Biotin production in microorganisms expressing genes for pantothenate kinases with reduced product inhibition by coenzyme a

Abstract

The invention relates to a method for producing biotin, dethiobiotin (DTB), or a mixture thereof, characterised in that a microbial production strain is cultivated which recombinantly expresses at least one coenzyme A feedback-resistant enzyme with the enzymatic activity of a pantothenate kinase (enzymatic activity of proteins of class EC 2.7.1.33), and biotin or DTB or a mixture thereof is subsequently isolated.

Description

[0001] Biotin production in microorganisms expressing genes for pantothenate kinases with reduced product inhibition by coenzyme A

[0002] The invention provides a process for the production of biotin, dethiobiotin (DTB), or a mixture thereof, characterized in that a microbial production strain is cultivated which recombinantly expresses at least one coenzyme A feedback-resistant enzyme with the enzymatic activity of a pantothenate kinase (enzymatic activity of proteins of class EC 2.7.1.33), and then biotin or DTB or a mixture thereof is isolated.

[0003] Biotin (D-biotin, vitamin B7, vitamin H, CAS number 58-85-5) is a water-soluble vitamin from the B complex. It plays an important role as a prosthetic group of enzymes in cellular metabolism, where it activates the otherwise inert CO2 in carboxylation reactions. Biotin-dependent enzymes include malonyl-CoA synthase, propionyl-CoA carboxylase, pyruvate carboxylase, and geranyl-CoA carboxylase. Biotin is a vitamin of great commercial importance as an additive in food supplements, animal feed, cosmetics, and the pharmaceutical sector.

[0004] Dethiobiotin (desthiobiotin, DTB, CAS number 533-48-2) is the biosynthetic precursor of biotin. Biotin is formed by the enzymatically catalyzed incorporation of sulfur into DTB. The microbial biosynthesis of biotin and its regulation are known (see review Sirithanakorn and Cronan, 2021, FEMS Microbiol. Rev. 45: fuab003). Biotin biosynthesis in microorganisms is strictly regulated, so that no biotin can be detected in a wild-type strain with a functional biotin biosynthesis pathway (see also the examples of the present invention). The functional biotin biosynthesis pathway is demonstrated by the fact that the wild-type strain can grow on a minimal medium (defined mineral salt medium without biotin) without biotin supplementation, while a biotin-auxotrophic mutant of the strain fails to grow under the same conditions.The lack of evidence of biotin production in a microorganism does not mean that it cannot produce biotin, but rather that.

[0005] Biotin biosynthesis is subject to strict regulation, and the biotin synthesized by the microorganism is immediately incorporated into the relevant enzymes as a cofactor and is not present in a free form. Furthermore, microorganisms can absorb biotin from the environment, and with a sufficient supply of biotin, e.g., from a culture medium, biotin synthesis, which is metabolically costly for the microorganism, is completely shut down. Thus, a WT strain will not produce its own biotin even with sufficient external supply.

[0006] The current state of the art in regulating biotin biosynthesis is described in detail by Sirithanakorn and Cronan. This strict regulation of biotin biosynthesis, which ensures that only as much biotin is produced as the microorganism needs for its viability, has so far prevented the development of an economically competitive biotechnological production process for biotin.

[0007] Pantothenic acid (vitamin B5, CAS number 79-83-4 for the R-form, CAS number 5999-54-2 for the racemate, CAS number 137-08-6 for the Ca salt) is the central biosynthetic precursor of coenzyme A (CoA), which serves in metabolism to activate acyl groups (e.g., as acetyl-CoA, succinyl-CoA, or malonyl-CoA in the citric acid cycle, fatty acid synthesis, or fatty acid oxidation). The biosynthesis of CoA is well known (see review by Leonardi and Jackowski, 2007, EcoSal Plus, 2). Coenzyme A is produced from pantothenic acid in five enzymatic steps. The first step of coenzyme A biosynthesis, the ATP-dependent phosphorylation of pantothenic acid to 4'-phosphopantothenate, is catalyzed by the enzyme pantothenate kinase (EC 2.7.1.33, referred to as CoaA, CoaA enzyme or PanK) according to equation (1).

[0008] Equation (1):

[0009] (R)-Pantothenate + ATP <=> (R)-4'-Phosphopantothenate + ADP + H +

[0010] Three types of CoaA enzymes are known in bacteria, see Hong et al. (2006), Structure 14: 1251-1261. Type I CoaA is represented, for example, by the enzyme from E. coli. The activity of type I CoaA is feedback-inhibited by coenzyme A or its CoA thioester. Type II CoaA enzymes are represented by the Staphylococcus aureus enzyme, which is not feedback-inhibited. Type III CoaA enzymes are represented by the Pseudomonas aeruginosa enzyme, which is also not feedback-inhibited. The microorganisms containing type II and type III CoaA enzymes are primarily pathogenic bacteria, whose genes one wants to avoid whenever possible in metabolic engineering. The influence of type I, type II, or type III CoaA enzymes on biotin synthesis has not yet been investigated.

[0011] In Escherichia coli, the enzyme pantothenate kinase, which represents type I CoaA, is encoded by the coaA gene. The activity of the CoaA enzyme is inhibited by coenzyme A, the end product of the biosynthesis pathway. Coenzyme A is thus an inhibitor of the CoaA enzyme. This form of product inhibition (feedback inhibition) is characteristic of many metabolic pathways in microorganisms such as E. coli, ensuring that a microorganism produces only as much of the metabolite (in this case, coenzyme A) as the organism needs. Pantothenate kinase is therefore the key enzyme for coenzyme A biosynthesis.

[0012] Rock et al. (2003), J. Bacteriol 185: 3410-3415, based on the protein structure of the E. coli CoaA enzyme, identified three amino acids that are essential for binding coenzyme A and thus mediate the inhibition of CoaA enzyme activity by coenzyme A (see Fig. 2 in Rock et al.). These three amino acids are arginine at position 106 (R106), histidine at position 177 (H177), and phenylalanine at position 247 (F247). Another amino acid that interacts with coenzyme A, lysine at position 101 of the protein sequence, is involved in binding the ATP substrate essential for enzymatic activity, and its mutation leads to inactive enzyme activity.

[0013] The aim of the investigations by Rock et al. was to produce mutants of the CoaA enzyme with reduced feedback inhibition of the enzyme activity by coenzyme A. The term "reduced feedback inhibition" is used in the present invention synonymously with the term "increased feedback resistance" (feedback resistance or feedback-resistant, abbreviated to fbr). In the present invention, correspondingly coenzyme A feedback-resistant mutants of pantothenate kinase are also referred to as fbr mutants of the CoaA

[0014] enzyme, or pantothenate kinase.

[0015] According to Rock et al. (Fig. 4A), the WT CoaA enzyme is characterized by its enzyme activity in the presence of 20 μM coenzyme A being only approximately 40% of the activity of the WT CoaA enzyme without added coenzyme A. Coenzyme A is thus an inhibitor of the WT CoaA enzyme. The so-called inhibitor constant IC 50indicates at which concentration of an inhibitor the activity of an enzyme is only 50% of the activity without inhibitor. According to Rock et al. Fig. 4A, the IC 50 for the CoaA enzyme from E. coli thus less than 20 μM

[0016] Coenzyme A

[0017] Point mutants of the CoaA enzyme, each mutated in one of the three amino acids R106 (fbr mutant CoaA [R106A]), H177 (fbr mutant CoaA [H177Q]), and F247 (fbr mutant CoaA [F247V]), were characterized by no longer exhibiting product inhibition against coenzyme A (see Fig. 2 and Fig. 4A in Rock et al.). The fbr mutants were characterized with regard to their enzymatic properties. Investigation of the physiological impact of overexpression of a feedback-resistant CoaA mutant, which is synonymous with deregulation of the coenzyme A biosynthesis pathway, resulted in the accumulation of phosphorylated metabolites such as 4'-phosphopantetheine as well as coenzyme A (Fig. 6 in Rock et al.) in E. coli cells. Thus, Rock et al. investigated how the expression of a feedback-resistant CoaA mutant affects the levels of various phosphorylated pantothenate-derived metabolites such as 4-phosphopantetheine and coenzyme A in E. coli.EP 3269 819 Bl uses a feedback-resistant CoaA mutant for.

[0018] Production of O-acetyl-homoserine. Studies on the production of biotin have not been conducted by Rock et al. or in

[0019] EP 3269 819 Bl. There is no mechanistic connection between the metabolic pathways to coenzyme A and biotin.

[0020] For commercial use, biotin is currently produced chemically. A thirteen-step synthesis starting from fumaric acid is known. With the trend away from chemically produced ingredients, driven by consumer demand, a biotechnological process for producing biotin is of great interest.

[0021] Known biotechnological approaches for the production of biotin production strains (see also Sirithanakorn and Cronan) primarily target the recombinant expression of the various biotin biosynthetic genes, namely the bioA, bioB, bioC, bioD, bioF, and bioH genes, or their functionally analogous genes. Furthermore, high-throughput screening (HTS) has enabled the isolation of microbial strains with enhanced biotin production through mutagenesis and selection (Bali et al., 2020, Metab. Eng. 60:97-107).

[0022] The state of the art thus reveals various metabolic

[0023] Engineering and HTS approaches to produce biotin from microbial sources have been used. All of these approaches have met with limited success, so chemical synthesis is currently still the method of choice for producing biotin for commercial purposes. Since known biotechnological approaches are currently unsuitable, there is a need for new genetic elements suitable for improving biotechnological processes for biotin production.

[0024] The object of the present invention was therefore to provide a process for the fermentative production of biotin or its biosynthetic precursor dethiobiotin.

[0025] The object is achieved by a process for the production of biotin, dethiobiotin (DTB), or a mixture thereof, characterized in that a microbial production strain is cultivated which recombinantly expresses at least one coenzyme A feedback-resistant enzyme with the enzymatic activity of a pantothenate kinase (enzymatic activity of the proteins of class EC 2.7.1.33), and then biotin or DTB or a mixture thereof is isolated.

[0026] As is known from the prior art (see above), it was also found in the present invention that an unmodified WT microorganism produces no detectable biotin (see Examples 5-7 of the present invention). Biotin and the biosynthetic precursor DTB were detectable neither intracellularly nor extracellularly (see Example 8). Surprisingly, however, it was found that deregulation of the coenzyme A biosynthesis pathway by expression of recombinant, feedback-resistant coaA mutants enables biotin production in a microorganism. This deregulation of the biotin biosynthesis pathway is of great interest for the development of biotechnological biotin production processes through metabolic engineering, particularly since WT microorganism strains cannot be utilized.

[0027] The gene recombinantly expressed in the production strain, which expresses a coenzyme A feedback-resistant enzyme with the enzymatic activity of a pantothenate kinase (enzymatic activity of proteins of class EC 2.7.1.33), may be a

[0028] 1. WT gene encoding a CoaA enzyme not inhibited by coenzyme A feedback (e.g. encoding a pantothenate kinase type II or III), wherein the WT gene is either homologously overexpressed in the strain of origin or heterologously expressed in a strain other than the one from which it was isolated, with heterologous expression being preferred, or

[0029] 2. mutated gene which in its wt form encodes a CoaA enzyme inhibited by coenzyme A feedback, which encodes a feedback-resistant CoaA enzyme due to the mutation.

[0030] Preferably, the method is characterized in that the gene expressing the coenzyme A feedback-resistant enzyme with pantothenate kinase activity is a mutated gene that, in its wt form, encodes a coenzyme A feedback-inhibited CoaA enzyme. It is preferred that the pantothenate kinase is a bacterial type I CoaA.

[0031] Gene and protein sequences of WT pantothenate kinase enzymes are accessible, for example, in the NCBI database under the search term "pantothenate kinase". Examples of pantothenate kinases covered by the invention are pantothenate kinases selected from Escherichia coli, Pantoea ananatis, Raoultella terrigena, Streptococcus pyogenes, Enterococcus faecalis, Pasturella multocida, Haemeophilus influenzae, Actinobacillus actinomycetemocomitans, Salmonella enterica, Klebsiella pneumoniae, Yersinia pestis, Vibrio cholerae, Mycobacterium tuberculosis, Corynebacterium diptheriae, and Streptomyces coelicolor. Protein and gene sequences of the selected pantothenate kinases can be found in sequence databases familiar to the person skilled in the art, such as the NCBI (National Center for Biotechnology Information).

[0032] It is an unexpected effect of the present invention that the homologous or heterologous expression of a pantothenate kinase which is feedback-inhibited in its wt form by coenzyme A and which has been mutated to a feedback-resistant enzyme, or the heterologous expression or homologous overexpression of a pantothenate kinase which is not feedback-inhibited in its wt form by coenzyme A, i.e. the deregulation of coenzyme A biosynthesis (see Examples 5-9), in microorganisms such as E. coli, P. ananatis and R. terrigena leads to the deregulation of biotin biosynthesis and thus the production of more biotin and its biosynthetic precursor DTB than is required for the viability of the microorganism. This results in freely available biotin and DTB. The freely available biotin and DTB are exported (secreted) from the cell, so that the two products can be easily isolated from the cell culture supernatant after separation of the biomass.

[0033] Preference is given to the homologous or heterologous expression, particularly preferably homologous expression, of a pantothenate kinase that is feedback-inhibited in its wt form by coenzyme A, which has been mutated to a feedback-resistant enzyme to deregulate biotin biosynthesis. In this case, the pantothenate kinase is particularly preferably CoaA from E. coli.

[0034] A great advantage of the present invention is that a process is provided for the fermentative production of biotin and / or DTB by using microorganism strains which are known feedback-resistant mutants of the CoaA

[0035] Enzyme expression can be used without requiring any changes to biotin metabolism. This was unknown at the time of writing and was not expected due to the unrelated metabolic pathways leading to coenzyme A and biotin.

[0036] Definitions: Metabolic engineering (also called "pathway design"), in contrast to biotransformation, is a biotechnology method in which the metabolic pathways of an organism are modified by optimizing or modifying genetic and regulatory processes. By supplementing the genome with enzyme genes, new or modified enzymes can be introduced into an organism, or genes of endogenous enzymes can be expressed in an increased or weakened form, thereby establishing new metabolic pathways in an organism or strengthening or weakening existing metabolic pathways. The aim of metabolic engineering is for the organism to produce a metabolic product either newly or a cellular metabolic product with an increased yield. A metabolic engineering process does not involve any starting materials specific for the metabolic product, such as an enzyme substrate, e.g.DTB is not used as a starting compound for the production of biotin, but merely a nutrient medium, also referred to as a culture medium, which is required for the growth of the organism in question and is composed of a C source (e.g., glucose), an N source (e.g., an ammonium salt or a complex amino acid mixture such as peptone or yeast extract), and other salts required for growth. Such nutrient media are known to the person skilled in the art from microbiological practice. The production strains disclosed in the present invention for the production of biotin and DTB originate from metabolic engineering approaches.

[0037] In contrast, biotransformation is defined as the conversion of one or more reactants into a product under enzymatic catalysis, whereby the enzyme substrate is added to a reaction mixture with the enzyme and is enzymatically converted.

[0038] In the context of the present invention, the enzymatic conversion of DTB to biotin would be a biotransformation.

[0039] The open reading frame (ORF, synonymous with cds, coding sequence) is the region of DNA or RNA that begins with a start codon and ends with a stop codon and encodes the amino acid sequence of a protein. The ORF is also called the coding region or structural gene.

[0040] A gene, or expression unit, is the DNA segment that contains all the basic information for producing biologically active RNA. A gene contains the DNA segment from which a single-stranded RNA copy is produced through transcription, and the expression signals involved in regulating this copying process. The expression signals include at least a promoter, a transcription start site, a translation start site, and a ribosome binding site (RBS). Further possible expression signals include a terminator and one or more operators.

[0041] For the purposes of this invention, bacterial proteins such as CoaA or PanK begin with a capital letter, while the sequences encoding these proteins (cds) are denoted by a lowercase letter (e.g., coaA or panK). Likewise, the promoters controlling the expression of these cds are denoted by a lowercase letter (e.g., tac promoter).

[0042] A gene construct is a DNA molecule produced by cloning that contains at least one expression unit and may also include other genetic elements, such as a selectable marker and replication origin. The gene construct can be a linear DNA molecule integrated into the genome or a circular DNA molecule in the form of a plasmid, also known as a vector. This vector is then referred to as an expression vector. When introduced into a suitable host strain (transformed), the genetic elements of the vector cause its extrachromosomal inheritance during cell growth and the production of the protein encoded by the plasmid.

[0043] A promoter is a nucleotide sequence located upstream of the 5' end of the CDS, which enables the expression of a CDS. The promoter is located upstream of the coding region in the direction of synthesis. The promoter contains regions that determine the start of transcription of the gene by RNA polymerase and can also mediate the specific interaction with DNA-binding proteins (transcription factors) that influence transcription intensity.

[0044] In principle, all promoters active in the host strain are suitable promoters. This includes, for example, all native promoters of the approximately 5000 genes in E. coli, but also non-native ones (e.g.

[0045] Promoters from other species of the family

[0046] Enterobacteriaceae') or "artificial" promoters such as the tac promoter. Preferred promoters are native promoters from a strain of the Enterobacteriaceae family and artificial promoters such as the tac promoter, particularly preferred are native promoters from E. coli and artificial promoters such as the tac promoter. The tac promoter is particularly preferred, as described, for example, in De Boer et al. (1983), Proc. Natl. Acad. Sci. USA 80: 21-25, Fig. 2 (referred to therein as PtacI).

[0047] An mRNA, also called messenger RNA, is a single-stranded ribonucleic acid (RNA) that carries the genetic information for building a protein. With an mRNA, the blueprint for a specific protein is available in a cell. The mRNA molecule carries the message from the genetic information (DNA) necessary for protein construction to the protein-building ribosomes. In a cell, it is formed as a transcript of a section of DNA belonging to a gene. The genetic information stored in the DNA is not altered in the process.

[0048] Homologous genes or homologous DNA sequences are understood to mean that the nucleotide sequences of these genes or DNA segments are at least 70%, preferably at least 80% and particularly preferably at least 90% identical.

[0049] The degree of DNA identity is determined using the "nucleotide blast" program, available at http: / / blast.ncbi.nlm.nih.gov / , which is based on the blastn algorithm. The preset parameters were used as algorithm parameters for the alignment of two or more nucleotide sequences. The preset general parameters are: Max target sequences = 100; Short queries = "Automatically adjust parameters for short input sequences"; Expect Threshold = 10; Word size = 28; Automatically adjust parameters for short input sequences = 0. The corresponding preset scoring parameters are: Match / Mismatch Scores = 1.00-2.00; Gap Costs = Linear.

[0050] Homologous protein sequences mean that the amino acid sequences of these proteins or protein segments are at least 70%, preferably at least 80% and particularly preferably at least 90% identical.

[0051] The program "protein blast" on the website http: / / blast.ncbi.nlm.nih.gov / is used to compare protein sequences. This program uses the blastp algorithm. The preset parameters were used as algorithm parameters for aligning two or more protein sequences. The preset general parameters are: Max target sequences = 100; Short queries = "Automatically adjust parameters for short input sequences"; Expect Threshold = 10; Word size = 3; Automatically adjust parameters for short input sequences = 0. The preset scoring parameters are: Matrix = BLOSUM62; Gap Costs = Existence: 11; Extension: 1; Compositional adjustments = Conditional compositional score matrix adjustment.

[0052] Recombinantly expressed proteins are those produced using genetically modified microorganisms or genetically modified cell cultures.

[0053] Homologous expression means that a DNA segment (e.g. gene, cds or gene fragment) is recombinantly expressed in the microorganism from which it was isolated.

[0054] Heterologous expression means that a DNA segment (e.g. gene, cds or gene fragment) is recombinantly expressed in a different microorganism than the one from which it was isolated.

[0055] The abbreviation WT (Wt) stands for wild type. A wild-type gene is the form of a gene that has arisen naturally through evolution and is present in the wild-type genome. The DNA sequence of Wt genes is publicly available in databases such as NCBI.

[0056] Mutants are defined as the states of a gene that can be interconverted by changes in the nucleotide sequence of the DNA. The gene naturally occurring in a microorganism is referred to as the wild-type gene, and the variants derived from it are referred to as mutated genes. The mutants of the present invention are point mutants, in which the DNA sequence of the gene is altered in such a way that only one amino acid of the protein sequence changes.

[0057] Recombinant expression of the CoaA enzyme is achieved by culturing the production strain. Culture can be performed on a laboratory scale using shake flask culture or on an industrial scale using fermentation, and pantothenate kinase activity can be assayed using an aliquot of the resulting culture broth. "Culture" is the generic term for both shake flask culture and fermentation. Fermentation is preferred for cell cultivation on an industrial scale.

[0058] Fermentation is a process step for the production (cultivation) of cell cultures on an industrial scale, in which a preferred microbial production strain is induced to grow under defined conditions of culture medium, temperature, pH, oxygen supply, and medium mixing. If all components of the fermentation are determined at the beginning of the cultivation and then remain unchanged, it is referred to as batch fermentation.

[0059] If media components such as

[0060] If glucose (C source) or a complex amino acid mixture such as yeast extract (N source) is continuously added as a so-called feed, this is referred to as fed-batch fermentation (so-called feed process). Fed-batch fermentation can optimize the formation of biomass and target product. The aim of fermentation is, depending on the configuration (genetic makeup) of the production strain, the production of a protein / enzyme or a metabolite, in each case with the highest possible yield for further use. The product biotin can be produced by fermentation. The end product of fermentation is a fermenter broth consisting of the biomass of the cells of the production strain (fermenter cells) and the fermentation medium (fermentation supernatant) freed of the biomass, which was formed during fermentation from the culture medium and the metabolites secreted by the fermenter cells.In contrast to the culture medium, which is defined by its chemical composition, the composition of the fermentation medium is not clearly defined due to the unpredictable formation of metabolic products. In the present invention, the product biotin is produced by fermentation. Biotin and / or DTB can either be used directly from the fermenter broth without further processing steps or can be enriched or purified using known methods. Such methods are familiar to the person skilled in the art, for example, from processes for

[0061] Isolation of amino acids is known. These include, for example, filtration, centrifugation, extraction, adsorption, ion exchange chromatography, precipitation, and crystallization.

[0062] The use according to the invention of feedback-resistant pantothenate kinases for the production of biotin and / or DTB comprises the use of a recombinant microorganism strain, wherein the microorganism strain is also referred to as a microbial production strain.

[0063] Production strain is a microorganism strain that can produce the desired product, in this case biotin and / or DTB, and by definition comprises a microorganism, referred to as host strain, and at least one gene construct.

[0064] A production strain for the production of biotin and / or DTB is characterized in that it contains a gene construct which comprises an expression unit, at least comprising the cds of a feedback-resistant pantothenate kinase, preferably the cds of a feedback-resistant mutant of a pantothenate kinase which is feedback-sensitive in its WT form, each functionally linked to a promoter.

[0065] The production strain is preferably characterized in that the gene construct is an expression vector.

[0066] Any microorganism capable of a biotin biosynthesis pathway that is amenable to recombinant DNA techniques is suitable as a host strain. The biotin biosynthesis pathway is genetically defined by the genes bioA (7,8-diaminononanoate transaminase, enzymatic activity of proteins of class EC 2.6.1.62), bioB (biotin synthase, EC 2.8.1.6), bioC (malonyl-CoA O-methyltransferase, EC 2.1.1.197), bioD (dethiobiotin synthase, EC 6.3.3.3), bioF (8-amino-7-oxononanoate synthase, EC 2.3.1.47), and bioH (pimeloyl-[acyl-carrier protein] methyl ester esterase, EC 3.1.1.85) or by genes functionally analogous to these genes. An overview of the biotin biosynthesis pathway is provided in the KEGG Pathway Database under the entry "Biotin Metabolism." Microorganisms with a biotin biosynthesis pathway are also able to grow in a biotin-free culture medium without biotin supplementation.

[0067] The gene expressing a coenzyme A feedback-resistant enzyme with the enzymatic activity of a pantothenate kinase (enzymatic activity of proteins of class EC 2.7.1.33) is preferably a bacterial gene, particularly preferably a bacterial gene from the family Enterobacteriaceae and especially preferably the coaA gene of Escherichia coli.

[0068] In a preferred embodiment of the invention, the coenzyme A feedback-resistant mutants of pantothenate kinase are mutants of the E. coli coaA gene with a cds selected from SEQ ID NO: 3, encoding a protein with

[0069] SEQ ID NO: 4 or SEQ ID NO: 5, encoding a protein with

[0070] SEQ ID NO: 6 or SEQ ID NO: 7, encoding a protein with

[0071] SEQ ID NO: 8 or a nucleotide sequence that is at least 70%, particularly preferably at least 80%, and especially preferably at least 90% identical thereto, which encodes a protein with pantothenate kinase activity that is coenzyme A feedback-resistant. The method is preferably characterized in that the coenzyme A feedback-resistant enzyme with the enzymatic activity of a pantothenate kinase has the amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8 or an amino acid sequence that is at least 70%, particularly preferably at least 80%, and especially preferably at least 90% identical thereto. It is particularly preferred that the amino acid sequence of the enzyme comprises the mutation R106A.

[0072] In a particularly preferred embodiment, the coenzyme A feedback-resistant mutant of pantothenate kinase is the mutant of the E. coli coaA gene with the cds SEQ ID NO: 3. The method is accordingly preferably characterized in that the enzyme has the amino acid sequence of SEQ ID NO: 4.

[0073] In a further preferred embodiment of the invention, the amino acid sequence of the WT protein of the pantothenate kinase which is then mutated to CoA feedback-resistant contains a conserved amino acid sequence motif selected from the following peptide sequences: i) Peptide sequence GSVAVGKST (TS)(AS)R (VLI)LQX (LI)L, as indicated, for example, in the amino acid sequence SEQ ID NO: 2 from position 95 to 112, where T at position 104 of SEQ ID NO: 2 can also be an S, A at position 105 of SEQ ID NO: 2 can also be an S, V at position 107 of SEQ ID NO: 2 can also be an L or

[0074] I and L at position 111 of SEQ ID NO: 2 can also be an I, or ii) peptide sequence AP(VI)YSH(X)(X)YD, as given, for example, in the amino acid sequence SEQ ID NO: 2 from positions 172 to 181, where V at position 174 of SEQ ID NO: 2 can be an I, the amino acids L at position 178 and I at position 179 of SEQ ID NO: 2 are not conserved, or

[0075] Peptide sequence RFL (KAS)F (RL), as for example in the

[0076] Amino acid sequence SEQ ID NO: 2 from position 243 to 248, where K at position 246 of SEQ ID NO: 2 can be an S or A and the amino acid R at position 248 of SEQ ID

[0077] NO: 2 can be an L. A conserved amino acid sequence motif (peptide sequence motif) is defined as a short amino acid sequence within the protein sequence of a protein that occurs in many homologous sequences of other microorganisms and denotes a sequence section that is important for the function of the protein. A conserved amino acid sequence motif can also contain individual amino acids that are replaced by one or two other amino acids in homologous sequences. In peptides i) to iii), these amino acids are explicitly named and preferred. Non-conserved amino acids can be any amino acid and are designated by the placeholder X and are not preferred. Peptide ii), for example, contains two non-conserved amino acids. The amino acid sequence is represented in a one-letter code, as is familiar to the person skilled in the art.

[0078] Particularly preferably, the WT protein sequence then comprises

[0079] CoA feedback-resistant mutated pantothenate kinase

[0080] Amino acid sequence motif selected from the peptide sequences GSVAVGKST (TS)(AS)R (VLI)LQX (LI)L, as indicated, for example, in the amino acid sequence SEQ ID NO: 2 from position 95 to 112, and AP(VI)YSH(X)(X)YD, as indicated, for example, in the amino acid sequence SEQ ID NO: 2 from position 172 to 181. Particularly preferably, the WT protein sequence of the pantothenate kinase then mutated to CoA feedback-resistant comprises the amino acid sequence motif GSVAVGKST (TS)(AS)R(VLI)LQX (LI)L, as indicated, for example, in the amino acid sequence SEQ ID NO: 2 from position 95 to 112.

[0081] The pantothenate kinase enzyme activity test can be carried out as follows: i) Preparation of the enzyme to be tested:

[0082] An enzyme produced by cultivation in a shake flask or in a fermentation can be used in the reaction as follows: as an aliquot from the not further processed

[0083] Culture broth or as an aliquot of the cell suspension after re-isolation of the cells from the culture broth, e.g. by centrifugation or in the form of an aliquot of the cell homogenate a) after mechanical disruption of the cell suspension or b) in the form of chemically permeabilized cells (e.g. by chloroform) or as a cell extract after separation of particulate components from the cell homogenate or as e.g. chromatographically purified enzyme.

[0084] The total protein concentration obtained in each case can be determined, for example, with a commercially available Qubit 3.0 fluorometer from Thermo Fisher Scientific using the "Qubit® Protein Assay Kit" according to the manufacturer's instructions. ii) Determination of pantothenate kinase enzyme activity: The test is carried out according to a test described by Strauss and Begley (2002), J. Biol. Chem. 277: 48205-48209, in which the pantothenate kinase enzyme reaction according to equation (1) (consumes ATP to form ADP) is linked to the reaction of pyruvate kinase, equation (2) (consumes phosphoenolpyruvate in the test by reaction with the phosphoenolpyruvate in the pantothenate kinase

[0085] reaction formed ADP with formation of pyruvate and ATP) and lactate dehydrogenase, equation (3) (reduces pyruvate to lactate with consumption of NADH).

[0086] (2) Phosphoenolpyruvate + ADP -> Pyruvate + ATP

[0087] (3) Pyurvate + NADH -> lactate + NAD

[0088] Thus, for each molecule of ADP from the pantothenate kinase reaction, one molecule of NADH is stoichiometrically consumed, which allows the determination of pantothenate kinase activity in the following assay.

[0089] A solution buffered to pH 7.6 with Tris-HCl contains the following test components (final concentration in the solution in parentheses): ATP (1.5 mM), NADH (0.3 mM), phosphoenolpyruvate (0.5 mM), MgCl2x 7 H2O (10 mM), KCl (20 mM), pyruvate kinase (5 units / ml), lactate dehydrogenase (5 units / ml), and pantothenate kinase from i). The amount of pantothenate kinase used from i) depends on the degree of purification. If culture broth, cell suspension of the reisolated cells, cell homogenate, or cell extract is used, at least 0.1 mg of the enzyme fractions prepared in i) is used. In the case of purified enzyme, at least 5 pg of the purified enzyme fraction is used. The assay volume is 1 ml. The temperature at which the assay is performed is 25°C. The test mixture is placed in a 1 ml cuvette in a spectrophotometer set to a wavelength of 340 nm.The reaction is initiated by the addition of Ca pantothenate (160 μM final concentration). The measurement time is 5 minutes. The decrease in absorbance at 340 nm (consumption of NADH) indicates the activity of pantothenate kinase according to equations (1) to (3). A ΔE at 340 nm of at least 0.02 is required after five minutes.

[0090] Measurement duration (ΔE = E0- E 5min : Decrease of extinction E0starting from time 0 min to extinction E 5min after completion of the measurement after 5 min) is set as the detection limit. If the ΔE at 340 nm is less than 0.02 under the stated test conditions, then the mixture does not contain active pantothenate kinase.

[0091] The feedback-resistant mutant of a pantothenate kinase and its use according to the invention is characterized in that it

[0092] I) has the enzyme activity of an enzyme designated EC 2.7.1.33 in the KEGG database, which catalyses the reaction of pantothenic acid to 4'-phosphopantothenate according to equation (1),

[0093] II) their enzyme activity is not feedback-inhibited by coenzyme A,

[0094] III) their recombinant expression in a host strain leads to the production of free biotin and / or DTB, which can be isolated from the cell culture supernatant.

[0095] The WT gene of the feedback-resistant mutant of a

[0096] Pantothenate kinase can originate from the same microorganism strain as the host strain (homologous pantothenate kinase) or it can be a foreign gene (heterologous pantothenate kinase), either synthetically produced (including the resulting adaptation of the cds to expression in the host strain through so-called codon optimization) or isolated from strains other than the host strain. The homologous gene of a pantothenate kinase is preferred.

[0097] Preferred variants of pantothenate kinase (CoaA) exhibit feedback inhibition by coenzyme A that is reduced by at least a factor of 5 compared to the corresponding wild-type enzyme. Particularly preferred CoaA variants exhibit feedback inhibition by coenzyme A that is reduced by at least a factor of 10 compared to the corresponding wild-type enzyme. Particularly preferred CoaA variants exhibit no feedback inhibition by coenzyme A.

[0098] The factor of reduced feedback inhibition by coenzyme A is determined by a Coenzyme A enzyme assay as described above, for example. The enzyme assay is carried out without and in the presence of various concentrations of coenzyme A, and the respective enzyme activity is determined. For the WT enzyme, a reference relationship is thus established between enzyme activity and the coenzyme A concentration in the enzyme assay (referred to as the control). The enzyme activity of a WT enzyme without added coenzyme A is defined as 100% activity, and the coenzyme A concentration at which 50% activity is still measured is referred to as the IC. 50 (Concentration of the inhibitor CoA in the test at which 50% of the enzyme activity is still measured without the addition of CoA). Analogously, the IC 50 for each mutant. The factor of reduced feedback inhibition is determined by measuring the IC 50 the mutant by the IC 50of the WT enzyme. For example, if a WT CoaA enzyme has an IC 50 for coenzyme A of 20 μM and the CoaA mutant an IC 50 of 100 μM, the factor of reduced feedback inhibition is five. This is referred to as IC 50 For a mutant with 200 μM coenzyme A, the factor of reduced feedback inhibition is ten. For a mutant with a 20-fold higher coenzyme A concentration than the IC 50 of the WT enzyme (corresponding to 400 μM coenzyme A in the chosen example) no IC50 can be determined because the enzyme activity is still >50% of the activity without inhibitor, this mutant is defined as not feedback-inhibitable by coenzyme A and shows no feedback inhibition by coenzyme A.

[0099] The mutated pantothenate kinase enzymes with reduced feedback inhibition known from Rock et al., 2002, represent preferred mutants of the CoaA enzyme for the production of production strains in the context of this invention: CoaA[R106A]: Arginine of the E. coli CoaA enzyme at position 106 replaced by alanine, designated as fbr mutant CoaA-R106A.

[0100] CoaA[H177Q]: histidine of the E. coli CoaA enzyme at position 177 replaced by glutamine, referred to as fbr mutant CoaA-H177Q.

[0101] CoaA[F247V]: phenylalanine of the E. coli CoaA enzyme at position 247 replaced by valine, referred to as fbr mutant CoaA-

[0102] F247V.

[0103] Microorganism strains suitable as production strains include bacterial strains selected from the families Enterobacteriaceae, Corynebacteriaceae, Bacillaceae or Gammaproteobacteria, as well as yeasts (e.g. Saccharomyces cerevisiae, Yarrowia lipolytica) or fungi (e.g. Aspergillus niger). The process is preferably characterized in that the microbial production strain is a bacterial strain, particularly preferably a strain of the family Enterobacteriaceae. The bacterial strain is preferably selected from the group consisting of Corynebacterium ssp. (such as particularly preferably C. glutamicum), Pseudomonas ssp. (such as particularly preferably P. mutabilis), Bacillus ssp. (such as particularly preferably B. subtilis), Pantoea ssp. (such as particularly preferably P. ananatis), Raoultella ssp. (such as particularly preferably R. terrigena) and Escherichia ssp. (such as particularly preferred E. coli).The method is preferably characterized in that the microbial production strain is a strain of the species Escherichia coli, Raoultella terrigena, or Pantoea ananatis, and especially preferably of the species Escherichia coli. In a particularly preferred embodiment, the microorganism is the strain E. coli K12 W3110, commercially available under strain number DSM 5911 from the DSMZ.

[0104] German Collection of Microorganisms and Cell Cultures GmbH.

[0105] A production strain containing a gene construct comprising an expression unit for a feedback-resistant mutant of a pantothenate kinase and thus for biotin and DTB

[0106] Production is characterized in that the expression unit preferably contains at least one cds selected from the group of feedback-resistant mutants a), b) or c), particularly preferably selected from a) or c) and especially preferably the mutant a) of the coaA gene: a) The production strain comprises the cds of a modified coaA-

[0107] A gene in which the codon of a conserved arginine is mutated in such a way that the resulting point mutant still has pantothenate kinase activity but is no longer

[0108] Coenzyme A is inhibited. Preferably, the conserved arginine is arginine at position 106 of the amino acid sequence of SEQ ID NO: 2, whereby the arginine can be mutated to all other 19 of the 20 natural amino acids, provided that the mutant still has pantothenate kinase activity but is no longer inhibited by coenzyme A. Particularly preferably, the conserved arginine is arginine at position 106 of the amino acid sequence of SEQ ID NO: 2, whereby the arginine is mutated to alanine (mutation R106A). b) The production strain comprises the cds of an altered coaA-

[0109] gene in which the codon of a conserved histidine is mutated in such a way that the resulting point mutant still

[0110] Pantothenate kinase activity is no longer present

[0111] Coenzyme A is inhibited. The conserved histidine is preferably histidine at position 177 of the

[0112] Amino acid sequence of SEQ ID NO: 2, wherein the histidine can be mutated to any of the other 19 of the 20 natural amino acids, provided that the mutant still possesses pantothenate kinase activity but is no longer inhibited by coenzyme A. Particularly preferably, the conserved histidine is histidine at position 177 of the amino acid sequence of SEQ ID NO: 2, wherein the histidine is mutated to glutamine.

[0113] (Mutation H177Q). c) The production strain comprises the cds of a modified coaA-

[0114] gene in which the codon of a conserved phenylalanine is mutated in such a way that the resulting point mutant still

[0115] Pantothenate kinase activity is no longer present

[0116] Coenzyme A is inhibited. Preferably, the conserved phenylalanine is phenylalanine of position 247 of the amino acid sequence of SEQ ID NO: 2, whereby the phenylalanine can be mutated to all other 19 of the 20 natural amino acids, provided that the mutant still

[0117] Pantothenate kinase activity, but no longer by

[0118] Coenzyme A is inhibited. Particularly preferably, the conserved phenylalanine is phenylalanine at position 247 of the amino acid sequence of SEQ ID NO: 2, wherein the phenylalanine is mutated to valine (mutation F247V).

[0119] The cds responsible for the feedback-resistant mutant of

[0120] The cds encoding pantothenate kinase is either present extrachromosomally on a vector in the microorganism strain (plasmid-encoded) or is integrated into the genome of the microorganism strain (genome-encoded). It is preferred that the cds encoding the feedback-resistant mutant of pantothenate kinase is present extrachromosomally on a vector in the microorganism strain (plasmid-encoded). As described in Example 1 of the present invention, the cds of the E. coli coaA-WT gene (coaA-wt, SEQ ID NO:

[0121] 1), encoding a CoaA enzyme with SEQ ID NO: 2, and the three feedback-resistant point mutants described in Rock et al. 2002, namely coaA-R106A (SEQ ID NO: 3), encoding a CoaA enzyme with SEQ ID NO: 4, coaA-H177Q (SEQ ID NO: 5), encoding a CoaA enzyme with SEQ ID NO: 6, and coaA-F247V (SEQ ID NO: 7), encoding a CoaA enzyme with SEQ ID NO: 8, were cloned into the vector pKKj-SC101, also described in Example 1. This resulted in the vectors pcoaA-wt (Fig.

[0122] 2), pcoaA-R106A, pcoaA-H177Q, and pcoaA-F247V. The gene constructs were prepared according to the state of the art, preferably using conventional recombinant DNA techniques, as described in Example 1 and as are familiar to the person skilled in the art. In pcoaA-wt, pcoaA-R106A, pcoaA-H177Q, and pcoaA-F247V, the coaA-cds are each functionally linked to the tac promoter, so that their expression occurs under the control of the tac promoter.

[0123] The production of a production strain is carried out in a known manner by transforming the gene construct according to the invention into a preferred microorganism (host strain), preferably the gene construct pcoaA-R106A, pcoaA-H177Q or pcoaA-F247V, particularly preferably the gene construct pcoaA-R106A or pcoaA-F247V and especially preferably the gene construct pcoaA-R106A.

[0124] Preferred host strains for producing a production strain are selected from strains of the species Escherichia coli, Pantoea ananatis, or Raoultella terrigena, particularly preferably the strains E. coli K12 W3110 DSM 5911, Pantoea ananatis DSM30080, or Raoultella terrigena DSM2687. These strains are commercially available from DSMZ GmbH.

[0125] In a particularly preferred embodiment, the host strain is the strain E. coli K12 W3110 DSM 5911. Preferred production strains are the strains disclosed in Example 2 of the present invention:

[0126] E. coli W3110 x pcoaA-R106A

[0127] E. coli W3110 x pcoaA-H177Q

[0128] E. coli W3110 x pcoaA-F247V

[0129] R. terrigena x pcoaA-R106A

[0130] P. ananatis x pcoaA-R106A

[0131] Particularly preferred production strains are the strains:

[0132] E. coli W3110 x pcoaA-R106A

[0133] E. coli W3110 x pcoaA-H177Q

[0134] E. coli W3110 x pcoaA-F247V

[0135] The production strain is particularly preferred:

[0136] E. coli W3110 x pcoaA-R106A

[0137] The production of biotin and / or its biosynthetic precursor DTB is carried out by cultivating a production strain according to the invention in a culture medium.

[0138] Shake flask culture is used to cultivate microorganisms on a laboratory scale, in contrast to production-scale fermentation. While shake flask culture also requires a specific medium and pH, and cultures are carried out in the presence of oxygen and with constant agitation (shaking), more defined conditions regarding the medium, temperature, pH, oxygen supply, and medium mixing can be adjusted and regulated in the fermenter. Culture on a smaller scale, for example, in a shake flask, can also be used as a pre-culture for inoculating a larger-scale culture, for example, in a fermenter.

[0139] The production scale in fermentation typically starts at 0.5 L batch volume and can reach 100,000 L or more. The production scale in shake flask culture typically ranges from 10 ml to 1000 ml batch volume. Yield, as used herein, is defined as the amount of product obtained by cultivating a production strain. The yield can be expressed as the absolute amount of product (mmol or g, or mg) or as the volume yield (concentration) in the amount of product relative to the volume (mM or g / L, or mg / L).

[0140] In principle, the fermentation products can be present in the fermenter cells and / or in the fermentation medium. As disclosed in Example 8 of the present invention, biotin and DTB are found exclusively in the fermentation medium within the detection limit. Therefore, only the biotin and DTB produced by secretion into the fermentation medium are considered.

[0141] The process is preferably characterized in that the biotin content in the fermentation supernatant, based on the biotin content in the fermentation batch, is above 70%, preferably above 80%, and particularly preferably above 90%. The same applies to DTB. The biotin / DTB content in the fermentation supernatant corresponds to the extracellular (secreted) biotin / DTB. The biotin / DTB content in the fermentation batch is the sum of the extracellular and intracellular biotin / DTB content, also referred to as the total biotin / DTB content. The extracellular portion of biotin and DTB is determined as in

[0142] Example 3 (sample preparation). The intracellular content of biotin and DTB is determined as described in

[0143] Example 8 describes the detection limit of 0.1 mg / L biotin and 0.1 mg / L DTB, respectively.

[0144] The expression of a feedback-resistant mutant of pantothenate kinase in a production strain according to the invention is characterized by the fact that both biotin and DTB are synthesized in the cells of the microorganism and secreted from the cells. This extracellular production or accumulation, i.e. the biosynthesis of biotin and DTB followed by their secretion (export, secretion) from the production strain into the culture medium, offers the great advantage that the volume in which biotin and DTB can accumulate is not limited to the small volume of the cell contents (cystosol). This prevents high intracellular product concentrations from leading to toxic

[0145] effects.

[0146] The extracellular production of biotin and DTB also has the advantage of simplified product isolation, since the

[0147] Products can be isolated directly from the fermentation supernatant without the need for prior, laborious mechanical or chemical disruption of the cells. How biotin and DTB are secreted from the production strain is unknown. It may be a passive mechanism, with the products diffusing through the cell membranes, or one or more transport proteins may be involved in the secretion.

[0148] As disclosed in the examples of the present invention, fermentation or shake flask cultivation of a recombinant production strain according to the invention comprising a host strain transformed with a

[0149] Gene construct comprising a cds encoding a coenzyme A feedback-resistant pantothenate kinase, the production of biotin and / or DTB, while the non-transformed host strain, despite a naturally present biotin

[0150] biosynthetic pathway cannot produce biotin or DTB in detectable quantities. This was not expected given the current state of technology.

[0151] The process for the fermentative production of biotin and / or DTB is preferably characterized in that the fermentation volume is at least 0.5 L, with a production scale of at least 10 L being particularly preferred, of at least 1000 L being particularly preferred, and a fermentation volume of at least 10,000 L being especially preferred. The content of biotin or DTB can be quantified from the culture broth. For this purpose, the culture broth can be cultured with a cell density OD 600 / ml of at least 1.0 / ml, e.g. an aliquot of 1 ml is taken, then all solid components are separated, e.g. by centrifugation for five minutes at maximum speed in a tabletop centrifuge and the supernatant is quantified by LC-MS calibrated for biotin and DTB, as described in Example 3.

[0152] In a preferred embodiment, the biotin

[0153] Yield at the end of fermentation after a

[0154] Fermentation time of up to 65 h at least 10 mg / L, particularly preferably at least 20 mg / L, and especially preferably at least 50 mg / L, while the non-transformed host strain produces no detectable biotin, with the detection limit being approximately 0.1 mg / L biotin or DTB. Using the production strains according to the invention thus enables the previously unknown fermentative production of biotin without optimizing the biotin biosynthesis pathway for applications in the food, feed, and pharmaceutical sectors.

[0155] In an alternatively preferred embodiment, the process is characterized in that the DTB yield at the end of the fermentation after a fermentation time of up to

[0156] 65 h is at least 10 mg / L, particularly preferably at least 20 mg / L and especially preferably at least 50 mg / L.

[0157] Since DTB is a biosynthetic precursor of biotin, in a preferred embodiment the process is characterized in that dethiobiotin is produced, which is then converted in a biotransformation to biotin, which is then isolated from the biotransformation mixture.

[0158] DTB can be used directly in biotransformation or can be enriched or isolated beforehand, for example, to be used in a more concentrated form for biotransformation. DTB is particularly preferably used in biotransformation in enriched or isolated form, and especially preferably in isolated form.

[0159] Media for cultivating the production strain in shake flasks and by fermentation are familiar to experts in microbial cultivation. They typically consist of a carbon source (C source), a nitrogen source (N source), and additives such as vitamins, salts, and trace elements, as well as a sulfur source (S source), which optimize cell growth and biotin or DTB production. Among the possible media additives that can increase biotin or DTB production is pimelic acid.

[0160] (Heptanedioic acid, 1,5-pentanedicarboxylic acid, CAS number 111-16-0).

[0161] C sources are those that can be used by the production strain for biotin or DTB product formation. These include all forms of monosaccharides, including C6 sugars (hexoses) such as glucose, mannose, fructose, or galactose, as well as C5

[0162] Sugars (pentoses) such as xylose, arabinose, or ribose, as well as all conceivable di- and polysaccharides formed from them, such as sucrose, lactose, maltose, maltodextrin, starch, or the monomers or oligomers released from them by hydrolysis (enzymatic or chemical). Other usable carbon sources other than sugars or carbohydrates are acetic acid (or acetate salts derived therefrom), ethanol, glycerol, citric acid (and their salts), or pyruvate (and its salts). Gaseous carbon sources such as carbon dioxide or carbon monoxide are also conceivable.

[0163] Preferred C sources for cultivating the production strain are glucose, fructose, sucrose, mannose, xylose and arabinose, among which glucose and sucrose are particularly preferred and glucose is especially preferred.

[0164] N sources are those that can be used by the production strain for biomass formation. Examples of N sources that can be used for this purpose include: - Ammonia, gaseous or in aqueous solution as NH4OH or its salts such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium acetate or ammonium nitrate and / or the known nitrate salts such as KNO3, NaNO3, ammonium nitrate, Ca(NO3)2, Mg(NO3)2, as well as other N sources such as

[0165] Urea and / or

[0166] - complex amino acid mixtures such as yeast extract, proteose peptone, malt extract, soy peptone, casamino acids, corn steep liquor (liquid or dried as so-called CSD) and / or

[0167] - NZ-amines and / or

[0168] - Yeast Nitrogen Base.

[0169] The addition of a sulfur source, either as a single addition in batch form or as a continuous feed, is necessary for the efficient production of biotin or DTB. The continuous addition can be carried out as a pure feed solution or in a mixture with another feed component such as glucose. The process is preferably characterized in that it is carried out in the presence of at least one compound selected from a salt of sulfates, sulfites, dithionites, thiosulfates and sulfides, whereby the use of the respective acids is also conceivable given a given stability. Preferred sulfur sources are salts of sulfates, sulfites, thiosulfates and sulfides, particularly preferably salts of sulfates and thiosulfates. The process is particularly preferably characterized in that it is carried out in the presence of sulfate salts.Particularly preferred as the salt of the sulfate is a compound selected from the group consisting of sodium sulfate, ammonium sulfate and mixtures thereof.

[0170] Further sulfur sources are selected from the group of amino acids cysteine, methionine, hypotaurine and taurine, preferably selected from cysteine ​​and methionine, particularly preferably from L-cysteine ​​and D,L-methionine, and especially preferred is D,L-methionine.

[0171] The cultivation of microorganism cells can be carried out in the so-called batch mode, i.e., to obtain biomass, the culture medium is inoculated with a starter culture of the production strain (microorganism cells carrying one or more gene constructs), and then cell growth occurs without further feeding of nutrient sources. The cultivation can also be carried out in the so-called fed-batch mode (also called feed-in cultivation mode), i.e., a process for obtaining biomass in which, after an initial growth phase in batch mode, additional nutrient sources are added (feed). The feed can consist of the C source, the N source, the sulfur source, one or more vitamins or trace elements important for production, or a combination of the above. The feed components can be added together as a mixture or separately in individual feed lines. In addition, other

[0172] Media components as well as additives specifically increasing biotin or DTB production, such as pimelic acid, can be added to the feed. The feed can be supplied continuously or in portions (discontinuously), or in a combination of continuous and discontinuous

[0173] Feed. The process according to the invention for the fermentative production of biotin or DTB with a microbial production strain is preferably characterized in that the fermentative process is a process in the feed-

[0174] Batch mode.

[0175] Preferred carbon sources in the feed are glucose, sucrose, and glucose- or sucrose-containing plant hydrolysates, as well as mixtures of the preferred carbon sources in any desired ratio. Glucose is a particularly preferred carbon source in the feed. The carbon source is preferably added to the culture in such a way that the carbon source content in the fermenter does not exceed 10 g / L during the production phase. A maximum concentration of 2 g / L is preferred, particularly preferably 0.5 g / L, and especially preferably 0.1 g / L.

[0176] Preferred N sources in the feed are ammonia, gaseous or in aqueous solution as NH4OH and its salts ammonium sulfate, ammonium phosphate, ammonium acetate and ammonium chloride, furthermore urea, KNO3, NaNO3 and ammonium nitrate, yeast extract, proteose peptone, malt extract, soy peptone, casamino acids, corn steep liquor as well as NZ amines and yeast nitrogen base, among which particularly preferred are ammonia or ammonium salts, yeast extract, soy peptone or corn steep liquor (liquid or in dried form).

[0177] Preferred sulfur sources in the feed are salts of sulfates, sulfites, thiosulfates and sulfides, among which particularly preferred are salts of sulfates and thiosulfates and especially preferred are salts of sulfate, such as sodium sulfate and ammonium sulfate.

[0178] Further media additives include salts of the elements phosphorus, chlorine, sodium, magnesium, nitrogen, potassium, calcium, iron, and, in trace amounts (i.e., in μM concentrations), salts of the elements molybdenum, boron, cobalt, manganese, zinc, copper, and nickel. Furthermore, organic acids (e.g., acetate, citrate), amino acids (e.g., isoleucine), and vitamins (e.g., vitamin B1, vitamin B6) can be added to the medium.

[0179] Further media additives include all types of fatty acids, monocarboxylic acids as well as dicarboxylic acids in the form of the free

[0180] Fatty acid or one of its salts, preferably the C7 dicarboxylic acid pimelic acid.

[0181] Cultivation takes place under pH and temperature conditions that favor growth and biotin or DTB production of the production strain. The pH range is preferably from pH 5 to pH 9. A pH range of pH 5.5 to pH 8 is particularly preferred. A pH range of pH 6.0 to pH 7.5 is especially preferred.

[0182] The preferred temperature range for the growth of the production strain is 20°C to 40°C. The temperature range from 25°C to 37°C is particularly preferred, and from 29°C to 35°C is especially preferred.

[0183] The growth of the production strain can be carried out facultatively without

[0184] The cultures can be grown with oxygen (anaerobic cultivation) or with oxygen (aerobic cultivation). Aerobic cultivation with oxygen is preferred.

[0185] During aerobic cultivation of the strain according to the invention for biotin production, a saturation of the oxygen content of preferably at least 5% (v / v), particularly preferably at least 15% (v / v), and especially preferably at least 30% (v / v) is established. The regulation of the oxygen saturation in the culture is carried out automatically according to the state of the art via a combination of gas supply and

[0186] Stirring speed .

[0187] The oxygen supply can be ensured by introducing compressed air or pure oxygen. Aerobic cultivation with the introduction of compressed air is preferred. The compressed air supply range for aerobic cultivation is preferably 0.05 vvm to 10 vvm (vvm: introduction of compressed air into the fermentation mixture expressed in liters of compressed air per liter of fermentation volume per minute). A compressed air supply of 0.2 vvm to 8 vvm is particularly preferred, particularly preferably 0.4 to 6 vvm, and especially preferably 0.8 to 5 vvm.

[0188] The maximum stirring speed is preferably 2500 rpm, particularly preferably a maximum of 2000 rpm and especially preferably a maximum of 1800 rpm.

[0189] The cultivation time is preferably between 10 h and 200 h. A cultivation time of 20 h to 120 h is particularly preferred. A cultivation time of 30 h to 100 h is especially preferred.

[0190] In the process according to the invention for the fermentative production of biotin or DTB, in step i the claimed microbial production strain is cultivated and in step ii the

[0191] Fermentation supernatant isolated. This means that fermentation batches obtained using the described method contain biotin or DTB accumulated extracellularly in the fermentation supernatant. Biotin or DTB can be used directly or isolated from the fermentation medium. Various analytical methods for identifying, quantifying, and determining the purity of biotin or DTB are available, including spectrophotometry, NMR, gas chromatography, HPLC, mass spectroscopy, gravimetry, or a combination of these analytical methods.

[0192] The figures show the plasmids used in the examples.

[0193] Fig. 1: pKKj-SC101.

[0194] Fig. 2: pcoaA-wt.

[0195] Abbreviations used in the figures:

[0196] TetR: gene that confers resistance to tetracycline.

[0197] Ptac: tac promoter pSClOl ORI: origin of replication. coaA: coaA (pantothenate kinase gene) cds

[0198] EcoRI: cleavage site for the restriction enzyme EcoRI

[0199] Ndel: cleavage site for the restriction enzyme Ndel

[0200] Pstl: cleavage site for the restriction enzyme Pstl

[0201] Styl: cleavage site for the restriction enzyme Styl

[0202] The following examples serve to further illustrate the invention. Example 1: Preparation of coaA expression vectors

[0203] Vector pKKj-SC101:

[0204] The vector pKKj-SC101 (Fig. 1) was used to generate coaA expression vectors. pKKj-SC101 was generated from the vector pKKj. pKKj is disclosed in EP 2 670837 A1 (Wacker) and is a derivative of the expression vector pKK223-3. The DNA sequence of pKK223-3 is disclosed in the GenBank gene database under accession number M77749.1. Approximately 1.7 kb was removed from the 4.6 kb plasmid (bp 262–1947 of the DNA sequence disclosed in M77749.1), resulting in the 2.9 kb expression vector pKKj.

[0205] Vector pKKj-tet: Plasmid DNA from the vector pKKj was cut with BspHI, and the 1.9 kb vector fragment was isolated. The tetracycline resistance gene was isolated from the plasmid pACYC184 as a 1.4 kb gene fragment by PCR. The DNA sequence of pACYC184 is disclosed in the GenBank gene database under accession number X06403.1. The 1.4 kb PCR product comprised the sequence disclosed in X06403.1 from nt 1434 to nt 2870. The 1.4 kb PCR product was cloned into the 1.9 kb vector fragment. Correct cloning was verified by DNA sequencing (Eurofins Genomics). The vector pKKj-tet was created, in which the tetracycline resistance gene has the same orientation as in the vector pKKj-SC101 described below (Fig. 1).

[0206] Vector pKKj-SC101: Plasmid DNA of the vector pKKj-tet was cut with Style and NdeI, and the 2.4 kb vector fragment was isolated. The replication origin SC101 was generated by gene synthesis (Eurofins Genomics) as a 1.4 kb DNA fragment. The DNA sequence of the replication origin SC101 is disclosed in the GenBank gene database under the

[0207] Accession number K00042.1. The 1.4 kb DNA fragment comprised nt 477 to nt 1816 of the sequence disclosed in K00042.1. The 1.4 kb DNA fragment was cloned into the 2.4 kb vector fragment in reverse complementary form to the orientation disclosed in K00042.1. This resulted in the 3.8 kb vector pKKj-SClOl (Fig. 1).

[0208] Vectors pcoaA, pcoaA-R106A, pcoaA-H177Q and pcoaA-F247V:

[0209] Plasmid DNA of the vector pKKj-SC101 was cut with EcoRI and PstI and the 3.8 kb linearized vector was isolated.

[0210] The cds of the coaA variants to be cloned were prepared as 950 nt DNA fragments by PCR ("Phusion™ High-Fidelity" DNA Polymerase, Thermo Scientific™) with the primers coaa-13f (SEQ

[0211] ID NO: 9) and coaa-14r (SEQ ID NO: 10) and

[0212] Isolated by agarose gel electrophoresis (QIAquick® Gel Extraction Kit, Qiagen).

[0213] Template DNA for the PCR reactions were genomic DNA of E. coli K12 strain W3110 for the coaA WT gene (coaA-wt, SEQ ID NO: 1) as well as synthetic genes (produced at Eurofins Genomics) for the mutants coaA-R106A (SEQ ID NO: 3), coaA-H177Q (SEQ ID NO: 5) and coaA-F247V (SEQ ID NO: 7).

[0214] The respective 950 nt coaA DNA fragments were cloned into the 3.8 kb pKKj-SC101 vector fragment using the NEBuilder® cloning kit (NEB New England Biolabs). This resulted in the coaA expression vectors pcoaA-wt, pcoaA-R106A, pcoaA-H177Q, and pcoaA-F247V. The vector map for pcoaA-wt is shown in Fig. 2, representative of all four expression vectors.

[0215] Example 2: Production of production strains

[0216] The starting strains (host strains) for the production of production strains were the microorganism strains Escherichia coli K12 W3110, strain number DSM 5911, Raoultella terrigena, strain number DSM2687, and Pantoea ananatis, strain number DSM30080, commercially available from the DSMZ German Collection of Microorganisms and Cell Cultures GmbH. E. coli W3110 was transformed in a known manner with the vectors pcoaA-wt, pcoaA-R106A, pcoaA-H177Q, and pcoaAA-F247V, and transformants were selected on LBtet plates. LBtet plates contained 10 g / L tryptone (GIBCO™), 5 g / L yeast extract (BD Biosciences), 5 g / L NaCl, 15 g / L agar, and 15 mg / L tetracycline (Sigma-Aldrich). One transformant was selected as the production strain. The production strains were designated E. coli W3110 x pcoaA-wt, E. coli W3110 x pcoaA-R106A, E. coli W3110 x pcoaA-H177Q, and E. coli W3110 x pcoaA-F247V and were used for shake flask cultivation and fermentation and analysis of the

[0217] Production of biotin and dethiobiotin. In an analogous manner, R. terrigena and PP.. ananatis were transformed with the vector pcoaA-R106A. The production strains received the

[0218] Designation R. terrigena x pcoaA-R106A and P. ananatis x pcoaA-R106A.

[0219] Example 3: Cultivation of production strains in

[0220] Shake flasks and analysis of biotin, DTB and pantothenic acid

[0221] Pre-culture: E. coli W3110 was pre-cultured in LB medium

[0222] A preculture of each of the production strains E. coli W3110 x pcoaA-wt, E. coli W3110 x pcoaA-R106A, E. coli W3110 x pcoaA-H177Q, and E. coli W3110 x pcoaA-F247V was established in LBtet medium (10 g / L tryptone, 5 g / L yeast extract, 5 g / L NaCl, 15 mg / L tetracycline). Cultivation was carried out overnight at 37°C and 120 rpm.

[0223] Precultures of R. terrigena and P. ananatis were also prepared in LB medium, and of the production strains R. terrigena x pcoaA-R106A and P. ananatis x pcoaA-R106A in LBtet medium. Cultivation was carried out overnight at 30°C and 120 rpm.

[0224] Main culture: 0.5 ml of the respective preculture was transferred into a 300 ml Erlenmeyer flask (with baffle) containing 30 ml BS20 medium (WT strains E. coli W3110, R. terrigena, P. ananatis) or BS20 medium containing 15 mg / L tetracycline for all strains transformed with a plasmid.

[0225] Composition of BS20 medium: 4 g / L K2HPO4, 4 g / L KH2PO4, 10 g / L NH4 sulfate, 2 g / L Na2SO4, 1 g / L NH4Cl, 10 g / L yeast extract (Sigma-Aldrich), 3 g / L Na3Citrate x 2 H2O, 0.03 g / L FeSO4x 7 H2O, 0.05 g / L CaCl2x 2 H2O, 1 g / L MgSO4x 7 H2O, 20 g / L glucose, 0.1 g / LD,L-methionine, 5 mg / L thiamine (Sigma-Aldrich), 50 mg / L pyridoxal phosphate (Sigma-Aldrich) and 3 ml / L trace element solution.

[0226] Composition of the trace element solution: 0.15 g / L Na2Mo04x 2 H2O, 2.5 g / L H3BO3, 0.7 g / L CoCl2x 6 H2O, 0.25 g / L CuSO4x 5 H2O, 1.6 g / L MnCl2x 4 H2O, 0.3 g / L ZnSO4x 7 H2O.

[0227] The main cultures of the E. coli strains were incubated for 24 h at 37°C and 140 rpm in a chest shaker (Infors). The main cultures of the R. terrigena and P. ananatis strains were incubated for 24 h at 30°C and 140 rpm in a chest shaker (Infors). After 24 h, 1 ml samples were taken, and the cell density was determined as OD. 600 / ml value (optical density of the main culture, measured photometrically at 600 nm) measured with a Genesys™ 10S UV-Vis spectrophotometer from Thermo-Scientific™, and the content of biotin, dethiobiotin and, if applicable, pantothenic acid was determined by LC-MS (liquid chromatography coupled with mass spectrometry).

[0228] Sample preparation for quantification of biotin or DTB by LC-MS:

[0229] 1 ml of culture broth from the shake flask culture was centrifuged for 5 min at 13000 rpm (Heraeus™ Fresco™ 21 centrifuge).

[0230] The cell culture supernatant was isolated and analyzed by LC-MS for the

[0231] Content of biotin, dethiobiotin and pantothenic acid was examined.

[0232] LC-MS analysis of biotin, dethiobiotin and pantothenic acid:

[0233] For the quantitative determination of the compounds analyzed in the examples, an LC-MS method calibrated for biotin, dethiobiotin and pantothenic acid was used.

[0234] (Liquid chromatography combined with mass spectrometry) was used, with the reference substances used for calibration being commercially available (Sigma-Aldrich). The LC-MS instrument used was a UHPLC 1290 Infinity II (liquid chromatography) combined with a 6470 triple quadrupole mass spectrometer, both from Agilent. The ACQUITY® UPLC HSS T3 column (2.1 x 100 mm, particle size: 1.8 μm) from Waters™ was used for liquid chromatography. The mobile phases used were: Mobile phase A: 0.1% (v / v) formic acid in H2O. Mobile phase B: 0.1% (v / v) formic acid in acetonitrile. Running conditions for liquid chromatography: 0.5 min isocratic 0% eluent B, then gradient in 1 min to 15% eluent B, then 1.5 min isocratic 15% eluent

[0235] B, then a 2-min gradient to 100% mobile phase B, followed by 2 min isocratically with 100% mobile phase B. The flow rate was 0.4 ml / min. The analytes biotin, dethiobiotin, and pantothenic acid, separated by liquid chromatography, were quantified by mass spectrometry using state-of-the-art multi-reaction monitoring (MRM). The detection limit for biotin, DTB, and pantothenic acid was 0.1 mg / L each.

[0236] An introduction to the theoretical principles of mass spectrometry is available from the manufacturer Agilent (https: / / www.agilent .com / cs / library / slidepresentation / public / 5 991-5857 Agilent MS Theory DEE.pdf).

[0237] For the quantification of the signals from the mass spectrometry, the molecular weights listed in Table 1 for the respective analyte ("precursor ion") and for the signals generated during the mass spectrometry were used.

[0238] Molecular fragments ("product ion") were used. The polarity "+" in Table 1 means that positively charged ions were measured.

[0239] Tab. 1: Product ions used for quantification by MRM Multi Reaction Monitoring:

[0240]

[0241] Example 4: Utilization of pantothenate

[0242] The strains E. coli W3110, E. coli W3110 x pcoaA-wt, and E. coli W3110 x pcoaA-R106A were grown as described in Example 3, with the main cultures of the three strains each supplemented with 8 mg / L Ca pantothenate. After 24 h of incubation at 37°C, the cell density OD 600 / ml and the content of pantothenic acid in the culture supernatant was determined by LC-MS (Table 2).

[0243] Tab.2: Cell density and pantothenic acid content in

[0244] Culture supernatant after 24 h cultivation of the strains E. coli W3110, E. coli W3110 x pcoaA-wt and E. coli

[0245] W3110 x pcoaA-R106A in shake flask

[0246] Example 5: Production of biotin and dethiobiotin in E. coli strains

[0247] The strains E. coli W3110, E. coli W3110 x pcoaA-wt, E. coli W3110 x pcoaA-R106A, E. coli W3110 x pcoaA-H177Q and E. coli W3110 x pcoaA-F247V were grown as described in Example 3. After 24 h of incubation at 37°C, the cell density OD 600 / ml, the content of biotin and dethiobiotin in

[0248] Culture supernatant determined by LC-MS (Table 3).

[0249] Tab. 3: Cell density, biotin and dethiobiotin content in

[0250] Culture supernatant after 24 h cultivation of the strains E. coli W3110, E. coli W3110 x pcoaA-wt, E. coli W3110 x pcoaA-R106A, E. coli W3110 x pcoaA-H177Q and E. coli W3110 x pcoaA-F247V in shake flasks

[0251] Example 6: Production of biotin and dethiobiotin in R. terrigena strains

[0252] The cultivation of the strains R. terrigena and R. terrigena x pcoaA-

[0253] R106A was carried out as described in Example 3. After 24 h of incubation at 30°C, the cell density OD 600 / ml, the

[0254] Content of biotin and dethiobiotin in the culture supernatant by LC-

[0255] MS determined (Table 4).

[0256] Tab. 4: Cell density, biotin and dethiobiotin content in

[0257] Culture supernatant after 24 h cultivation of the strains R. terrigena and R. terrigena x pcoaA-R106A in

[0258] Shake flask Example 7: Production of biotin and dethiobiotin in P. ananatis strains

[0259] The cultivation of the strains P. ananatis and P. ananatis x pcoaA-

[0260] R106A was carried out as described in Example 3. After 24 h of incubation at 30°C, the cell density OD 600 / ml, the

[0261] Content of biotin and dethiobiotin in the culture supernatant by LC-

[0262] MS determined (Table 5).

[0263] Tab. 5: Cell density, biotin and dethiobiotin content in

[0264] Culture supernatant after 24 h cultivation of the strains P. ananatis and P. ananatis x pcoaA-R106A in

[0265] Shake flask

[0266] Example 8: Extraction of biotin and DTB from cells of the

[0267] Shake flask culture

[0268] Shake flask culture of the

[0269] Production strain E. coli W3110 x pcoaA-R106A from Example 5 with a biotin content of 1.2 mg / L and DTB of 0.7 mg / L (Table 2). 30 ml of the shake flask culture was incubated for 10 min at

[0270] The cells were centrifuged at 6300 rpm (Thermo Scientific™ Multifuge X1R, TX-400 rotor), and the supernatant was discarded. The cell pellet was resuspended in 10 ml of water, centrifuged for 5 min at 6300 rpm, and the supernatant was discarded. The cell pellet was suspended in 2 ml of water. A cell extract was prepared from the cell suspension using a FastPrep-24™ 5G cell homogenizer from MP Biomedicals™. The cell pellet, suspended in 2 ml of water, was lysed in two 1 ml aliquots in 1.5 ml tubes pre-filled by the manufacturer with glass beads ("Lysing Matrix B") (3 x 20 seconds at a shaking frequency of 6000 rpm with a 30 seconds break between each interval). The resulting cell homogenates were combined and centrifuged for 5 min at 13,000 rpm (Heraeus™ Fresco™ 21 centrifuge) to produce a cell extract. The cell extract was analyzed for biotin and DTB content by LC-MS.Neither biotin nor DTB could be detected, with a detection limit of the LC-MS analysis of 0.1 mg / L for biotin and DTB, respectively.

[0271] Example 9: Production of biotin or DTB by fermentation

[0272] Pre-culture 1:

[0273] 20 ml of LBtet medium was inoculated with the production strain E. coli W3110 x pcoaA-R106A in a 100 ml Erlenmeyer flask and incubated for 7 h on a shaker (150 rpm, 34°C). 20 ml of LB medium was inoculated with the WT strain in a 100 ml Erlenmeyer flask.

[0274] E. coli W3110 and inoculated for 7 h on a shaker (150 rpm,

[0275] 34°C).

[0276] Pre-culture 2:

[0277] Subsequently, precultures 1 were completely transferred to 100 ml of BS20 medium, whereby the medium for preculture 2 of the E. coli W3110 x pcoaA-R106A strain was supplemented with 15 mg / L tetracycline (for the composition of the BS20 medium, see Example 3). The cultures were each shaken in an Erlenmeyer flask (1 L volume) at 34°C for 17 h at 150 rpm (Infors chest shaker). After this incubation, the cell densities OD 600 / ml between 3 and 5.

[0278] Main crop:

[0279] The fermentation was carried out in a fermenter of the type "DASGIP® Parallel Bioreactor Systems for Microbiology" from Eppendorf. A culture vessel with a total volume of 1.81 was used. The culture medium consisted of 600 ml

[0280] BS20 medium and 15 mg / L tetracycline (strain E. coli W3110 x pcoaA-R106A), or from 600 ml BS20 medium (strain E. coli

[0281] W3110).

[0282] The pH in the fermenter was initially adjusted to 7.0 by pumping in a 25% NH4OH solution. During fermentation, the pH was maintained at 7.0 by automatic correction with 25% NH4OH or 4 M H3PO4. Foam control was achieved by automatically dosing 4% v / v Struktol J673 in H2O (Schill & Seilacher).

[0283] For inoculation, 60 ml of the respective preculture 2 was pumped into the fermenter vessel. The initial volume was thus 660 ml. The cultures were initially stirred at 400 rpm and aerated with compressed air purified through a sterile filter at an aeration rate of 2 vvm (vvm: compressed air input into the fermentation mixture expressed in liters of compressed air per liter of fermentation volume per minute). Under these initial conditions, the oxygen probe was calibrated to 100% saturation before inoculation.

[0284] The target oxygen saturation during fermentation was set to 15%. Once the oxygen saturation dropped below the target value, a regulation cascade was initiated to return the oxygen saturation to the target value. First, the gas supply was continuously increased (to a maximum of 5 vvm) and then the stirring speed was continuously increased (to a maximum of 1,500 rpm). Fermentation was carried out at a temperature of 34°C.

[0285] Once the glucose content in the fermenter had dropped from an initial 20 g / L to approximately 2 g / L, a 56% (w / w) glucose solution was continuously added. The feed rate was adjusted so that the glucose concentration in the fermenter no longer exceeded 2 g / L. Glucose was determined using a glucose analyzer from YSI (Yellow Springs, Ohio, USA). The fermentation time was 65 h. 24 h, 42 h, and 65 h after

[0286] At the start of the fermentation, samples were taken from the fermentation mixture, the cell density OD 600 / ml and the content of biotin or DTB in the culture supernatant was determined by LC-MS.

[0287] Results for the strain E. coli W3110 x pcoaA-R106A are in

[0288] Tab. 6 summarizes. After a fermentation time of 65 h, the biotin yield was 20.1 mg / L and the DTB yield was 28.6 mg / L. In the parallel fermentation of the strain

[0289] Neither biotin nor DTB could be detected in E. coli W3110 (Table 7).

[0290] Tab. 6: Time course of cell density, biotin and DTB

[0291] Content of the fermentation of the strain E. coli W3110 x pcoaA-R106A

[0292] Tab. 7: Time course of cell density, biotin and DTB

[0293] Content of the fermentation of the strain E. coli W3110

Claims

Patent claims:

1. A process for the production of biotin, dethiobiotin (DTB) or a mixture thereof, characterized in that a microbial production strain is cultivated which recombinantly expresses at least one coenzyme A feedback-resistant enzyme with the enzymatic activity of a pantothenate kinase (enzymatic activity of proteins of class EC 2.7.1.33, CoaA), and then biotin, DTB or a mixture thereof is isolated.

2. The method according to claim 1, characterized in that the microbial production strain is a bacterial strain.

3. The method according to one or more of claims 1 or 2, characterized in that the microbial production strain is a strain of the species Escherichia coli, Raoultella terrigena or Pantoea ananatis.

4. The method according to one or more of claims 1 to 3, characterized in that the microbial production strain is a strain of the species Escherichia coli.

5. Process according to one or more of claims 1 to 4, characterized in that the cultivation of the cells on an industrial scale is carried out by fermentation.

6. The method according to one or more of claims 1 to 5, characterized in that the gene expressing the coenzyme A feedback-resistant enzyme with the activity of a pantothenate kinase is a mutated gene which, in its wt form, encodes a coenzyme A feedback-inhibited CoaA enzyme.

7. The method according to one or more of claims 1 to 6, characterized in that the mutated gene which expresses a coenzyme A feedback-resistant enzyme with the enzymatic activity of a pantothenate kinase is a bacterial gene.

8. The method according to one or more of claims 1 to 7, characterized in that the mutated gene which expresses a coenzyme A feedback-resistant enzyme with the enzymatic activity of a pantothenate kinase is a bacterial gene from the Enterobacteriaceae family.

9. The method according to one or more of claims 1 to 8, characterized in that the mutated gene expressing a coenzyme A feedback-resistant enzyme with the enzymatic activity of a pantothenate kinase is the coaA gene of Escherichia coli.

10. The method according to one or more of claims 1 to 9, characterized in that the coenzyme A feedback-resistant enzyme with the enzymatic activity of a pantothenate kinase has the amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 8 or an amino acid sequence which is at least 70% identical thereto.

11. The method according to claim 10, characterized in that the enzyme has the amino acid sequence of SEQ ID NO:

4.

12. Process according to one or more of claims 1 to 11, characterized in that the biotin content in the fermentation supernatant is more than 70% based on the biotin content in the fermentation batch.

13. Process according to one or more of claims 1 to 12, characterized in that the biotin yield at the end the fermentation after a fermentation period of up to 65 h is at least 10 mg / L.

14. Process according to one or more of claims 1 to 13, characterized in that the DTB yield at the end of the fermentation after a fermentation time of up to 65 h is at least 10 mg / L.

15. The method according to one or more of claims 1 to 14, characterized in that DTB is produced and converted in a biotransformation to biotin, which is then isolated from the biotransformation mixture.

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