved in the transfer of sulfur to thiazole in the thiamine biosynthetic pathway. The ATP binding site is located between the second and third domains (residues 183-184) and within the third domain (residues 265-296).

[0024] Figure 4 Bar graphs showing thiamine titer (mg / L) or growth (OD) after 24 h of batch cultivation of E. coli strains expressing the native thiamine genes ThiF, ThiS, ThiG, ThiI and P. putida ThiO in a synthetic operon, where ThiI (from E. coli) was co-expressed in WT (BS07082) or truncated (BS07083) form. The truncated forms correspond to Figure 3 The rhodanese domain structure explained in . The bar graph shows the median of 4 replicates and the error bars show the standard deviation.

[0025] Figure 5 Table-top fed-batch fermentations of strains overexpressing ThiI and ThiO or ThiH (fed with HMP) at 31°C and 15% DO are shown. Strain BS06232 contains plasmids encoding ThiFSGH (pBS2819) and ThiI (pBS02896), while BS06285 contains plasmids encoding ThiFSGO (pBS2919) and ThiI (pBS2896). Thiamine was quantified using the thiochrome assay (n=2).

[0026] Figure 6 Table-top fed-batch fermentations of strains overexpressing ThiI and ThiO in the same operon (fed with HMP) at 31°C and 15% DO are shown. Strain BS06232 contains plasmids encoding ThiFSGH (pBS2819) and ThiI (pBS2896), while BS06375 contains only the plasmid encoding ThiFSGIO (pBS2967). Thiamine was quantified using the thiamine assay (n=2).

[0027] Figure 7Table-scale fed-batch fermentations of strains with a native thiamine pathway and overexpressing ThiI (fed with HMP) at 31°C and 15% DO are shown. Strain BS05897 contains a plasmid encoding ThiCEFSGH / ThiMD (pBS140), while BS06245 contains a ThiI plasmid (pBS2896) in addition to pBS140. Thiamine was quantified using the thiochrome assay (n=2).

[0028] Figure 8 Table-top fed-batch fermentations of strains with either native or synthetic thiamine pathways and no added precursors (de novo) are shown at 31°C and 15% DO. Strain BS05897 contains the ThiCEFSGH operon (pBS140), while BS06468 has the ThiFSGIOCE operon (pBS3001). HMPs are the sum of the precursors HMP, HMP-P, and HMP-PP, while THZs are the sum of the precursors THZ, THZ-P, and cTHZ. Thiamine was quantified using the thiochrome assay, while all precursors were quantified by LC-MS.

[0029] Fig. 9 Small-scale experiments using natural (BS05897) or synthetic (BS06468) thiamine pathways were shown, with no precursor added (left) and with 500 μM HMP (middle) or 500 μM HMP+500 μM THZ (right). The strains were cultured for 24 hours at 37°C in a minimum FIT medium (300 μL) plus yeast extract (1 g / L) with 2 g / L glucose. Thiamine was quantified using the thiochrome assay (n=3). In the observation, there was no difference in growth between the two strains with no precursor or only one precursor, while the OD of the natural pathway strain was about 20% higher than the synthetic pathway with two precursors.

[0030] Fig.10 The performance of the different modules of the de novo strain BS06468 with a synthetic thiamine operon is shown. The experiments were performed at 31°C and 15% DO without the addition of precursors (de novo) or with the addition of HMP ( Figure 1 Module 2) or HMP plus THZ ( Figure 1 Table-scale fed-batch fermentation of module 3 of . Precursors were added to give a maximum yield of 15 mg thiamine per gram of glucose. Thiamine was quantified using the thiochrome assay, while thiamine monophosphate (TMP) and thiamine diphosphate (TPP) were quantified by LC-MS (n=1).

[0031] Incorporation by Reference

[0032] All publications, patents, and patent applications mentioned 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 in a terminology herein and that of an incorporated reference, the terminology herein shall control. DETAILED DESCRIPTION

[0033] Provided herein are genetically modified host cells with increased thiamine production achieved by expressing one or more heterologous ThiO enzymes (which have a higher turnover value for converting glycine to dehydroglycine (DHG) than ThiH of E. coli) and one or more heterologous ThiI enzymes (which catalyze the transfer of sulfur from IscS to the sulfur carrier protein ThiS, thereby increasing the level of active ThiS thiocarboxylating protein in the host cell), thereby improving thiamine production in the genetically modified host cell compared to the unmodified parental host cell. In addition, it has been demonstrated that the rhodanese domain of ThiI is sufficient to produce thiamine.

[0034] definition

[0035] Any EC number used herein refers to the NC-IUBMB's Enzyme Nomenclature 1992, Academic Press, San Diego, CA, 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. This nomenclature is supplemented and updated regularly; see, e.g., http: / / enzyme.expasy.org / . The term "PEP" as used herein refers to phosphoenolpyruvate.

[0036] As used herein, the term "thiamine" is used interchangeably with the compound thiamine itself, but may also be used interchangeably with thiamine monophosphate (TMP) and / or thiamine pyrophosphate (TPP) where appropriate.

[0037] The term "TMP-phosphatase" as used herein refers to a thiamine monophosphate phosphatase that dephosphorylates thiamine monophosphate to thiamine. A bifunctional TH2 protein from Arabidopsis has been shown to have this activity (see also WO2017103221).

[0038] As used herein, the term "ThiK" refers to thiamine kinase, which catalyzes the phosphorylation of thiamine to thiamine monophosphate (TMP).

[0039] The term "ThiL" as used herein refers to 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. It has a high specificity for ATP as a phosphate donor.

[0040] As used herein, the term "ThiM" refers to hydroxyethylthiazole kinase, which catalyzes the phosphorylation of the hydroxyl group of 4-methyl-5-β-hydroxyethylthiazole.

[0041] As used herein, the term "ThiD" refers to a hydroxymethylpyrimidine or phosphomethylpyrimidine kinase, which catalyzes the phosphorylation of hydroxymethylpyrimidine phosphate (HMP-P) to HMP-PP, and the phosphorylation of HMP to HMP-P. ThiD is inactive with pyridoxal, pyridoxamine, or pyridoxine.

[0042] 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 riboside (AIR) in a free radical S-adenosyl-L-methionine (SAM)-dependent reaction.

[0043] The term "ThiE" as used herein refers to thiamine phosphate synthase protein, which condenses 4-methyl-5-[2-(phosphono)ethyl]-1,3-thiazole-2-carboxylic acid (cTHZ-P) and 2-methyl-4-amino-5-hydroxymethylpyrimidine pyrophosphate (HMP-PP) to form thiamine monophosphate (TMP).

[0044] The term "ThiF" as used herein refers to the sulfur carrier protein ThiS adenylyltransferase. ThiF catalyzes the adenylation of the carboxyl terminus of ThiS, followed by ThiI-persulfide-catalyzed displacement of AMP to yield ThiS-ThiI acyl disulfide ThiS.

[0045] The term "ThiS" as used herein refers to a sulfur carrier protein, the C-terminal thiocarboxylation of which is carried out in two steps: first acylation and adenylation by ThiF, and then thiocarboxylation by ThiI.

[0046] 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 generate the thiazole phosphate portion of thiamine. Sulfur is provided by the thiocarboxylate portion of the carrier protein ThiS.

[0047] As used herein, the term "ThiH" refers to 2-imidoacetic acid synthase protein, which catalyzes the free radical SAM-mediated cleavage of tyrosine to 2-imidoacetic acid and 4-cresol.

[0048] The term "ThiI" as used herein refers to a tRNA sulfur transferase protein that catalyzes the ATP-dependent transfer of sulfur to tRNA to produce 4-thiouridine at position 8 of tRNA, which functions as a near-UV light sensor. It also catalyzes the transfer of sulfur to the sulfur carrier protein ThiS, forming ThiS-thiocarboxylate. This is a step in the thiazole synthesis in the thiamine biosynthetic pathway. Sulfur is provided by IscS in the form of persulfide.

[0049] The term "Dxs protein" as used herein refers to 1-deoxy-D-xylulose-5-phosphate synthase, which catalyzes the acyloin condensation reaction between pyruvate and C atoms 2 and 3 of glyceraldehyde 3-phosphate to produce 1-deoxy-D-xylulose-5-phosphate (DXP).

[0050] The term "ThiO" as used herein refers to glycine oxidase, which catalyzes the FAD-dependent oxidative deamination of various amines and D-amino acids to generate the corresponding α-keto acids, ammonia / amines, and hydrogen peroxide, including the formation of dehydroglycine from glycine. It is essential for thiamine biosynthesis in organisms that normally lack ThiH, because glycine oxidation catalyzed by ThiO also generates the glycine imine intermediate (dehydroglycine) required for the biosynthesis of the thiamine pyrophosphate thiazole ring.

[0051] The term "IscR" as used herein refers to the HTH-type transcriptional regulator IscR, which regulates the transcription of several operons and genes involved in the biogenesis of Fe-S clusters and Fe-S-containing proteins. The transcriptional repressor of the iscRSUA operon, which is involved in the assembly of Fe-S clusters into Fe-S proteins.

[0052] The term "host cell" refers to any cell type susceptible to transformation, transfection, transduction, etc., with a nucleic acid construct or expression vector comprising a polynucleotide of the present invention. Host cells include any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication.

[0053] The term "polynucleotide construct" refers to a single-stranded or double-stranded polynucleotide isolated from a naturally occurring gene or modified to contain a nucleic acid segment not found in nature, or synthetic, and comprises a polynucleotide encoding a polypeptide and one or more control sequences.

[0054] The terms "nucleotide sequence" and "polynucleotide" are used interchangeably herein.

[0055] The term "coding sequence" refers to a nucleotide sequence that directly specifies the amino acid sequence of a polypeptide. The boundaries of a coding sequence are usually determined by an open reading frame, which begins with a start codon (e.g., ATG, GTG, or TTG) and ends with a stop codon (e.g., TAA, TAG, or TGA). The coding sequence can be genomic DNA, cDNA, synthetic DNA, or a combination thereof.

[0056] The term "control sequence" as used herein refers to the nucleotide sequence necessary for the expression of a polynucleotide encoding a polypeptide. The control sequence can be a native sequence (i.e., from the same gene or organism) or a heterologous sequence or an exogenous sequence (i.e., from a different gene or organism) of a polynucleotide encoding a polypeptide. Control sequences include, but are not limited to, a leader sequence, a polyadenylation sequence, a propeptide coding sequence, a promoter sequence, a signal peptide coding sequence, a translation terminator (stop) sequence, and a transcription terminator (stop) sequence. In order to operate, the control sequence must generally include a promoter sequence and transcription and translation termination signals. The control sequence can be provided together with a joint to introduce a specific restriction site, thereby facilitating the connection of the control sequence to the coding region of the polynucleotide encoding the polypeptide.

[0057] The term "expression vector" refers to a single-stranded or double-stranded, linear or circular DNA molecule that contains a polynucleotide encoding a polypeptide and is operably linked to a control sequence that provides its expression. Expression vectors include expression cassettes for integrating genes into host cells and plasmids and / or chromosomes containing such genes.

[0058] The term "expression" includes any step involved in the production of the polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0059] The term "overexpression" includes situations where one or more components of a cell may be present at higher than normal cellular levels (i.e., higher concentrations known to be normally present in the cell type expressing the gene and / or protein complex of interest). For example, a gene encoding a protein may become overexpressed, or may be amplified in certain cells (i.e., its gene copy number may increase), resulting in an increase in the number of component molecules within these cells.

[0060] As used herein, the terms "heterologous" or "recombinant" or "genetically modified" and their grammatical equivalents interchangeably refer to entities "derived from a different species or cell". For example, a heterologous or recombinant polynucleotide gene is a gene that is not naturally present in the host cell, i.e., the gene is from a species or cell type different from the host cell. As used herein, the terms referring to microbial host cells refer to microbial host cells that contain and express heterologous or recombinant polynucleotide genes.

[0061] The term "metabolic pathway" as used herein is intended to mean that two or more enzymes act sequentially in a living cell to convert a chemical substrate into a chemical product. The enzyme is characterized by catalytic activity, which can change the chemical structure of the substrate. The enzyme can have a variety of substrates and produce a variety of products. The enzyme can also rely on cofactors, which can be inorganic chemical compounds or organic compounds, such as proteins, such as enzymes (coenzymes). Reducing the CPR of certain cytochrome P450 enzymes is an example of an enzyme cofactor. The term "effective biosynthetic metabolic pathway" refers to a metabolic pathway that occurs in a living recombinant host, as described herein.

[0062] As used herein, the term "in vivo" means within a living cell or organism, including, for example, an animal, a plant, or a microorganism.

[0063] As used herein, the term "in vitro" means outside a living cell or organism, including but not limited to, for example, in a microtiter plate, test tube, flask, beaker, tank, reactor, and the like.

[0064] As used herein, the term "substrate" or "precursor" refers to any compound that can be converted into a different compound. For example, Air can be a substrate for phosphomethylpyrimidine synthase and can be converted into thiamine, i.e., hydroxymethylpyrimidine phosphate (HMP-P), a precursor of thiamine. For clarity, substrates and / or precursors include compounds generated in situ by enzymatic reactions in cells or exogenously provided compounds, such as exogenously provided organic molecules, which host cells can metabolize into desired compounds.

[0065] As used herein, the term "endogenous" or "native" refers to a gene or polypeptide in a host cell that originates from the same host cell.

[0066] As used herein, the term "deletion" refers to the manipulation of a gene so that it is no longer expressed in the host cell.

[0067] As used herein, the term "attenuation" refers to the manipulation of a gene or any mechanism involved in gene expression such that the expression of the gene is reduced compared to the expression in the absence of the manipulation.

[0068] The term "substantially" or "approximately" or "about" as used herein refers to a reasonable deviation around a value or parameter so that the value or parameter does not change significantly. These deviation terms should be interpreted as including deviations of values, wherein the deviation does not negate the meaning of the value of the deviation. For example, with respect to a reference value, a degree term may include a range of values ​​of the value plus or minus 10%. For example, a deviation may include a specified value plus or minus a certain percentage of the value, such as plus or minus 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% of the specified value.

[0069] As used herein, the term "and / or" is intended to mean an inclusive "or". The phrase X and / or Y is intended to mean X or Y as well as X and Y. Furthermore, the phrase X, Y and / or Z is intended to mean X, Y and Z individually or in any combination of X, Y and Z.

[0070] The term "isolated" used herein with respect to compounds refers to any compound that is placed in a form or environment different from the form or environment in which it exists in nature by human intervention. Isolated compounds include, but are not limited to, compounds disclosed herein, wherein the compound is increased or decreased relative to the ratio of other components with which it is associated in nature. In an important embodiment, the amount of the compound is increased relative to other components with which the compound is associated in nature. In one embodiment, the compounds disclosed herein can be separated into pure or substantially pure forms. In this case, substantially pure compounds refer to compounds that are separated from other foreign or unwanted materials that exist from the beginning of the production of the compound or that are produced during the manufacturing process. This substantially pure compound preparation contains less than 10%, for example, less than 8%, for example, less than 6%, for example, less than 5%, for example, less than 4%, for example, less than 3%, for example, less than 2%, for example, less than 1%, for example, less than 0.5% of weight percentage of other foreign or unwanted substances that are usually associated with natural or recombinantly expressed compounds. In one 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).

[0071] The term "% identity" as used herein refers to the relationship between two amino acid sequences or two nucleotide sequences. For sequence comparison, usually one sequence is used as a reference sequence to which a test sequence is compared. When a sequence comparison algorithm is used, the test sequence and the reference sequence are input into a computer, subsequence coordinates are specified (if necessary), and sequence algorithm program parameters are specified. "% identity" for amino acid sequences as used herein refers to the percent identity between two amino acid sequences obtained when using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453), which is implemented in the Needle program of the EMBOSS software package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), preferably version 5.0.0 or higher. The parameters used are a gap open penalty of 10, a gap extension penalty of 0.5, and an EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The Needle output labeled "longest identity" (obtained using the -nobrief option) was used as the percent identity, calculated as follows:

[0072]

[0073] The term "% identity" used herein for nucleotide sequences refers to the percentage identity between two deoxyribonucleotide sequences obtained using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, above) implemented in the Needle program of the EMBOSS software package (The European Molecular Biology Open Software Suite, Rice et al., 2000, above), preferably 5.0.0 or higher versions. The parameters used are gap opening penalty 10, gap extension penalty 0.5 and EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The label "longest identity" (obtained using the -nobrief option) output by Needle is used as percentage identity, and the calculation method is as follows:

[0074]

[0075] Other examples of algorithms suitable for determining percent sequence identity and sequence similarity include the BLAST and BLAST 2.0 algorithms 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)).

[0076] The protein sequences of the invention can also be used as "query sequences" to search sequence databases, for example, to identify other family members or related sequences. Such searches can be performed using the BLAST program. Software for performing BLAST analysis 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 is used for nucleotide sequences. The BLAST program uses the default values:

[0077] Cost of opening a gap: default = 5 nucleotides / 11 protein

[0078] Cost of extending a gap: default = 2 nucleotides / 1 protein

[0079] Penalty for nucleotide mismatch: default value = -3

[0080] Reward for nucleotide match: default value = 1

[0081] Expected value: Default value = 10

[0082] Word length: default = nucleotide 11 / meg 28 / protein 3.

[0083] In addition, the degree of local identity between an amino acid sequence query or a nucleic acid sequence query and a retrieved homologous sequence is determined by the BLAST program. However, only sequence fragments with a matching rate above a certain threshold are compared. Therefore, the program only calculates the identity of these matching fragments. Therefore, the identity calculated in this way is called local identity.

[0084] The term "mature polypeptide" or "mature enzyme" as used herein refers to a polypeptide in its final active form after translation and any post-translational modifications (e.g., N-terminal processing, C-terminal truncation, glycosylation, phosphorylation, etc.). It is known in the art that a host cell can produce a mixture of two or more different mature polypeptides (i.e., having different C-terminal and / or N-terminal amino acids) expressed by the same polynucleotide.

[0085] The term "cDNA" refers to a DNA molecule that can be prepared by reverse transcription from a mature spliced ​​mRNA molecule 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 and is processed through a series of steps, including splicing, before it can appear as mature spliced ​​mRNA.

[0086] The terms "include" and "comprising" and variations such as "include", "including", "comprising" and "comprising" used throughout the specification and appended items should be interpreted as inclusive. These words are intended to convey that additional elements or integers not specifically listed may be included as long as the context permits.

[0087] The articles "a" and "an" are used herein to refer to one or more than one (ie, one or at least one) of the grammatical object of the article. For example, "an element" can mean one element or a plurality of elements.

[0088] Terms such as "preferably," "usually," "particularly," and "typically" are not used herein to limit the scope of the subject invention or to imply that certain features are critical, necessary, or even important to the structure or function of the subject invention. Instead, these terms are merely intended to highlight alternative or additional features that may or may not be utilized in a particular embodiment of the invention.

[0089] The term "cell culture" as used herein refers to a culture medium comprising a plurality of host cells disclosed herein. The cell culture may include a single host cell strain or may include two or more different host cell strains. The culture medium may be any culture medium that may include a recombinant host, such as a liquid culture medium (i.e., a culture solution) or a semisolid culture medium, and may include additional components, such as one or more of the following: (i) trace metals; (ii) vitamins; (iii) salts (e.g., phosphates, magnesium salts, potassium salts, zinc salts, iron salts); (iv) nitrogen sources (e.g., YNB, ammonium sulfate, urea, yeast extract, ammonium nitrate, ammonium chloride, malt extract, peptone and / or amino acids); (v) carbon sources (e.g., dextrose, sucrose, glycerol, glucose, maltose, molasses, starch, cellulose, xylan, pectin, lignocellulose hydrolysate and / or acetate); (vi) nucleobases; (vii) aminoglycosides; and / or (viii) antibiotics (e.g., G418 and hygromycin B).

[0090] As used herein, the term "radical SAM" refers to a SAM prepared using [4Fe-4S] +A superfamily of enzymes that reductively cleave S-adenosyl-L-methionine (SAM) to generate a free radical (usually 5'-deoxyadenosine radical) as a key intermediate. The vast majority of known free radical SAM enzymes have a cysteine-rich motif that matches or is similar to CxxxCxxC.

[0091] Genetically modified host cells

[0092] The genetically modified host cells provided herein have increased thiamine production, in some embodiments, they express one or more heterologous ThiO enzymes, which convert glycine in the host cell into dehydroglycine (DHG), thereby improving thiamine production in the genetically modified host cell compared to the unmodified parental host cell. The genetically modified host cells provided herein have increased thiamine production, which express one or more other heterologous ThiI enzymes, which catalyze the transfer of sulfur from IscS to the sulfur carrier protein ThiS in the host cell, thereby improving thiamine production in the genetically modified host cell compared to the unmodified parental host cell.

[0093] In some embodiments, the increase in the ability of the genetically modified host cell to produce thiamine can be at least 50%, such as at least 100%, such as at least 150%, such as at least 200%. In other embodiments, the genetic modification of the host cell with increased thiamine production provided herein includes one or more mutations in a natural polynucleotide construct encoding one or more proteins, the protein selected from the group consisting of protein cAMP receptor protein (CRP), carbohydrate inhibition resistance protein (CRR) and adenylate cyclase protein (CyaA), as described in patent application PCT / EP2022 / 069711.

[0094] In some embodiments, the genetically modified host cell with increased thiamine production is a cell in which one or more heterologous ThiO enzymes are bacterial ThiO enzymes. In another embodiment, the genetically modified host cell with improved thiamine production is a cell in which one or more heterologous ThiI enzymes are bacterial ThiI enzymes. In a further embodiment, the genetically modified host cell with increased thiamine production is a cell in which one or more heterologous bacterial ThiO enzymes are BsThiO enzymes from Bacillus subtilis and / or PpThiO enzymes from Pseudomonas putida. In other embodiments, the heterologous bacterial ThiO enzymes from Bacillus subtilis and / or PpThiO enzymes from Pseudomonas putida are 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 ThiO enzymes contained in SEQ ID NO 27 and SEQ ID NO 29. In one embodiment, the bacterial ThiI enzyme is an E. coli ThiI enzyme having SEQ ID NO: 23. In another embodiment, the bacterial ThiO gene is a BsThiO gene from Bacillus subtilis having SEQ ID NO: 28 and / or a PpThiO gene from Pseudomonas putida having SEQ ID NO: 30.

[0095] In some embodiments, the gene encoding:

[0096] a) the ThiO enzyme 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 enzyme contained in SEQ ID NO: 27 or 29 or the genomic DNA thereof encoding the ThiO enzyme contained in SEQ ID NO: 28 or 30;

[0097] b) the ThiI enzyme 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 enzyme contained in SEQ ID NO:23 or the genomic DNA encoding the ThiI enzyme contained in SEQ ID NO:24.

[0098] In yet another embodiment, said:

[0099] a) the BsThiO 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 BsThiO gene contained in SEQ ID NO: 28;

[0100] b) the PpThiO 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 PpThiO gene contained in SEQ ID NO:30.

[0101] In one embodiment, the ThiI gene is a truncated ThiI gene comprising a rhodanese domain that transfers sulfur from IscS as persulfide to the sulfur carrier protein ThiS, forming ThiS-thiocarboxylic acid in the thiamine biosynthetic pathway. In a further embodiment, the rhodanese domain 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 rhodanese domain contained in SEQ ID NO: 34. In another embodiment, the ThiI enzyme is a truncated ThiI enzyme comprising a rhodanese domain that transfers sulfur from IscS as persulfide to the sulfur carrier protein ThiS, thereby forming ThiS-thiocarboxylic acid in the thiamine biosynthetic pathway. In a further embodiment, the rhodanese domain 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 rhodanese domain contained in SEQ ID NO:33.

[0102] In some embodiments, the genetically modified host cell also includes an effective metabolic pathway that includes one or more natural or heterologous pathway elements that produce thiamine. In one embodiment, one or more pathway elements include one or more free radical SAM enzymes. In another embodiment, one or more pathway elements are selected from:

[0103] a) one or more phosphate synthases selected from phosphomethylpyrimidine synthases (ThiC); which catalyze the synthesis of the hydroxymethylpyrimidine phosphate (HMP-P) moiety of thiamine from aminoimidazole riboside (AIR) in a free radical S-adenosyl-L-methionine (SAM)-dependent reaction;

[0104] b) hydroxymethylpyrimidine / methylpyrimidine phosphate kinase (ThiD), which catalyzes the phosphorylation of hydroxymethylpyrimidine phosphate (HMP-P) to HMP-PP and the phosphorylation of HMP to HMP-P;

[0105] c) adenylyltransferase (ThiF), catalyzing the adenylation of the carboxyl terminus of the sulfur carrier protein ThiS, followed by the replacement of AMP by ThiI-persulfide to yield ThiS-ThiI acyl disulfide ThiS;

[0106] d) ThiS, the C-terminal thiocarboxylation is carried out in two steps: first, acyladenylation by ThiF, then thiocarboxylation by ThiI;

[0107] e) conversion by 2-imidoacetic acid synthase (ThiH), catalyzing the free radical-mediated cleavage of tyrosine into 2-imidoacetic acid and 4-cresol;

[0108] f) Thiazole synthase (ThiG), catalyzes the rearrangement of 1-deoxy-D-xylulose 5-phosphate (DXP) to produce the thiazole phosphate moiety of thiamine;

[0109] g) hydroxyethylthiazole kinase (ThiM), catalyzes the rearrangement of 1-deoxy-D-xylulose 5-phosphate (DXP) to generate the thiazole phosphate moiety of thiamine;

[0110] h) thiamine monophosphate (TMP) phosphatase, which dephosphorylates thiamine monophosphate to thiamine;

[0111] i) thiamine kinase (ThiK), catalyzing the phosphorylation of thiamine to thiamine phosphate;

[0112] j) Thiamine monophosphate kinase (ThiL), catalyzes the ATP-dependent phosphorylation of thiamine monophosphate (TMP) to form thiamine pyrophosphate (TPP);

[0113] k) Thiamine phosphate synthase (ThiE), which condenses 4-methyl-5-[2-(phosphonoyloxy)ethyl]-1,3-thiazole-2-carboxylic acid (cTHZ-P) and 2-methyl-4-amino-5-hydroxymethylpyrimidine pyrophosphate (HMP-PP) to form thiamine monophosphate (TMP);

[0114] l) HTH-type transcriptional regulator (IscR), which regulates the transcription of several operons and genes involved in the biosynthesis of Fe-S clusters and Fe-S-containing proteins.

[0115] In further embodiments, the one or more pathway elements are encoded by one or more genes selected from the group consisting of:

[0116] a) ThiC, which is at least 70%, such as 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% identical to the ThiC contained in SEQ ID NO: 2;

[0117] b) a ThiD which is at least 70%, such as 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% identical to the ThiD contained in SEQ ID NO: 4;

[0118] c) ThiF, which is at least 70%, such as 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% identical to the ThiF contained in SEQ ID NO: 6;

[0119] d) ThiS, which is at least 70%, such as 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% identical to the ThiS contained in SEQ ID NO: 8;

[0120] e) a ThiH which is at least 70%, such as 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% identical to the ThiH contained in SEQ ID NO: 10;

[0121] f) ThiG, which is at least 70%, such as 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% identical to the ThiG contained in SEQ ID NO: 12;

[0122] g) ThiM, which is at least 70%, such as 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% identical to the ThiM contained in SEQ ID NO: 14;

[0123] h) a TMP phosphatase that is at least 70%, such as 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% identical to the TMP phosphatase comprised in SEQ ID NO: 16;

[0124] i) ThiK, which is at least 70%, such as 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% identical to the Thik contained in SEQ ID NO: 18;

[0125] j) a ThiL which is at least 70%, such as 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% identical to the ThiL comprised in SEQ ID NO: 20;

[0126] k) ThiE, which is at least 70%, such as 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% identical to the ThiE contained in SEQ ID NO: 22;

[0127] 1) an IscR that is at least 70%, such as 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% identical to the IscR comprised in SEQ ID NO:32.

[0128] In some embodiments, one or more pathway elements are encoded by one or more genes selected from the group consisting of:

[0129] a) a gene that 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 contained in SEQ ID NO: 2 or its genomic DNA encoding the ThiC enzyme contained in SEQ ID NO: 1;

[0130] b) a gene that 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 contained in SEQ ID NO:4 or its genomic DNA encoding the ThiD enzyme contained in SEQ ID NO:3;

[0131] c) a gene that 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 contained in SEQ ID NO: 6 or its genomic DNA encoding the ThiF enzyme contained in SEQ ID NO: 5;

[0132] d) a gene that 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 contained in SEQ ID NO: 8 or its genomic DNA encoding the ThiS enzyme contained in SEQ ID NO: 7;

[0133] e) a gene that 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 contained in SEQ ID NO: 10 or its genomic DNA encoding the ThiH enzyme contained in SEQ ID NO: 9;

[0134] f) a gene that 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 contained in SEQ ID NO: 12 or its genomic DNA encoding the ThiG enzyme contained in SEQ ID NO: 11;

[0135] g) a gene that 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 contained in SEQ ID NO: 14 or its genomic DNA encoding the ThiM enzyme contained in SEQ ID NO: 13;

[0136] h) a gene that 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 contained in SEQ ID NO: 16 or its genomic DNA encoding the TMPase phosphatase contained in SEQ ID NO: 15;

[0137] i) a gene that 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 contained in SEQ ID NO: 18 or its genomic DNA encoding the ThiK enzyme contained in SEQ ID NO: 17;

[0138] j) a gene that 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 contained in SEQ ID NO: 20 or its genomic DNA encoding the ThiL enzyme contained in SEQ ID NO: 19;

[0139] k) a gene that 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 contained in SEQ ID NO: 22 or its genomic DNA encoding the ThiE enzyme contained in SEQ ID NO: 23;

[0140] l) a gene that 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 contained in SEQ ID NO:24 or its genomic DNA encoding the ThiI enzyme contained in SEQ ID NO:23;

[0141] m) a gene that 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 contained in SEQ ID NO:32 or its genomic DNA encoding the IscR enzyme contained in SEQ ID NO:31.

[0142] In some embodiments, the genetically modified host cell is a cell in which the endogenous natural ThiH gene is deleted, destroyed and / or weakened. In a further embodiment, the deletion, destruction and / or weakening is caused by introducing a deletion by completely removing the gene or by introducing one or more stop codons or frameshift mutations to cause translation knockout, thereby preventing the expression of active peptides. In another embodiment, the deletion, destruction and / or weakening includes translation knockout or frameshift mutations. In another embodiment, the deletion, destruction and / or weakening includes translation knockout or frameshift mutations. In a further embodiment, the deletion, destruction and / or weakening is a point mutation in the promoter, RBS region and / or protein coding sequence of the protein coding sequence.

[0143] In some embodiments, one or more genes and / or polypeptides of the thiamine pathway 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 thiamine pathway.

[0144] In another embodiment, the host cell further comprises a transport molecule that promotes the transport of a precursor or product of the thiamine pathway. In one embodiment, the host cell is further genetically modified to provide an increased amount of substrate in the thiamine pathway. In another embodiment, the host cell is further genetically modified to show increased tolerance to one or more substrates, intermediates or products in the thiamine pathway. In a further embodiment, one or more additional natural or endogenous genes of the host cell are deleted, destroyed and / or weakened. In a further embodiment, one or more genes in the thiamine pathway are overexpressed. In another embodiment, the host cell is further genetically modified to provide an increased amount of substrate in the thiamine pathway.

[0145] Host cells

[0146] The host cell disclosed herein can be any host cell suitable for carrying and expressing the thiamine pathway. Such cells can be prokaryotic or eukaryotic cells. Suitable prokaryotic host cells can be selected from Escherichia coli, Bacillus, Brevibacterium, Burkholderia, Campylobacter, Corynebacterium, Serratia, Lactobacillus, Lactococcus, Acinetobacter, Acetobacter or Pseudomonas putida. Particularly useful prokaryotic host cells are Escherichia coli, Corynebacterium, Bacillus, Serratia or Pseudomonas putida, such as Escherichia coli, Corynebacterium glutamicum, Bacillus subtilis, Serratia marcescens, Pseudomonas putida and / or Pseudomonas putida variabilis. Useful eukaryotic host cells include mammalian, insect, plant, fungal or archaeal cells. Among eukaryotic host cells, fungal cells of the genera Saccharomyces, Kluyveromyces, Candida, Pichia, Debaryomyces, Hansenula, Yarrowia, Zygosaccharomyces, Schizosaccharomyces, and Ashwagandha are particularly useful, such as Kluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Kluyveromyces, Saccharomyces benthamiana, Saccharomyces oviformis, Yarrowia lipolytica, Pichia pastoris, or Ashwagandha.

[0147] Polynucleotide constructs and expression vectors

[0148] Also provided herein are polynucleotide constructs containing genes encoding natural or mutant ThiC, ThiD, ThiF, ThiS, ThiH, ThiG, ThiM, ThiE, TMP phosphatase, ThiK, ThiL, ThiI, IscR and ThiO genes, the polynucleotide constructs being operably linked to one or more control sequences, the polynucleotide constructs comprising mutations to delete, disrupt and / or attenuate gene transcription or translation or the activity and / or function of the encoded gene. The control sequences direct the expression of the encoded ThiC, ThiD, ThiF, ThiS, ThiH, ThiG, ThiM, ThiE, TMP phosphatase, ThiK, ThiL, ThiI, IscR or ThiO genes in a host cell containing the polynucleotide constructs. The expression conditions should be compatible with the control sequences. The control sequence can be a gene encoding a ThiC, ThiD, ThiF, ThiS, ThiH, ThiG, ThiM, ThiE, TMP phosphatase, ThiK, ThiL, ThiI, IscR or ThiO gene and / or a gene heterologous or native to the host cell. In some embodiments, the control sequence and the gene encoding ThiC, ThiD, ThiF, ThiS, ThiH, ThiG, ThiM, ThiE, TMP phosphatase, ThiK, ThiL, ThiI, IscR or ThiO are both heterologous to the host cell and are optionally heterologous to each other. In one embodiment, the polynucleotide construct is an expression vector comprising a gene encoding ThiC, ThiD, ThiF, ThiS, ThiH, ThiG, ThiM, ThiE, TMP phosphatase, ThiK, ThiL, ThiI, IscR or ThiO operably linked to one or more control sequences.

[0149] The polynucleotides can be manipulated in a variety of ways to alter the expression of ThiC, ThiD, ThiF, ThiS, ThiH, ThiG, ThiM, ThiE, TMP phosphatase, ThiK, ThiL, ThiI, IscR 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.

[0150] The control sequence can be a promoter, which is a polynucleotide that is recognized by a host cell to express a polynucleotide. The promoter comprises a transcriptional control sequence that mediates the expression of a polypeptide. The promoter can be any polynucleotide that shows transcriptional activity in a host cell, including mutant, truncated and hybrid promoters, and can be obtained from genes encoding extracellular or intracellular polypeptides that are homologous or heterologous to the host cell. The promoter can also be an inducible promoter. The selection of promoters suitable for expression in yeast is well known and fully understood by those skilled in the art.

[0151] The control sequence can also be a transcription terminator, which is recognized by the host cell to terminate transcription. The terminator is operably connected to the 3' end of the polynucleotide encoding the polypeptide. Any terminator that is functional in the host cell can be used. The control sequence can also be an mRNA stabilizing region downstream of the promoter and upstream of the gene coding sequence, which increases the expression of the gene.

[0152] The control sequence can also be a leader sequence, i.e., an untranslated region of an mRNA that is important for translation in the host cell. The leader sequence is operably linked to the 5' end of the polynucleotide encoding the polypeptide. Any leader sequence that is functional in the host cell can be used.

[0153] The control sequence may also be a polyadenylation sequence; a sequence operably linked to the 3' terminus of a polynucleotide which, when transcribed, is recognized by the host cell as a signal to add polyadenylic acid residues to transcribed mRNA. Any polyadenylation sequence that functions in the host cell may be used.

[0154] 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 or ThiO genes relative to host cell growth. Examples of regulatory systems are those that cause gene expression to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound.

[0155] In addition to the polynucleotide constructs disclosed herein, various nucleotide sequences can be linked together to produce a recombinant expression vector, which may include one or more convenient restriction sites to allow insertion or replacement of the polynucleotide sequence encoding the ThiC, ThiD, ThiF, ThiS, ThiH, ThiG, ThiM, ThiE, TMP phosphatase, ThiK, ThiL, ThiI, IscR or ThiO gene at these sites. The recombinant expression vector may be any vector (e.g., a plasmid or virus or chromosome) that can be conveniently subjected to recombinant DNA procedures and can result in the expression of the ThiC, ThiD, ThiF, ThiS, ThiH, ThiG, ThiM, ThiE, TMP phosphatase, ThiK, ThiL, ThiI, IscR or ThiO gene. The choice of vector generally depends 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, such as a plasmid (linear or closed circular plasmid), an extrachromosomal element, a minichromosome or an artificial chromosome. The vector may comprise any means for ensuring self-replication. Alternatively, the vector may be integrated into the genome when introduced into the host cell and replicated together with the chromosome into which it is integrated. In addition, a single vector or plasmid or two or more vectors or plasmids (which together comprise the total DNA to be introduced into the host cell genome) or a transposon may be used.

[0156] The vector may contain one or more selectable markers, allowing easy selection of transformed, transfected, transduced or similar cells. A selectable marker is a gene whereby the product provides biocide or virus resistance, heavy metal resistance, prototrophy to auxotrophy, etc.

[0157] The vector can also include elements that allow the vector to be integrated into the host cell genome (itself being a vector) or allow the vector to replicate autonomously in the cell independently of the genome. Alternatively, the vector can include additional polynucleotides for guiding the integration of the precise position in the chromosome into the host cell genome by homologous recombination. In order to increase the possibility of integration at the precise position, the integration element should include a sufficient number of nucleic acids, such as 100 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 with the corresponding target sequence, to improve the possibility of homologous recombination. The integration element can be any sequence homologous to the target sequence in the host cell genome. In addition, the integration element can be a non-coding or encoded polynucleotide. On the other hand, the vector can be integrated into the genome of the host cell by non-homologous recombination.

[0158] For autonomous replication, the vector may also 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 replicon that mediates autonomous replication that functions in the cell. The term "origin of replication" or "plasmid replicon" refers to a polynucleotide that enables a plasmid or vector to replicate in vivo.

[0159] As described above, more than one copy of a gene encoding a thiamine pathway element can be inserted into a host cell to increase thiamine production. An increase in gene copy number can be obtained by integrating one or more additional copies of the gene into the host cell genome or by including an amplifiable selectable marker gene with the gene, such that cells containing amplified copies of the selectable marker gene (and thus additional copies of the polynucleotide) can be selected by culturing the cells in the presence of an appropriate selection agent. The procedures used to connect the above elements to construct recombinant expression vectors of the present disclosure are well known to those skilled in the art (see, e.g., Sambrook et al., 1989, supra).

[0160] In a separate embodiment, a host cell comprises a polynucleotide construct and / or a vector as disclosed herein.

[0161] Culture

[0162] Further provided herein are cell cultures comprising the genetically modified host cells disclosed herein and a growth medium. Suitable growth media for relevant prokaryotic or eukaryotic host cells are well known in the art.

[0163] A method of producing a chemical compound.

[0164] The present disclosure also describes a method of producing thiamine, comprising

[0165] a) culturing a cell culture as disclosed herein under conditions that allow the host cells to produce thiamine; and

[0166] b) optionally recovering and / or isolating the thiamine.

[0167] The cell culture can be cultured in a nutrient medium under conditions suitable for producing the thiamine disclosed herein and / or increasing the cell count using methods known in the art. For example, the culture can be cultured by shake flask culture or by small-scale or large-scale fermentation (including continuous fermentation, batch fermentation, fed-batch fermentation or solid-state fermentation) in a suitable medium under conditions that allow the growth and / or propagation of the host cells in a laboratory or industrial fermentor, and optionally recovering and / or isolating the host cells.

[0168] Cultivation can be carried out in a suitable nutrient medium comprising a carbon source and a nitrogen source and an inorganic salt, using procedures known in the art. Suitable culture medium can be obtained from commercial suppliers, or can be prepared according to published formulas (e.g., from the catalog of the American Type Culture Collection). The selection of appropriate culture medium can be based on the selection of host cells and / or based on the regulatory requirements of host cells. Such culture medium is available in the art. If desired, the culture medium can contain additional components that are conducive to the conversion of the expression host rather than other potential contamination microorganisms. Thus, in one embodiment, a suitable nutrient medium may include one or more of the following: (i) trace metals; (ii) vitamins; (iii) salts (e.g., phosphates, magnesium salts, potassium salts, zinc salts, iron salts); (iv) nitrogen sources (e.g., YNB, ammonium sulfate, urea, yeast extract, ammonium nitrate, ammonium chloride, malt extract, peptone and / or amino acids); (v) carbon sources (e.g., glucose, sucrose, glycerol, glucose, maltose, molasses, starch, cellulose, xylan, pectin, lignocellulosic hydrolyzate and / or acetate); (vi) nucleobases; (vii) aminoglycosides; and / or (viii) antibiotics (e.g., G418 and hygromycin B).

[0169] The host cell can be cultured over a period of about 0.5 to about 30 days. The culture process can be a batch process, a continuous process or a fed-batch process, suitably at a temperature in the range of 0-100°C or 0-80°C, for example, about 0°C to about 50°C and / or a pH of about 2 to about 10. Preferred fermentation conditions are a temperature in the range of about 25°C to about 55°C and a pH of about 3 to about 9. Appropriate conditions are generally selected based on the choice of the host cell. Therefore, in some embodiments, the methods disclosed herein include one or more elements selected from the following:

[0170] a) Cultivation of cell cultures under aerobic or anaerobic conditions

[0171] b) culturing the host cell under mixing;

[0172] c) culturing the host cell at a temperature between 25°C and 50°C;

[0173] d) culturing the host cell at a pH of 3-9; and

[0174] e) culturing the host cells for 10 hours to 120 days.

[0175] The cell cultures disclosed herein can be recovered and / or separated using methods known in the art. For example, the compound can be recovered from the nutrient medium by conventional procedures, including but not limited to centrifugation, filtration, spray drying or lyophilization. In certain embodiments, the method includes a recovery and / or separation step, comprising separating the liquid phase of the cell culture from the solid phase of the cell culture to obtain a supernatant containing thiamine, and subjecting the supernatant to one or more steps selected from the following:

[0176] a) contacting the supernatant with one or more adsorption resins to obtain at least a portion of the thiamine produced, then optionally recovering the thiamine from the resins in a concentrated solution, and then isolating the thiamine by crystallization or solvent evaporation;

[0177] b) contacting the supernatant with one or more ion exchange or reverse phase chromatography columns to obtain at least a portion of the thiamine, and then optionally recovering the thiamine from the resin in a concentrated solution, and then isolating the thiamine by crystallization or solvent evaporation;

[0178] c) extracting the thiamine from the supernatant, for example by liquid-liquid extraction into an immiscible solvent, and then optionally isolating the thiamine by crystallization or solvent evaporation; and

[0179] Thiamine is thereby recovered and / or isolated.

[0180] The methods disclosed herein provide thiamine production that is generally higher than when thiamine is produced by methods using host cells without reducing the formation of CRP-cAMP complexes and / or increasing degradation and / or reducing cAMP binding, in some embodiments at least 10% higher, such as at least 50% higher, such as at least 100% higher, such as at least 150% higher, such as at least 200% higher.

[0181] In one embodiment, the thiamine yield and / or titer obtained by the methods disclosed herein is at least 50 mg / L to 800 mg / L thiamine. In other embodiments, the thiamine 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.

[0182] The methods disclosed herein may also include one or more steps of mixing thiamine with one or more carriers, agents, adjuvants, additives and / or excipients, optionally pharmaceutical grade carriers, agents, adjuvants, additives and / or excipients.

[0183] The methods disclosed herein may also include one or more in vitro steps in the process of producing thiamine. It may also include one or more in vivo steps performed in another cell other than the host cell disclosed herein. For example, precursors and / or intermediates in the thiamine pathway may be produced and separated from another cell and then fed to the cell culture disclosed herein to be converted into thiamine. Therefore, in one embodiment, the methods disclosed herein also include feeding one or more exogenous thiamine precursors to the host cell culture. In a preferred embodiment, feeding one or more exogenous thiamine precursors to the host cell culture includes feeding THZ, HMP or THZ to the cell culture.

[0184] Fermentation composition

[0185] The present disclosure also describes a fermentation composition comprising a cell culture disclosed herein and thiamine - contained in the cells or in the culture medium. In the fermentation composition, the genetically modified host cells can be lysed and / or disintegrated in whole or in part. 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 decomposed. In addition, 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 the solid cell material may have been separated and / or removed from the liquid phase of the fermentation composition.

[0186] The fermentation composition may also contain one or more compounds

[0187] a) a precursor or product of an efficient metabolic pathway that produces thiamine;

[0188] b) nutritional supplements, including; and

[0189] Wherein the concentration of thiamine is at least 1 mg / L composition. Specifically, the fermentation composition may comprise thiamine in a concentration 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.

[0190] Suitable supplemental nutrients may include one or more of the following: (i) trace metals; (ii) vitamins; (iii) salts (e.g., phosphates, magnesium salts, potassium salts, zinc salts, and iron salts); (iv) nitrogen sources (e.g., YNB, ammonium sulfate, urea, yeast extract, ammonium nitrate, ammonium chloride, malt extract, peptone, and / or amino acids); (v) carbon sources (e.g., dextrose, sucrose, glycerol, glucose, maltose, molasses, starch, cellulose, xylan, pectin, lignocellulose hydrolysate, and / or acetate); (vi) nucleobases; (vii) aminoglycosides; and / or (viii) antibiotics (e.g., G418, hygromycin B, spectinomycin, and / or kanamycin).

[0191] Composition and use

[0192] The disclosure also describes a composition comprising a fermentation composition disclosed herein and one or more carriers, reagents, adjuvants, additives and / or excipients. Suitable carriers, reagents, adjuvants, additives and / or excipients include formulation additives, stabilizers, fillers, etc. The composition and one or more carriers, reagents, adjuvants, additives and / or excipients can be suitably formulated into dry solid forms, such as by using methods known in the art, such as spray drying, spray cooling, freeze drying, quick freezing, granulation, micronization, encapsulation or microencapsulation. Compositions and one or more carriers, medicaments, adjuvants, additives and / or excipients can also be formulated into liquid stable forms using methods known in the art, such as adding one or more stabilizers, such as sugar and / or polyols (e.g., sugar alcohols) and / or organic acids (e.g., lactic acid) to the fermentation composition.

[0193] The compositions disclosed herein can be further refined into pharmaceutical preparations, dietary supplements, cosmetics, food preparations, feed preparations and / or analytical or diagnostic reagents, optionally using one or more steps of the method for producing thiamine described herein, including mixing thiamine with one or more pharmaceutical grade carriers, medicaments, adjuvants, additives and / or excipients. In one embodiment, the pharmaceutical composition is a pharmaceutical preparation obtainable by the method disclosed herein. The pharmaceutical preparation can be a dry preparation, optionally in the form of a powder, tablet, capsule, hard chewable tablet and / or soft tablet or chewing gum. Alternatively, the pharmaceutical preparation can be in the form of a liquid pharmaceutical solution. Such pharmaceutical preparations can be used as a medicament in a method for treating and / or alleviating a disease and / or medical condition, particularly for treating a disease and / or medical condition in a mammal, particularly for treating a nutritional deficiency. Therefore, the present disclosure further describes a method for preventing, treating and / or alleviating a disease and / or medical condition, comprising administering a therapeutically effective amount of a pharmaceutical composition disclosed herein to a mammal in need of treatment and / or alleviating. Diseases and / or medical conditions that can be treated or alleviated by a pharmaceutical composition include, but are not limited to, diseases and / or medical conditions associated with a lack or insufficient body intake of thiamine. The pharmaceutical preparation can be administered parenterally, for example topically, epidermally, sublingually, buccally, nasally, intradermally, intravenously and / or intramuscularly. The pharmaceutical composition can also be administered enterally via the gastrointestinal tract.

[0194] The present disclosure also describes a kit comprising:

[0195] a) a genetically modified host cell as described herein; and / or

[0196] b) instructions for use of the genetically modified host cell; and / or

[0197] c) a nucleic acid construct as described herein; and / or

[0198] d) instructions for use of the nucleic acid construct; and / or

[0199] e) Host cells that can be genetically modified using the methods described herein.

[0200] In some embodiments, the kit includes a genetically modified cell capable of producing thiamine, wherein the genetically modified cell expresses a pathway enzyme that produces thiamine. In one embodiment, the genetically modified cell expresses thiamine synthase (ThiC) and one or more thiamine pathway enzymes or factors optionally selected from ThiD, ThiF, ThiS, ThiH, ThiG, ThiM, ThiE, TMP phosphatase, ThiK, ThiL, ThiI, IscR, and ThiO.

[0201] This application contains a sequence listing prepared in Patentl described below, but also submitted electronically in ST26 format, which sequence listing is incorporated herein by reference in its entirety.

[0202] Sequence Listing

[0203] This application contains a sequence listing prepared in Patentl described below, but also submitted electronically in ST26 format, which sequence listing is incorporated herein by reference in its entirety.

[0204]

[0205]

[0206] Working Example

[0207] Strains and plasmids The E. coli strains used in this article are as follows:

[0208]

[0209]

[0210]

[0211] 1 Commercially available strains, for example from the Coli Genetic Stock Center; http: / / cgsc2.biology.yale.edu / KeioList.php

[0212] 2 WO2017103221A1

[0213] 3 WO2019012058A1

[0214] 4 The method is described elsewhere: https: / / pubmed.ncbi.nlm.nih.gov / 24050148 /

[0215] The plasmids used in this article are as follows:

[0216]

[0217]

[0218] Analytical procedures

[0219] Procedure I: Optical density measurement

[0220] To measure the optical density (OD) of the cell culture at 600nm cuvette OD (cOD600), dilute the culture 10-fold with dH2O to a final volume of 1 ml and transfer to a 1.5 ml clear cuvette with a 10 mm path length. Measure the diluted culture on mySPEC (VWR) at 600nm and 10 mm path length. If the diluted culture measures cOD600>0.4, dilute the culture further 10-fold and re-measure.

[0221] Procedure II: Thiochrome Assay for Thiamine Quantification

[0222] To quantify thiamine in small-scale culture supernatant samples, the thiochrome assay was performed according to the assay described in Part III of the method in WO2017103221A1. Specifically, the supernatant of each culture was diluted with >20 thiamine standards ranging from 0 μM (mg / L) to 60 μM (15.9 mg thiamine / L), which were prepared in Milli-Q water. 50 μL of supernatant and each thiamine standard were then added to the wells of a 96-well microtiter plate. 100 μl of 4M potassium acetate was added to each well. The sample was then oxidized by adding 50 μl of freshly prepared 3.8 mM potassium ferrocyanide (dissolved in 7 M NaOH). The solution was mixed by pipetting and quenched by adding 50 μl of fresh 0.06% H202 (dissolved in saturated KH2PO4). The samples were neutralized with 6M HCl and fluorescence was measured at 444 nm after excitation at 365 nm. The thiamine concentration of each well / sample was estimated based on comparison with the standard curve included in the derivatization plate.

[0223] Procedure III: LC-MS method for quantification of thiamine and precursors

[0224] Chemicals and reagents

[0225] Commercial standards of the analytes of interest were purchased from the suppliers listed in Table 1 below. Water (H2O) was purchased from Honeywell, methanol (MeOH), ascorbic acid, and ammonium bicarbonate (NH4HCO3) were purchased from Sigma-Aldrich. Ammonium hydroxide was purchased from Carl Roth. Stock solutions of the analytes and internal standards 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 with 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 each calibration curve sample to correspond to a final concentration of 0.5 ng mL-1. An internal standard working standard solution 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 standardization.

[0226] Sample preparation

[0227] Prior to analysis, samples from the bioreactor were diluted and an internal standard mixture (ISTD MIX) was added to correct for possible technical variations. In order to be able to quantify the analytes as accurately as possible, two different dilution factors were applied. For analytes with lower concentrations, a dilution factor of 1:50 was applied, while for analytes with higher concentrations, a dilution factor of 1:1500 was used for quantification. The 1:1500 dilution was prepared in two steps. First, a 1:15 dilution was achieved by pipetting 980 μL of H2O:MeOH (50:50, v / v) + 0.1% ascorbic acid into an Eppendorf tube and adding 70 μL of supernatant. The tube was then vortexed thoroughly to mix. A final 1:1500 dilution was achieved by pipetting 980 μL of H2O:MeOH (50:50, v / v) + 0.1% ascorbic acid into a glass vial and then adding 10 μL of the 1:15 diluted sample as well as 10 μL of ISTD MIX. The solution was vortexed again to mix. A 1:50 dilution was achieved by pipetting 970 μL of H2O:MeOH (50:50, v / v) + 0.1% ascorbic acid into a glass vial, followed by the addition of 20 μL of the original sample and 10 μL of ISTDMIX. The solution was vortexed again to mix.

[0228] Liquid chromatography-tandem mass spectrometry

[0229] After sample preparation, randomization was performed 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 to quantify the analytes, and the fragmentor voltage, collision energy, and accelerator voltage were optimized for each ion transition.

[0230] The analytes were separated by chromatography before entering the mass spectrometer. An XBridge PremierBEH C18 VanGuardFIT column (4.6 mm × 100 mm, particle size 2.5 m, Waters Corporation) was used with 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 at 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 were collected using MassHunterAcquisition software (version 10.0, Build 10.0.142) from Agilent Technologies, and the retention time of each analyte is shown in Table 1.

[0231] Table 1. Abbreviations and suppliers of commercial standards (including retention times).

[0232]

[0233] Data processing

[0234] All data were processed using Agilent Technologies' MassHunter quantitative analysis software (version B.09.00, Build 9.0.647.0). The peak areas of the target analytes were normalized according to the peak area of ​​the internal standard 13C4-THI. Data quality was ensured by evaluating the technical replicates of specific fermentation samples as well as commercial standards. The quantitative concentration of THI in these samples needs to be kept within ±10% for the data set to be approved.

[0235] Molecular Biology Techniques

[0236] Standard techniques are used for DNA isolation, amplification, purification and cloning (restriction digestion, ligation), transformation, etc. 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, New York. And M. Green, J. Sambrook (2012) Molecular Cloning: a laboratory manual. 4th edition, Cold Spring Harbor Laboratory Press, CSH, New York, both of which are incorporated herein by reference in their entirety.

[0237] Example 1 - Construction of strains carrying IscR and CyaA mutations

[0238] Using the ΔThiP mutant BS00734 as the starting strain, the PCR cassette of Arabidopsis phosphatase was introduced into the chromosome KO-176 position by the "clonal amplification" method described in PCT / EP2022 / 069711. The resulting strain BS04565 was used as the starting point for IscR mutagenesis. This transcription factor regulates the expression of dozens of genes involved in the biosynthesis of FeS clusters. By mutating IscR to facilitate the formation of apoproteins, we aim to increase the yield of thiamine. Therefore, using the DNA oligonucleotides shown in Table 3, the iscR in BS04565 was mutated by multiple automated genome engineering (MAGE), as described in "Methods in Enzymology", 498, 409-426, 2011", to produce strain BS04701.

[0239] CRP is activated as a transcriptional regulator by binding to its allosteric activator cyclic adenosine monophosphate (cAMP), which is produced by the enzyme adenylate cyclase (encoded by cyaA). Under low glucose conditions, adenylate cyclase is activated by the phosphorylation state of the glucose-specific enzymes IIA or EIIA (encoded by crr). In order to effectively eliminate cAMP, the cyaA gene is completely removed from the chromosome of strain BS04701 using methods known in the art and replaced with a short FRT sequence. One method to achieve cyaA gene removal is to generate a DNA fragment carrying a kanamycin resistance gene, which is flanked by homologous regions of cyaA, and transform strain BS04701 (carrying a λ Red recombinase gene expressed by an inducible promoter) with this DNA cassette. A protocol for this method can be found in Datsenko, KA and Wanner, BL, "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.

[0240] Table 3: Oligonucleotides used to introduce and examine mutations

[0241]

[0242]

[0243] Amplify the region by PCR followed by Sanger sequencing using the oligonucleotides in Table 3 to verify successful introduction of the desired mutation.

[0244] Example 2 - Construction of strains carrying genes providing thiamine production

[0245] ThiEMD was introduced into chromosomes using the cloning integration method described in PCT / EP2022 / 069711. The promoter of ThiEMD is apFAB70 (SEQ ID NO: 44), and each gene has a specific RBS sequence (SEQ ID NO: 46).

[0246] pBS2896: Phusion U polymerase (Thermo Fischer Scientific) was used according to the manufacturer's protocol, by amplifying genes from the chromosome and ribosome binding site of Escherichia coli, and using primers containing uracil for USER restriction enzyme recognition, the IPTG inducible (promoter SEQ ID NO:45) transgenic encoding ThiIWT (SEQ ID NO:24) was cloned into plasmid pBS2896. Similarly, the plasmid backbone carrying starting point pSC101 and tetracycline resistance box and promoter sequence was amplified. USER enzyme (New England Biolabs) and T4 ligase (Thermo Fischer Scientific) were used to digest and connect DNA fragments according to the manufacturer's protocol. These mixtures were introduced into BS05801 by electroporation, and the transformed cells were grown overnight at 37 degrees Celsius on the selective LB agar supplemented with tetracycline.

[0247] pBS2819: Using the USERLON method described above for the construction of pBS2896, plasmids encoding genes thiF, thiS, thiG and thiH (SEQ ID Nos. 6, 8, 12, 10, respectively) were cloned into a plasmid backbone carrying a spectinomycin resistance cassette and a p15A origin of replication. Each gene was preceded by a synthetic ribosome binding site (RBS) sequence.

[0248] pBS2919: Using the USER cloning method described above for the construction of pBS2896, a plasmid encoding the genes thiF, thiS, thiG and ThiO from Pseudomonas putida (SEQ ID Nos. 6, 8, 12, 30, respectively) was cloned into a plasmid backbone carrying a spectinomycin resistance cassette and a p15A origin of replication, driven by the constitutive promoter apFAB71 (SEQ ID No: 43). Each gene was carried out by a synthetic RBS sequence.

[0249] pBS2921: Using the USER cloning method described above for the construction of pBS2896, a plasmid encoding the Bacillus subtilis genes thiF, thiS, thiG and ThiO (SEQ ID Nos. 6, 8, 12, 28, respectively) was cloned into a plasmid backbone carrying a spectinomycin resistance cassette and a p15A origin of replication, driven by the constitutive promoter apFAB71 (SEQ ID No: 43). Each gene was carried out by a synthetic RBS sequence.

[0250] pBS3120: Using the USER cloning method as described above for the construction of pBS2896, a plasmid encoding the genes thiF, thiS, thiG, ThiI (1,449 bp WT form) and ThiO (SEQ ID Nos. 6, 8, 12, 24, 30, respectively) from Pseudomonas putida was cloned into a plasmid backbone carrying a spectinomycin resistance cassette and a p15A origin of replication, driven by the constitutive promoter apFAB71 (SEQ ID No: 43). Each gene was carried out by a synthetic RBS sequence.

[0251] pBS3121: Using the USER cloning method as described above for the construction of pBS2896, a plasmid encoding the genes thiF, thiS, thiG, ThiI (300 bp truncated form) and ThiO (SEQ ID Nos. 6, 8, 12, 34 and 36 enzymes, respectively) from Pseudomonas putida was cloned into a plasmid backbone carrying a spectinomycin resistance cassette and a p15A origin of replication, driven by the constitutive promoter apFAB71 (SEQ ID No: 43). Each gene was carried out by a synthetic RBS sequence.

[0252] Transformation: Plasmid pBS2819 (alone or together with pBS2896), pBS2919 (alone or together with pBS2896), pBS2921 (alone or together with pBS2896), pBS3120 or pBS3121 were introduced into the background strain BS05801 by electroporation transformation protocols well known in the art, and the transformed strains were selected on selective agar plates containing spectinomycin (pBS2819, pBS2919, pBS2921, pBS3120 or pBS3121) and / or tetracycline (pBS2896).

[0253] Example 3: Replacement of ThiH with ThiO and balanced expression of ThiI to increase thiamine titers compared to ThiH or ThiO alone.

[0254] The BS05801 strain can produce thiamine when fed with precursors HMP and THZ, because ThiD converts HMP to HMP-PP, while ThiM converts THZ to cTHZ-P. Both the precursors HMP-PP and cTHZ-P are linked by ThiE to produce thiamine monophosphate (TMP) and thiamine (a single copy phosphatase gene in the chromosome converts >98% of TMP to thiamine). In a standard small-scale culture (see below), the addition of 500 μM HMP and 500 μM THZ to BS05801 produces a titer of about 20 mg / L thiamine.

[0255] When strain BS05801 was fed with HMP alone, the ability to produce THZ could be probed based on thiamine quantification by simultaneously expressing genes involved in THZ formation (ThiF, ThiS, ThiG, and ThiH). Previous studies in our laboratory have demonstrated that to maximize THZ production, not only is it necessary to overexpress ThiF, ThiS, ThiG, and ThiH, but their expression must also be carefully balanced using specific RBS sequences (e.g., a sequence comprising SEQ ID NO:46) (data not shown). Our best strain (BS05850) that overexpressed these four genes produced approximately 6 mg / L thiamine from 2 g / L glucose and 500 μM HMP ( Figure 2 However, ThiI co-expression (BS06232) increased THZ production by 30% to 9 mg / L thiamine ( Figure 2 Interestingly, although ThiO from a different organism could be substituted for ThiH (BS06278 or BS06280) to produce ~4.5 mg / L thiamine, in both cases (BS06285 and BS06287), co-expression of ThiI increased the titer by >300% to ~13 mg / L. This improvement was >30% higher than the combination of native ThiH and ThiI (~9 mg / L). The final OD values ​​of the four strains containing ThiO were very similar, but ~30% lower than the thiH control - / + ThiI ( Figure 2 Middle panel), indicating that the ThiO strains had higher specific biomass yields. In fact, the four ThiO strains had 75% and 100% higher specific cell productivity, as shown by OD normalized titers ( Figure 2 Bottom panel). All strains were cultured in 400 μL mMOPS with 500 μM HMP (without or with IPTG). ThiI expression was induced by IPTG at the concentration that produced maximal activity for each strain, while ThiFSGH or ThiFSGO expression was constitutive. Strains were cultured in deep-well plates with shaking at 275 rpm for 24 h at 37 °C, after which thiamine production was assessed using the thiochrome assay. Thiamine titers were obtained as shown in Table 1. Figure 2 The bars represent the median thiamine production (height) and the black dots represent the thiamine production of individual replicate cultures. A total of 4 replicates were used for each strain.

[0256] Example 4: Production of Thiamine Using the Rhodanese Domain of ThiI

[0257] ThiI is a multifunctional gene that is not only involved in thiamine biosynthesis (transferring sulfur from IscS to the sulfur carrier protein ThiS to form ThiS-thiocarboxylate), but also catalyzes the ATP-dependent transfer of sulfur to tRNA as part of a modification (4-thiouridine) that serves as a near-UV light sensor in E. coli. ThiI is a very long protein that in E. coli consists of 482 amino acid residues that form different functional domains. Figure 3 The four domains present in ThiI in E. coli and other organisms are shown. The first three domains are involved in binding to ferredoxin, ATP, and tRNA, while the fourth domain is thought to be specialized for thiazole biosynthesis.

[0258] To demonstrate that ThiIWT is not required for the production of THZ (measured as thiamine, thanks to the co-expression of ThiD and ThiE on the chromosome), ThiI was truncated at the N-terminus to remove the first three domains, leaving a 98 amino acid truncated version (residue I384 was changed to methionine, and the rest of the C-terminus remained intact until the last amino acid P482). ThiIWT or the truncated version was then introduced into an operon already containing the E. coli genes ThiF, ThiS, ThiG and P. putida ThiO with defined RBS sequences, resulting in an operon of five genes that was constitutively expressed and did not require the addition of IPTG. In both cases, the RBS sequence of ThiI was also optimized, resulting in the ThiIWT (BS07082) or truncated (BS07083) versions. Both strains were cultured in 400 μL mMOPS in deep-well plates with HMP and shaken at 275 rpm for 24 h at 37 °C, after which thiamine production was assessed using the thiochrome assay. Figure 4 The thiamine titer obtained from 2 g / L glucose and 500 μM HMP is shown to be approximately 11 mg / L. The bar graph shows the median thiamine titer and the final OD value (height), indicating that the two strains have very similar production and growth patterns. A total of 4 replicates were used for each strain. Therefore, the thiamine domains of E. coli ThiI and other organisms are sufficient for thiamine production.

[0259] Example 5: Benchtop Fed-Batch Fermentation of Strains Overexpressing ThiI and ThiO

[0260] Small-scale, overexpressed ThiI and two heterologous ThiOs showed approximately 30% higher thiamine titers than native ThiH, but a 20% reduction in growth ( Figure 2). This effect was independent of ThiI, as the final OD of the ThiFSGO strain without ThiI was even lower at 30% compared to the reference strain ThiFSGH. This data suggests that overexpression of ThiO leads to growth defects. In contrast to the small-scale data, one of the strains with the best ThiO+thiI combination (BS06285) performed worse than the reference strain (BS06232) in 0.5 L fed-batch fermentation ( Figure 5 ). BS06285 growth stopped at 48 hours and the average thiamine titer reached about 270 mg / L, which was 60% lower than the titer of BS06232 at 48 hours (480 mg / L) and 300% lower at 72 hours (about 700 mg / L). The fermentation data indicated that ThiO expression needs to be properly adjusted to avoid problems.

[0261] Example 6: Optimizing ThiO expression levels

[0262] Next, the expression level of ThiO was optimized by combining ThiI and ThiO in one operon to avoid having two separate plasmids for ThiFSGO and ThiI. Therefore, the thiI gene was introduced between ThiG and ThiO, resulting in a synthetic thiamine operon ThiFSGIO in a single plasmid (pBS2967). The best strain (BS06375) with the new operon was then fermented, resulting in an average thiamine titer of about 650 mg / L at 72 h, which is very similar to the reference strain BS06232 ( Figure 6 ). BS06375 grew about 25% higher than BS0232 (240 vs. 200 OD). Strains with a synthetic thiamine operon appear to have proper ThiO co-expression, as growth and production were not affected.

[0263] Example 7: Overexpression of ThiI and the native thiamine pathway containing ThiH

[0264] ThiC or ThiC+ThiE were introduced into the synthetic operon ThiFSGIO. The start ATG sequences of all five downstream genes of thiC overlapped with the bases at the end of the upstream gene, indicating that the co-expression of the thiamine genes was coupled and optimal for catalysis. In small-scale experiments, we found that the plasmid configuration that produced the most thiamine de novo from glucose was ThiFSGIOCE (pBS3001).

[0265] The background strain BS05801 contains a single copy of the synthetic operon ThiEMD and a phosphatase on the chromosome ( Figure 1, module 3) were transformed with synthetic (pBS3001) or natural thiamine operon plasmids (pBS140), resulting in strains BS06468 and BS05897, respectively. The latter strain was also transformed with the ThiI plasmid, resulting in strain BS06245.

[0266] Fermentation strains with the native operon but without ThiI (BS05897) or with ThiI (BS06245) showed similar thiamine titers of about 450 mg / L at 72 h ( Figure 7 ). However, the replicates of one of the strains containing thiI had poor yields, indicating that thiI did not contribute to the improvement of thiamine production. This may be because ThiC drives the cells to have a higher demand for the biosynthesis of iron-sulfur (FeS) clusters from cysteine. Similarly, in the thiazole pathway, IscS and thiI mobilize sulfur from cysteine ​​for the thiazole moiety. In addition, the radical s-adenosylmethionine (SAM) enzyme thiH from the native pathway also requires FeS clusters and SAM, just like ThiC. Therefore, in the de novo thiamine strains, there may be competition between module 1 (ThiC) and module 2 (ThiI / IscS and thiH) for sulfur, cysteine, and methionine to make FeS clusters and SAM. Therefore, replacing ThiH with ThiO can improve thiamine titers in fermenters.

[0267] Therefore, this example shows that overexpression of ThiI with the native thiamine pathway containing thiH has no positive effect on thiamine production.

[0268] Example 8: Overexpression of ThiI and ThiO-containing thiamine synthesis pathways.

[0269] One replicate performed poorly due to the presence of thiI in BS06245 (indicating genetic instability), while the other replicate performed as well as both replicates of strain BS05897 ( Figure 7 ), we compared the latter (BS06245 rep2) carrying the native thiamine operon ThiCEFSGH / ThiMD without thiI with strain BS06468 with ThiFSGIOCE as the synthetic thiamine operon under the same fermentation conditions.

[0270] Figure 8 The results showed that both replicates of BS06468 showed a thiamine titer of about 750 mg / L at 72 h, which was 74% higher than the reference strain BS05897 of about 430 mg / L. There was almost no difference in growth, and there was little difference in the accumulation of two precursors, including 4-amino-5-hydroxymethyl-2-methylpyrimidine (HMP) and thiazole (THZ), which were about 20 mg / L and about 40 mg / L, respectively. Figure 8). Therefore, the use of a synthetic ThiI+ThiO combination might be able to achieve this improvement, possibly because the mechanism of ThiO is less demanding and less complex compared to ThiH. Therefore, as shown above, the expression of ThiO needs to be tuned to achieve optimal performance.

[0271] Thus, this example demonstrates that overexpression of ThiI and a synthetic thiamine pathway containing ThiO has a significant effect on thiamine production.

[0272] Example 9: Capacity of Thiamine Pathway Modules 1, 2 or 3.

[0273] To better understand the capacity of all modules 1, 2, or 3, strains BS05897 and BS06468 were fed with THZ, HMP, or HMP and THZ precursors, respectively, and thiamine production was quantified in small-scale experiments. Fig. 9 It was shown that regardless of the strain, the thiamine titer of module 2 was approximately 3-fold higher than that of module 1. In contrast, the titer of module 3 was 2-fold or 10-fold higher than that of module 2 in BS06468 or BS05897, respectively.

[0274] Overall, the above data suggest that module 1 (ThiC and ThiD, which form HMP-P and HMP-PP, respectively) is limiting, followed by module 2 (ThiFSGH or ThiFSIGO), which ultimately produces cTHZ-P. Module 3 (ThiEMD + phosphatase) does not limit module 1 or 2, as kinases are very efficient enzymes, in this case, they convert HMP to HMP-P and HMP-PP (ThiD) or THZ to THZ-P (ThiM). While ThiE is the enzyme responsible for linking HMP-PP and cTHZ-P or THZ-P to form thiamine monophosphate (TMP), phosphatases convert TMP to thiamine. Thiamine diphosphate (TPP) is formed by phosphorylation of TMP by the kinase ThiL, which is present in the chromosome but is not expressed at high levels to allow efficient phosphorylation of TMP to TPP.

[0275] In terms of strain performance, compared with BS05897, BS06468 had a de novo titer of about 20% higher (3.2 vs. 2.5 mg / L), a module 2 titer of about 30% higher (about 10 vs. 7 mg / L), but a module 3 titer of about 3-fold lower (22 vs. 66 mg / L). This large difference may be attributed to the higher overexpression of the pBS140 plasmid kinases ThiM and ThiD in strain BS05897.

[0276] To evaluate the actual performance of all modules under fermentation conditions, strain BS06468 was cultured de novo without added precursors and supplemented with HMP (module 2) or HMP + THZ (module 3). Similarly, strain BS06468 was transformed with plasmids encoding ThiE and ThiD and fermented in the presence of HMP and THZ to determine the full potential of module 3 in this strain.

[0277] Thus, this example demonstrates that the major limiting factor for thiamine biosynthesis is ThiC (module 1), followed by thiazole formation (module 2).

[0278] Example 10: Performance of the three modules of the best thiamine producing strain.

[0279] Fig.10 The de novo thiamine titer was shown to be approximately 700 mg / L at 72 hours. This is approximately 25% lower than when fed with HMP (~900 mg / g). The simultaneous addition of HMP and THZ precursors increased the thiamine titer to 2000 mg / L or 2 g / L, which is nearly 3 times and 2 times higher than modules 1 and 2, respectively. Therefore, as observed in the small-scale fed-batch experiments, module 3 is the most effective module, followed by module 2 and finally module 1. In addition, overexpression of ThiD and ThiE in strain BS06581 fermented in the presence of HMP and THZ achieved a thiamine titer of 2.9 g / L, indicating that "module 3" can be directly improved. Finally, we observed very low accumulation of TMP and TTP in all cases. For example, 12-17 mg / L TMP and 6-16 mg / L TTP were detected in the supernatant of strains fed with both precursors at 72 hours (module 3), indicating that phosphatase expression was optimal even at high levels of precursors. In other words, the microbial cell factory produced about 99% thiamine and about 0.1% thiamine in phosphorylated form.

[0280] Thus, this example demonstrates that an engineered synthetic pathway can support thiamine titers of at least about 3 g / L from added precursors (Module 3).

[0281] References

[0282] D1 Radical S-adenosylmethionine enzymes.Broderick JB,Duffus BR,Duschene KS,Shepard EM.Chem Rev.2014Apr23;114(8):4229-317.doi:10.1021 / cr4004709.

[0283] D2 Glycine oxidase from B.subtilis:role of histidine244andmethionine261.Boselli A,Rosini E,Marcone GL,Sacchi S,Motteran L,Pilone MS,Pollegioni L,Molla G.Biochimie.2007Nov;89(11):1372-80.doi:10.1016 / j.biochi.2007.04.019.

[0284] D3 Purification and Properties of Glycine Oxidase from P.putidaKT2440.Equar MY,Tani Y,Mihara H.J Nutr Sci Vitaminol(Tokyo).2015;61(6):506-10.doi:10.3177 / jnsv.61.506.

[0285] D4 Thiamine biosynthesis in Escherichia coli:isolation and initialcharacterisation of the ThiGH complex.Leonardi R,Fairhurst SA,Kriek M,LoweDJ,Roach PL.FEBS Lett.2003Mar27;539(1-3):95-9.doi:10.1016 / s0014-5793(03)00204-7.

[0286] D5 Structure of the Escherichia coliThiS-ThiF complex,a key componentof the sulfur transfer system in thiamin biosynthesis.Lehmann C,Begley TP,Ealick SE.Biochemistry.2006Jan10;45(1):11-9.doi:10.1021 / bi051502y.

[0287] D6 Thiamin biosynthesis in Escherichia coli.IdentificationofThiSThiOcarboxylate as the immediate sulfur donor in the thiazoleformation.Taylor SV,Kelleher NL,Kinsland C,Chiu HJ,Costello CA,Backstrom AD,McLafferty FW,Begley TP.J Biol Chem.1998J

[0288] D7 The rhodanese domain ofThiIis both necessary and sufficient forsynthesis ofthe thiazole moiety ofthiamine in Salmonella enterica.Martinez-Gomez NC,Palmer LD,Vivas E,Roach PL,Downs DM.J Bacteriol.2011Sep;193(18):4582-7.doi:10.1128 / JB.05325-1D8 US8329395B1.

[0289] D9(Microbial cell factories for the sustainable manufacturing of Bvitamins.Carlos G.Acevedo-Rocha,Luisa S.Gronenberg,Matthias Mack,FabianM.Commichau,Hans J.Genee.Cur.Opinion Biotech.2019,Vol.56,P18-29).

[0290] D10 WO2017103221

Claims

1. A genetically modified host cell with improved thiamine production, wherein the host cell expresses one or more heterologous ThiO enzymes that convert glycine in the host cell to dehydroglycine (DHG), thereby improving thiamine production in the genetically modified host cell compared to an unmodified parental host cell.

2. A genetically modified host cell with improved thiamine production, wherein the host cell expresses one or more heterologous ThiI enzymes that catalyze the transfer of sulfur from IscS to the sulfur carrier protein ThiS in the host cell, thereby improving thiamine production in the genetically modified host cell compared to an unmodified parent host cell.

3. The host cell of claim 1, wherein the one or more heterologous ThiO enzymes are bacterial ThiO enzymes. The host cell of claim 2 , wherein the one or more heterologous ThiI enzymes are bacterial ThiI enzymes.

5. The host cell according to claim 1 or 3, wherein the one or more heterologous bacterial ThiO enzymes are a BsThiO enzyme from Bacillus subtilis and / or a PpThiO enzyme from Pseudomonas putida.

6. A host cell according to any one of the preceding claims, wherein one or more heterologous: a) the ThiO enzyme is at least 70% identical, such as at least 80% identical, such as at least 90% identical, such as at least 95% identical, such as at least 96% identical, such as at least 97% identical, such as at least 98% identical, such as at least 99% identical, such as 100% identical to the ThiO enzyme contained in SEQ ID NO: 27 and / or 29; and / or b) the ThiI enzyme is at least 70% identical, such as at least 80% identical, such as at least 90% identical, such as at least 95% identical, such as at least 96% identical, such as at least 97% identical, such as at least 98% identical, such as at least 99% identical, such as 100% identical to the ThiI enzyme contained in SEQ ID NO: 23, 35, 37, 39 and / or 41.

7. The host cell according to claim 2 or 4, wherein the one or more bacterial ThiI enzymes are the E. coli ThiI enzyme having SEQ ID NO: 23 8. The host cell of claim 3 or 5, wherein the one or more heterologous bacterial ThiO enzymes are a BsThiO enzyme from Bacillus subtilis having SEQ ID NO: 27 and / or a PpThiO enzyme from Pseudomonas putida having SEQ ID NO:

29.

9. A host cell according to any one of the preceding claims, wherein the gene encodes: a) the ThiO enzyme is at least 50% identical to the gene contained in SEQ ID NO: 28 or 30, or the genomic DNA encoding the ThiO enzyme contained in SEQ ID NO: 27 and 29, such as at least 60% identical, such as at least 70% identical, such as at least 80% identical, such as at least 90% identical, such as at least 95% identical, such as at least 96% identical, such as at least 97% identical, such as at least 98% identical, such as at least 99% identical, such as 100% identical; b) the ThiI enzyme is at least 50% identical to the gene contained in SEQ ID NO: 24, 36, 38, 40 and / or 42 or the genomic DNA encoding the ThiI enzyme contained in SEQ ID NO: 23, 35, 37, 39 and / or 41, such as at least 50% identical, such as at least 60% identical, such as at least 70% identical, such as at least 80% identical, such as at least 90% identical, such as at least 95% identical, such as at least 96% identical, such as at least 97% identical, such as at least 98% identical, such as at least 99% identical, such as 100% identical.

10. The host cell according to any one of the preceding claims, wherein a) the BsThiO gene is at least 70% identical to the BsThiO gene contained in SEQ ID NO: 28, such as at least 80% identical, such as at least 90% identical, such as at least 95% identical, such as at least 96% identical, such as at least 97% identical, such as at least 98% identical, such as at least 99% identical, such as 100% identical; b) the PpThiO gene is at least 70% identical, such as at least 80% identical, such as at least 90% identical, such as at least 95% identical, such as at least 96% identical, such as at least 97% identical, such as at least 98% identical, such as at least 99% identical, such as 100% identical to the PpThiO gene contained in SEQ ID NO:

30. The host cell according to claim 7 or 9, wherein the ThiI gene is a truncated ThiI gene.

12. The host cell of claim 11, wherein the ThiI gene is a truncated ThiI comprising a rhodanese domain that transfers sulfur from the persulfide IscS to the sulfur carrier protein ThiS to form ThiS-thiocarboxylic acid in the thiamine biosynthetic pathway.

13. A host cell according to claim 12, wherein the rhodanese domain is at least 70% identical to the rhodanese domain contained in SEQ ID NO: 13, such as at least 80% identical, such as at least 90% identical, such as at least 95% identical, such as at least 96% identical, such as at least 97% identical, such as at least 98% identical, such as at least 99% identical, such as 100% identical.

14. The host cell of any one of the preceding claims, further comprising an efficient metabolic pathway comprising one or more native or heterologous pathway elements to produce said thiamine.

15. The host cell of claim 14, wherein the one or more pathway elements comprise one or more free radical SAM enzymes.

16. The host cell according to any one of the preceding claims, wherein one or more pathway elements are selected from: a) one or more phosphate synthases selected from phosphomethylpyrimidine synthases (ThiC); which catalyze the synthesis of the hydroxymethylpyrimidine phosphate (HMP-P) moiety of thiamine from aminoimidazole riboside (AIR) in a free radical S-adenosyl-L-methionine (SAM)-dependent reaction; b) hydroxymethylpyrimidine / methylpyrimidine phosphate kinase (ThiD), which catalyzes the phosphorylation of hydroxymethylpyrimidine phosphate (HMP-P) to HMP-PP and the phosphorylation of HMP to HMP-P; c) adenylyltransferase (ThiF), catalyzing the adenylation of the carboxyl terminus of the sulfur carrier protein ThiS, followed by the replacement of AMP by ThiI-persulfide to yield ThiS-ThiI acyl disulfide ThiS; d) ThiS, the C-terminal thiocarboxylation is carried out in two steps: first, acyladenylation by ThiF, then thiocarboxylation by ThiI; e) conversion by 2-imidoacetic acid synthase (ThiH), catalyzing the free radical-mediated cleavage of tyrosine into 2-imidoacetic acid and 4-cresol; f) Thiazole synthase (ThiG), catalyzes the rearrangement of 1-deoxy-D-xylulose 5-phosphate (DXP) to produce the thiazole phosphate moiety of thiamine; g) hydroxyethylthiazole kinase (ThiM), catalyzes the rearrangement of 1-deoxy-D-xylulose 5-phosphate (DXP) to generate the thiazole phosphate moiety of thiamine; h) thiamine monophosphate (TMP) phosphatase, which dephosphorylates thiamine monophosphate to thiamine; i) thiamine kinase (ThiK), catalyzing the phosphorylation of thiamine to thiamine phosphate; j) Thiamine monophosphate kinase (ThiL), catalyzes the ATP-dependent phosphorylation of thiamine monophosphate (TMP) to form thiamine pyrophosphate (TPP); k) Thiamine phosphate synthase (ThiE), which condenses 4-methyl-5-(β-hydroxyethyl)thiazole monophosphate (THZ-P) and 2-methyl-4-amino-5-hydroxymethylpyrimidine pyrophosphate (HMP-PP) to form thiamine monophosphate (TMP); l) HTH-type transcriptional regulator (IscR), which regulates the transcription of several operons and genes involved in the biosynthesis of Fe-S clusters and Fe-S-containing proteins.

17. The host cell of claims 14 to 16, wherein one or more pathway elements are encoded by one or more genes selected from the group consisting of: a) ThiC, which is at least 70%, such as 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% identical to the ThiC contained in SEQ ID NO: 2; b) a ThiD which is at least 70%, such as 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% identical to the ThiD contained in SEQ ID NO: 4; c) ThiF, which is at least 70%, such as 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% identical to the ThiF contained in SEQ ID NO: 6; d) ThiS, which is at least 70%, such as 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% identical to the ThiS contained in SEQ ID NO: 8; e) a ThiH which is at least 70%, such as 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% identical to the ThiH contained in SEQ ID NO: 10; f) ThiG, which is at least 70%, such as 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% identical to the ThiG contained in SEQ ID NO: 12; g) ThiM, which is at least 70%, such as 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% identical to the ThiM contained in SEQ ID NO: 14; h) a TMP phosphatase that is at least 70%, such as 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% identical to the TMP phosphatase comprised in SEQ ID NO: 16; i) ThiK, which is at least 70%, such as 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% identical to the Thik contained in SEQ ID NO: 18; j) a ThiL which is at least 70%, such as 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% identical to the ThiL comprised in SEQ ID NO: 20; k) ThiE, which is at least 70%, such as 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% identical to the ThiE contained in SEQ ID NO: 22; 1) an IscR that is at least 70%, such as 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% identical to the IscR comprised in SEQ ID NO:

32.

18. The host cell according to any one of the preceding claims, wherein the endogenous native ThiH gene is disrupted or inactivated.

19. The host cell according to claim 18, wherein the disruption / inactivation is caused by introducing a deletion by complete removal of the gene or by introducing one or more stop codons or frameshift mutations resulting in a translational knockout thereby preventing expression of the active peptide.

20. The host cell of claim 18 or 19, wherein the disruption / inactivation comprises a translational knockout or a frameshift mutation.

21. The host cell of claims 18 to 20, wherein the deletion, disruption and / or attenuation is a point mutation in the promoter of the protein coding sequence, the RBS region and / or the protein coding sequence.

22. The host cell of any one of the preceding claims, wherein one or more pathway elements are encoded by one or more genes selected from the group consisting of: a) a gene that 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 contained in SEQ ID NO: 2 or its genomic DNA encoding the ThiC enzyme contained in SEQ ID NO: 1; b) a gene that 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 contained in SEQ ID NO:4 or its genomic DNA encoding the ThiD enzyme contained in SEQ ID NO:3; c) a gene that 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 contained in SEQ ID NO: 6 or its genomic DNA encoding the ThiF enzyme contained in SEQ ID NO: 5; d) a gene that 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 contained in SEQ ID NO: 8 or its genomic DNA encoding the ThiS enzyme contained in SEQ ID NO: 7; e) a gene that 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 contained in SEQ ID NO: 10 or its genomic DNA encoding the ThiH enzyme contained in SEQ ID NO: 9; f) a gene that 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 contained in SEQ ID NO: 12 or its genomic DNA encoding the ThiG enzyme contained in SEQ ID NO: 11; g) a gene that 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 contained in SEQ ID NO: 14 or its genomic DNA encoding the ThiM enzyme contained in SEQ ID NO: 13; h) a gene that 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 contained in SEQ ID NO: 16 or its genomic DNA encoding the TMPase phosphatase contained in SEQ ID NO: 15; i) a gene that 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 contained in SEQ ID NO: 18 or its genomic DNA encoding the ThiK enzyme contained in SEQ ID NO: 17; j) a gene that 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 contained in SEQ ID NO: 20 or its genomic DNA encoding the ThiL enzyme contained in SEQ ID NO: 19; k) a gene that 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 contained in SEQ ID NO: 22 or its genomic DNA encoding the ThiE enzyme contained in SEQ ID NO: 21; and l) a gene that 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 contained in SEQ ID NO:32 or its genomic DNA encoding the IscR enzyme contained in SEQ ID NO:

31.

23. The host cell of any one of the preceding claims, wherein one or more genes and / or polypeptides of the thiamine pathway are heterologous to the host cell.

24. The host cell of any one of the preceding claims, further comprising at least 2 copies of one or more genes and / or polypeptides of the thiamine pathway.

25. The host cell of any preceding claim, further comprising a transport molecule that facilitates transport of the thiamine pathway precursor or product.

26. The host cell of any one of the preceding claims, wherein the host cell is further genetically modified to provide an increased amount of substrate in the thiamine pathway.

27. The host cell of any one of the preceding claims, wherein the host cell is further genetically modified to exhibit increased tolerance to one or more substrates, intermediates or products in the thiamine pathway.

28. A host cell according to any one of the preceding claims, wherein one or more additional native or endogenous genes of the host cell are deleted, disrupted and / or attenuated.

29. The host cell of any one of the preceding claims, wherein one or more genes in the thiamine pathway are overexpressed.

30. A host cell according to any one of the preceding claims, wherein the host cell is prokaryotic or eukaryotic.

31. The host cell of any one of the preceding claims, wherein the host cell is further genetically modified to provide an increased amount of substrate in the thiamine pathway.

32. A cell culture comprising a genetically modified host cell as claimed in any one of the preceding claims and a growth medium.

33. A method for producing thiamine, comprising: a) cultivating the cell culture of claim 32 under conditions that allow the host cells to produce said thiamine; and b) optionally recovering and / or isolating said thiamine.

34. The method of claim 33, further comprising supplying one or more exogenous thiamine precursors to the host cell culture.

35. The method of claim 34, wherein supplying one or more exogenous thiamine precursors to the host cell culture comprises supplying THZ, HMP, or THZ to the cell culture.

36. The method according to claims 33 to 35, further comprising one or more elements selected from the group consisting of: a) culturing the cell culture under aerobic or anaerobic conditions b) culturing the host cell under mixing; c) culturing the host cell at a temperature between 25°C and 50°C; d) culturing the host cell at a pH between 3-9; and e) culturing the host cell for 10 hours to 120 days.

37. The method according to claims 33 to 36, wherein the recovery and / or separation step comprises separating the liquid phase of the cell culture from the solid phase of the cell culture to obtain a supernatant comprising the thiamine, and subjecting the supernatant to one or more steps selected from the following: a) contacting the supernatant with one or more adsorption resins to obtain at least a portion of the thiamine produced, then optionally recovering the thiamine from the resins in a concentrated solution, and then isolating the thiamine by crystallization or solvent evaporation; b) contacting the supernatant with one or more ion exchange or reverse phase chromatography columns to obtain at least a portion of the thiamine, then optionally recovering the thiamine from the resin in a concentrated solution, and then isolating the thiamine by crystallization or solvent evaporation; and / or c) extracting the thiamine from the supernatant, for example by liquid-liquid extraction into an immiscible solvent, and then optionally isolating the thiamine by crystallization or solvent evaporation; d) thereby recovering and / or isolating said thiamine.

38. The method of any one of claims 33 to 37, wherein the production of thiamine is at least 10%, such as at least 20%, such as at least 50%, such as at least 100%, such as at least 200% higher than the production of the host cell without requiring a) expressing in the host cell one or more heterologous ThiO genes capable of converting glycine into dehydroglycine (DHG), and / or, b) overexpressing in the host cell one or more ThiI genes capable of catalyzing sulfur transfer to the sulfur carrier protein ThiS.

39. The method of claim 38, wherein the thiamine production and / or titer is at least 400 mg / L, optionally at least 800 mg / L, optionally at least 1000 mg / L, optionally at least 1500 mg / L, optionally at least 2000 mg / L, optionally at least 3000 mg / L, optionally at least 5000 mg / L, optionally at least 10000 mg / L, optionally at least 15000 mg / L, optionally at least 20000 mg / L.

40. The method of claims 33 to 39, wherein one or more steps of producing the thiamine are performed in vitro.

41. The method of claims 33 to 40, further comprising: The thiamine is mixed with one or more carriers, agents, adjuvants, additives and / or excipients, optionally with pharmaceutical grade carriers, agents, adjuvants, additives and / or excipients.

42. A fermented composition comprising the cell culture of claim 32 or a cell culture produced by the method of any one of claims 33 to 41 and said thiamine.

43. The fermentation composition of claim 42, 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.

44. A fermentation composition according to claim 42 or 43, wherein at least 50%, such as at least 75%, such as at least 95%, such as at least 99% of the solid cellular material has been separated from the composition.

45. The fermented composition of claims 42 to 44, further comprising one or more compounds selected from: a) a precursor or product of an efficient metabolic pathway for producing said thiamine; b) nutritional supplementation; and c) wherein the concentration of thiamine is at least 1 mg / L of the composition.

46. ​​A composition comprising the fermentation composition of claims 42 to 45 and one or more carriers, agents, adjuvants, additives and / or excipients.

47. The composition of claim 46, wherein the fermentation composition and one or more carriers, agents, adjuvants, additives and / or excipients are in dry solid form.

48. The composition of claim 46 or 47, wherein the fermented composition and the one or more carriers, agents, adjuvants, additives and / or excipients are in a liquid stable form.

49. The composition according to claims 46 to 48, wherein the fermented composition is refined into a pharmaceutical preparation, a dietary supplement, a cosmetic, a food preparation, a feed preparation and / or an analytical or diagnostic reagent.

50. The composition according to claim 49, wherein the pharmaceutical preparation is in the form of a powder, tablet, capsule, hard chewable tablet and / or soft tablet or chewing gum.

51. The composition of claim 49 or 50, wherein the pharmaceutical formulation is in the form of a liquid pharmaceutical solution.

52. A composition according to claims 46 to 51 for use as a medicament.

53. A composition according to claims 46 to 52 for use in the treatment of nutritional deficiencies.

54. A method of treating a disease in a mammal comprising administering to the mammal a therapeutically effective amount of a composition according to claims 46 to 53.

55. The method of claim 54, wherein the disease is or is associated with a nutritional deficiency.

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