Biotin production in microorganisms expressing pantothenate kinase genes with reduced inhibition of coenzyme A products

Abstract

The present invention relates to a method for producing biotin, devulcanized biotin (DTB) or a mixture thereof, characterized in that a microbial producing strain which recombinantly expresses at least one enzyme which is feedback resistant to CoA and has the enzymatic activity of pantothenate kinase (the enzymatic activity of EC 2.7. 1.33 class proteins) is cultured, and then biotin or DTB or a mixture thereof is isolated.

Description

Technical Field

[0001] The present invention provides a method for producing biotin, desthiobiotin (DTB) or a mixture thereof, characterized in that a microbial production strain that recombinantly expresses at least one enzyme that is feedback-resistant to coenzyme A and has the enzymatic activity of pantothenate kinase (enzymatic activity of a protein of class EC 2.7.1.33) is cultivated, and then biotin or DTB or a mixture thereof is isolated. Background of the Invention

[0003] Biotin (D-biotin, vitamin B7, vitamin H, CAS number 58-85-5) is a water-soluble vitamin from the vitamin B complex. As a prosthetic group in enzymes, it plays an important role in cellular metabolism, where it activates the essentially inert CO2 in carboxylation reactions. Examples of biotin-dependent enzymes include malonyl-CoA synthase, propionyl-CoA carboxylase, pyruvate carboxylase, and geranyl-CoA carboxylase. Biotin is a very economically important vitamin used as a food supplement and additive in feed, cosmetics, and the pharmaceutical industry.

[0004] Desthiobiotin (dethiobiotin, DTB, CAS No. 533-48-2) is the biosynthetic precursor of biotin. Biotin is formed by enzymatic incorporation of sulfur into DTB. The microbial biosynthesis of biotin and the regulation of its biosynthesis are known (see review by Sirithanakorn and Cronan, 2021, FEMS Microbiol. Rev. 45: fuab 003). The biosynthesis of biotin in microorganisms is strictly regulated so that no biotin can be detected in wild-type strains with a functional biotin biosynthetic pathway (see also the examples of the present invention). Evidence for a functional biotin biosynthetic pathway is provided by the fact that the wild-type strain can grow on a minimal medium (a chemically defined inorganic salt medium without biotin) to which no biotin is added, while the biotin-auxotrophic mutant of the strain cannot grow under the same conditions. Therefore, the absence of detectable biotin production in a microorganism does not mean that it is incapable of producing biotin; rather, it means that biotin biosynthesis is tightly regulated, and that biotin synthesized by the microorganism is immediately incorporated as a cofactor into the relevant enzymes and is not in free form. Furthermore, microorganisms can absorb biotin from the environment, and in the absence of an adequate supply of biotin, such as from the growth medium, biotin synthesis, which is metabolically expensive for the microorganism, will be completely shut down. Therefore, when there is an adequate external supply, the WT strain will not produce its own biotin.

[0005] Sirithanakorn and Cronan describe in detail the existing technology involved in regulating biotin biosynthesis.To date, this tight regulation of biotin biosynthesis (which ensures that only as much biotin is produced as the microorganism requires for its viability) has prevented the development of economically competitive biotechnological processes for the production of biotin.

[0006] 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 a key biosynthetic precursor of coenzyme A (CoA), which is used for the metabolism of activated 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 known (for review, see Leonardi and Jackowski, 2007, EcoSalPlus, 2). Coenzyme A is produced from pantothenic acid via five enzymatic stages. The first stage of Coenzyme A biosynthesis, ATP-dependent phosphorylation of pantothenic acid to form 4'-phosphopantothenic acid, is catalyzed by pantothenate kinase (EC 2.7.1.33, referred to as CoaA, CoaA enzyme, or PanK) according to Equation (1).

[0007] Formula (1):

[0008] (R)-Pantothenic acid + ATP <=> (R)-4'-P-cyclophospho-pantothenic acid + ADP + H +

[0009] Three types of CoaA enzymes are known in bacteria; see Hong et al. (2006), Structure 14:1251-1261. Type I CoaA is represented by, for example, the enzyme from Escherichia 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. Microorganisms with type II and type III CoaA enzymes are primarily pathogenic, and their genes are avoided as much as possible in metabolic engineering. No previous studies have investigated whether type I, type II, or type III CoaA enzymes affect biotin synthesis.

[0010] In E. coli, the pantothenate kinase enzyme, representing type I CoaA, is encoded by the coaA gene. The activity of the CoaA enzyme is inhibited by coenzyme A (the final product of the biosynthetic pathway). Therefore, coenzyme A is an inhibitor of the CoaA enzyme. In microorganisms such as E. coli, this form of product inhibition (feedback inhibition) is a feature of many metabolic pathways, and it ensures that the microorganism only produces as much metabolite (in this case, coenzyme A) as the organism requires. For coenzyme A biosynthesis, pantothenate kinase is therefore a key enzyme.

[0011] By observing the protein structure of the CoaA enzyme from Escherichia coli, Rock et al. (2003), J. Bacteriol 185: 3410-3415 identified three amino acids that are essential for the binding of CoA and thus mediate the inhibition of CoA enzyme activity by CoA (see Rock et al. Figure 2 These three amino acids are arginine at position 106 (R106), histidine at position 177 (H177), and phenylalanine at position 247 (F247). Another coenzyme A-interacting amino acid, lysine at position 101 of the protein sequence, is involved in binding the ATP substrate necessary for enzyme activity, and mutations in it result in an inactive enzyme.

[0012] The goal of Rock et al.'s research was to generate CoaA enzyme mutants with reduced feedback inhibition of enzyme activity by Coenzyme A. In the present invention, the expression "reduced feedback inhibition" is used synonymously with the expression "increased feedback resistance" (feedback resistance or anti-feedback is abbreviated as fbr). In the present invention, pantothenate kinase mutants that are feedback-resistant to Coenzyme A are therefore also referred to as CoaA enzymes or fbr mutants of pantothenate kinase.

[0013] According to Figure 4A of Rock et al., the WT CoaA enzyme is characterized in that its enzymatic activity in the presence of 20 μM CoA corresponds only to about 40% of the activity of the WT CoaA enzyme without any added CoA. CoA is therefore an inhibitor of the WT CoaA enzyme. The so-called inhibitor constant IC 50 represents the concentration of inhibitor at which the enzyme activity is only 50% of that in the absence of the inhibitor. According to Figure 4A of Rock et al., the IC of the CoaA enzyme from E. coli 50 Therefore less than 20 μM Coenzyme A.

[0014] Three point mutants of the CoaA enzyme, with amino acid mutations at R106 (fbr mutant CoaA [R106A]), H177 (fbr mutant CoaA [H177Q]), or F247 (fbr mutant CoaA [F247V]), are distinguished by the loss of product inhibition of CoA (see Rock et al. Figure 2and Figure 4A). The fbr mutants were characterized with respect to their enzymatic properties. Studies on the physiological effects of overexpression of feedback-resistant CoaA mutants, which are equivalent to deregulation of the CoA biosynthetic pathway, revealed the accumulation of phosphorylated metabolites such as 4'-phosphopantheine and CoA (Figure 6 of Rock et al.) in E. coli cells. Therefore, Rock et al. investigated how the expression of feedback-resistant CoaA mutants affects the content of various phosphorylated pantothenate-derived metabolites such as 4-phosphopanthenate and CoA in E. coli. EP 3 269 819 B1 used feedback-resistant CoaA mutants to produce O-acetylhomoserine. Studies on biotin production were not conducted by Rock et al. nor in EP 3 269 819 B1. There is no mechanistic connection between the metabolic pathways of CoA and biotin.

[0015] For commercial use, biotin is currently produced chemically. A thirteen-stage synthesis starting from fumaric acid is known. Due to the consumer-driven trend away from chemically produced ingredients, biotechnological processes for producing biotin are of great interest.

[0016] Known biotechnological methods for producing biotin-producing strains (see also Sirithanakorn and Cronan) focus primarily on the recombinant expression of various biosynthetic genes for biotin, i.e., genes bioA, bioB, bioC, bioD, bioF, and bioH, or functionally similar genes thereof. In addition, high-throughput screening (HTS) is used to isolate microbial strains with improved biotin production by mutagenesis and selection (Bali et al., 2020, MetAb. Eng. 60:97-107).

[0017] Thus, the prior art discloses various metabolic engineering and HTS methods for obtaining biotin from microbial production. Since all of these methods have met with limited success, chemical synthesis currently remains the method of choice for commercial production of biotin. The lack of applicability of the biotechnological methods known to date suggests a need for novel genetic elements capable of improving biotechnological processes for biotin production.

[0018] Therefore, an object of the present invention is to provide a method for fermentative production of biotin or its biosynthetic precursor desthiobiotin.

[0019] This object is achieved by a method for producing biotin, desthiobiotin (DTB) or a mixture thereof, characterized in that a microbial production strain is cultivated which recombinantly expresses at least one enzyme which is feedback-resistant to coenzyme A and has the enzymatic activity of pantothenate kinase (enzymatic activity of a protein of class EC 2.7.1.33), and then biotin or DTB or a mixture thereof is isolated.

[0020] As is known from the prior art (see above), it has also been found in the present invention that unmodified WT microorganisms do not produce detectable biotin (see Examples 5-7 herein). Biotin and the biosynthetic precursor DTB are neither detectable intracellularly nor extracellularly (see Example 8). However, it has been found that, surprisingly, the deregulation of the coenzyme A biosynthetic pathway by expressing recombinant feedback-resistant coaA mutants results in the production of biotin in microorganisms. This deregulation of the biotin biosynthetic pathway is of great interest for the development of biotechnological processes for the production of biotin by metabolic engineering, particularly since WT microbial strains cannot be used.

[0021] The gene that is recombinantly expressed in the production strain and expresses an enzyme that is feedback-resistant to Coenzyme A and has the enzymatic activity of pantothenate kinase (enzymatic activity of a protein of class EC 2.7.1.33) can be

[0022] 1. A WT gene encoding a CoaA enzyme that is not feedback inhibited by coenzyme A (e.g., encoding a type II or type III pantothenate kinase), wherein the WT gene is homologously overexpressed in the strain from which it is derived or heterologously expressed in a strain other than the strain from which it is isolated, preferably heterologously expressed, or

[0023] 2. A mutant gene which, in its Wt form, encodes a CoaA enzyme that is feedback inhibited by coenzyme A and, as a result of the mutation, encodes a feedback-resistant CoaA enzyme.

[0024] Preferably, the method is characterized in that the gene expressing an enzyme having feedback resistance to Coenzyme A and having pantothenate kinase activity is a mutant gene which, in its wt form, encodes a CoaA enzyme that is feedback inhibited by Coenzyme A. Preferably, the pantothenate kinase is type I bacterial CoaA.

[0025] The gene and protein sequences of WT pantothenate kinase are available, for example, in the NCBI database by searching the term "pantothenate kinase". Examples of pantothenate kinases relevant to the present invention are pantothenate kinases selected from the group consisting of 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. The protein and gene sequences of the selected pantothenate kinase can be found in sequence databases familiar to those skilled in the art, such as the sequence database of NCBI (National Center for Biotechnology Information).

[0026] The unexpected effect of the present invention is that homologous or heterologous expression of a pantothenate kinase whose Wt form is feedback-inhibited by coenzyme A but has been mutated to form an anti-feedback enzyme, or heterologous expression or homologous overexpression of a pantothenate kinase whose Wt form is not feedback-inhibited by coenzyme A, that is, in microorganisms such as Escherichia coli, P. ananatis and R. terrigena, the regulation and control of coenzyme A biosynthesis is released (see Examples 5-9) resulting in the regulation and control of biotin biosynthesis, thereby resulting in an amount of biotin and its biosynthetic precursor DTB being greater than the amount required for microbial survival. Thus, freely available biotin and DTB are formed. Freely available biotin and DTB are transferred out of the cell (exported, secreted), so that after removing the biomass, the two products can be easily separated from the cell culture supernatant.

[0027] For deregulation of biotin biosynthesis, homologous or heterologous expression of a pantothenate kinase is preferred, particularly preferably homologous expression, which in its wt form is feedback inhibited by coenzyme A and has been mutated to form a feedback-resistant enzyme. In this case, the pantothenate kinase is particularly preferably CoaA from Escherichia coli.

[0028] A major advantage of the present invention is that it provides a method for the fermentative production of biotin and / or DTB by using a microbial strain expressing a previously known feedback-resistant mutant of the CoaA enzyme without having to make any modifications to biotin metabolism. This was not known from the prior art and was undesirable because the metabolic pathways of Coenzyme A and biotin are unconnected. Summary of the Invention

[0029] definition:

[0030] In contrast to biotransformation, metabolic engineering (also known as "pathway design") 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 genes for enzymes, new or modified enzymes can be introduced into an organism, or genes for endogenous enzymes can be expressed at enhanced or reduced levels, thereby establishing new metabolic pathways in the organism or enhancing or reducing existing metabolic pathways. The goal of metabolic engineering is for an organism to produce new metabolites or endogenous metabolites in an increased yield. The metabolic engineering process does not use starting materials that are specific for the metabolites, such as enzyme substrates, such as DTB as a starting compound for the production of biotin; instead, it only uses a nutrient medium, also known as a growth medium, which is required for the growth of the organism in question and consists of a carbon source (e.g., glucose), a nitrogen 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 those skilled in the art from microbiological practice. The production strains disclosed in the present invention for the production of biotin and DTB are derived from metabolic engineering methods.

[0031] In contrast, biotransformation is defined as the conversion of one or more reactants into products under enzyme catalysis, with the enzyme substrate being added to the reaction along with the enzyme and being enzymatically converted. With respect to the present invention, the enzymatic conversion of DTB to biotin would be a biotransformation.

[0032] An open reading frame (ORF, synonymous with coding sequence or coding sequence) is a 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. An ORF is also called a coding region or structural gene.

[0033] A gene or expression unit is a portion of DNA that contains all the essential information for producing biologically active RNA. A gene consists of a DNA segment that produces a single-stranded RNA copy through transcription, as well as expression signals involved in regulating this copying process. Expression signals include at least a promoter, transcription initiation, translation initiation, and a ribosome binding site (RBS). A terminator and one or more operators are additional possible expression signals.

[0034] In the context of the present invention, bacterial proteins, such as CoaA or PanK, begin with an uppercase letter, while the sequence encoding the protein (cds) is identified by lowercase letters (e.g., coaA or panK). Similarly, the promoter that controls the expression of the cds is identified by lowercase letters (e.g., tac promoter).

[0035] Genetic construct refers to the DNA molecule produced by cloning, and it comprises at least one expression unit, and can also comprise other genetic elements beside it, such as selective marker and replication origin.Genetic construct can be the linear DNA molecule that is integrated into the genome, or it can be the circular DNA molecule of plasmid form, also synonymously referred to as vector.Then described vector is called expression vector.After being inserted (transformed) into suitable host strain, the genetic elements of vector produce the extrachromosomal inheritance of described vector during cell growth, and produce the protein encoded by cds.

[0036] A promoter is a nucleotide sequence that allows expression of a cds and is located upstream of the 5' end of the cds. In the direction of synthesis, the promoter precedes the coding region. A promoter contains a region that can define the start of transcription of a gene by RNA polymerase and mediate specific interactions with DNA-binding proteins (transcription factors) that influence transcription levels.

[0037] All promoters that are active in the host strain are generally suitable as promoters. These include, for example, all natural promoters of the approximately 5000 genes in E. coli, but also non-natural promoters (e.g., promoters from other species of the Enterobacteriaceae family) or "artificial" promoters such as the tac promoter. Preferred promoters are natural promoters and artificial promoters such as the tac promoter from strains of the Enterobacteriaceae family, particularly preferred natural promoters and artificial promoters such as the tac promoter from E. coli. Particularly preferred is the tac promoter, as described, for example, in De Boer et al. (1983), Proc. Natl. Acad. Sci. USA 80: 21-25, Figure 2 (where it is referred to as PtacI).

[0038] mRNA, also known as messenger RNA, is a single-stranded ribonucleic acid (RNA) that carries the genetic information for protein synthesis. mRNA provides the assembly instructions for specific proteins in the cell. mRNA molecules transfer the information necessary for protein synthesis from the genetic information (DNA) to the ribosomes responsible for protein synthesis. In the cell, it is formed as a transcript of the DNA segment corresponding to the gene. The genetic information stored in the DNA is not changed by this process.

[0039] Homologous genes or homologous DNA sequences are understood to mean that the nucleotide sequences of these genes or DNA parts are at least 70% identical, preferably at least 80% identical, particularly preferably at least 90% identical.

[0040] The degree of DNA identity was determined using the "nucleotide blast" program, which can be found at http: / / blast.ncbi.nlm.nih.gov / . The algorithm parameters used to align two or more nucleotide sequences were the default parameters. The default general parameters were: maximum target sequences = 100; short query = "Automatically adjust parameters for short input sequences"; expectation threshold = 10; word length = 28; Automatically adjust parameters for short input sequences = 0. The corresponding default scoring parameters were: match / mismatch score = 1, -2; gap cost = linear.

[0041] Homologous protein sequences are understood to mean that the amino acid sequences of these proteins or protein parts are at least 70% identical, preferably at least 80% identical, particularly preferably at least 90% identical.

[0042] Protein sequences were blasted using the protein blast program. http: / / blast.ncbi.nlm.nih.gov / Comparison. This program uses the blastp algorithm. The algorithm parameters used to align two or more protein sequences are the default parameters. The default general parameters are: Maximum target sequences = 100; Short query = "Automatically adjust parameters for short input sequences"; Expectation threshold = 10; Word length = 3; Automatically adjust parameters for short input sequences = 0. The default scoring parameters are: Matrix = BLOSUM62; Gap cost = Existence: 11 Extension: 1; Composition adjustment = Conditional composition score matrix adjustment.

[0043] Recombinant expression refers to the production of proteins with the aid of genetically modified microorganisms or genetically modified cell cultures.

[0044] Homologous expression is understood to mean that a portion of the DNA (eg a gene, cds or gene fragment) is expressed recombinantly in the microorganism from which it was isolated.

[0045] Heterologous expression is understood to mean that a portion of the DNA (eg a gene, cds or a gene fragment) is recombinantly expressed in a microorganism other than the microorganism from which it was isolated.

[0046] The abbreviation WT (Wt) refers to wild type. A wild-type gene refers to a form of a gene that occurs naturally through evolution and is present in a wild-type genome. The DNA sequence of the Wt gene is publicly available in databases such as NCBI.

[0047] Mutants define the state of a gene that can be transformed into another by changing the nucleotide sequence of the DNA. Genes naturally occurring in microorganisms are called wild-type genes, and variants derived from them are called mutant genes. The mutants of the present invention are point mutants, in which the DNA sequence of a gene is modified so that only one amino acid in the protein sequence is changed.

[0048] Recombinant expression of the CoaA enzyme is achieved by culturing the production strain. The culture can be grown on a laboratory scale by shake flasks or on an industrial scale by fermentation, and aliquots of the resulting culture broth are analyzed for pantothenate kinase activity, i.e., the term "culture" is a general term for growth in shake flasks or by fermentation. Preferably, the cells are cultured on an industrial scale by fermentation.

[0049] Fermentation is the process step for producing (culturing) cell cultures on an industrial scale, wherein preferably the microbial production strain is grown under defined conditions of culture medium, temperature, pH, oxygen supply and culture medium mixing. If all fermentation components are defined at the start of the culture and are not changed thereafter, the fermentation is referred to as batch fermentation.

[0050] If after fermentation starts, culture medium components such as glucose (carbon source) or complex amino acid mixtures such as yeast extract (nitrogen source) are continuously supplied as so-called feed, then fermentation is called fed-batch fermentation (so-called feed process). Fed-batch fermentation allows the optimization of the formation of biomass and target products. Depending on the configuration (genetic composition) of the production strain, the purpose of fermentation is to produce the highest possible yield of protein / enzyme or metabolites for further use. The product biotin can be produced by fermentation. The final product of fermentation is a fermentation broth composed of the biomass (fermentation cells) of the cells of the production strain and a biomass-free fermentation medium (fermentation supernatant) formed from the growth medium and the metabolites secreted by the fermentation cells during the fermentation process. In contrast to the growth medium defined by its chemical composition, the composition of the fermentation medium is not clearly defined because the formation of metabolites is unpredictable. In the present invention, the product biotin is produced by fermentation.

[0051] Biotin and / or DTB can further be used directly from the fermentor broth without further processing steps, or enriched or purified by known methods. Such methods are known to those skilled in the art, for example from methods used to separate amino acids. Examples include filtration, centrifugation, extraction, adsorption, ion exchange chromatography, precipitation, and crystallization.

[0052] The use of the feedback-resistant pantothenate kinase of the present invention for producing biotin and / or DTB includes the use of recombinant microbial strains, also referred to as microbial production strains. Production strains are microbial strains that can produce a desired product (in this case, biotin and / or DTB) and, by definition, comprise a microorganism (referred to as a host strain) and at least one genetic construct.

[0053] The production strain for producing biotin and / or DTB is characterized in that it contains a gene construct comprising an expression unit comprising at least the cds of a feedback-resistant pantothenate kinase, preferably the cds of a mutant of a feedback-resistant pantothenate kinase, which in its WT form is feedback-sensitive, in each case functionally linked to a promoter.

[0054] The production strain is preferably characterized in that the gene construct is an expression vector.

[0055] Suitable host strains are any microorganisms that contain a biotin biosynthetic pathway and can be produced by recombinant DNA technology. The biotin biosynthetic pathway is genetically defined by the genes bioA (7,8-diaminononanoate transaminase, enzymatic activity of the EC 2.6.1.62 protein class), bioB (biotin synthase, EC 2.8.1.6), bioC (malonyl-CoA O-methyltransferase, EC 2.1.1,197), bioD (desulfobiotin synthase, EC 6.3.3.3), bioF (8-amino-7-oxononanoate synthase, EC 2.3.1.47), and bioH (pimelate-[acyl carrier protein] methylester esterase, EC 3.1.1.85), or genes functionally similar to these genes. The KEGG pathway database provides an overview of the biotin biosynthetic pathway under the entry "biotin metabolism." Microorganisms with a biotin biosynthetic pathway are additionally able to grow in biotin-free growth media without the need for supplemental biotin.

[0056] The gene expressing an enzyme that is feedback-resistant to Coenzyme A and has pantothenate kinase enzymatic activity (enzymatic activity of EC 2.7.1.33 class protein) is preferably a bacterial gene, particularly preferably a bacterial gene derived from the family Enterobacteriaceae, particularly preferably the coaA gene from Escherichia coli.

[0057] In a preferred embodiment of the present invention, the pantothenate kinase mutant that is feedback-resistant to Coenzyme A is a mutant of the Escherichia coli coaA gene having a cds selected from SEQ ID NO: 3, encoding a protein having SEQ ID NO: 4 or SEQ ID NO: 5, encoding a protein having SEQ ID NO: 6 or SEQ ID NO: 7, encoding a protein having SEQ ID NO: 8, or a nucleotide sequence that is at least 70%, particularly preferably at least 80%, and particularly preferably at least 90% identical to a protein that has pantothenate kinase activity and is feedback-resistant to Coenzyme A. The method is preferably characterized in that the enzyme that is feedback-resistant to Coenzyme A and has pantothenate kinase activity has an 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 particularly preferably at least 90% identical to the amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8. Particularly preferably, the amino acid sequence of the enzyme contains the mutation R106A.

[0058] In a particularly preferred embodiment, the pantothenate kinase mutant having feedback resistance to coenzyme A is a mutant of the E. coli coaA gene having the cds SEQ ID NO: 3. Therefore, the method is preferably characterized in that the enzyme has the amino acid sequence of SEQ ID NO: 4.

[0059] In another preferred embodiment of the present invention, the amino acid sequence of the WT protein of pantothenate kinase, which is then mutated to become feedback-resistant to CoA, contains a conserved amino acid sequence motif selected from the following peptide sequences:

[0060] i) the peptide sequence GSVAVGKST(TS)(AS)R(VLI)LQX(LI)L, as specified, for example, from positions 95 to 112 in the amino acid sequence SEQ ID NO: 2,

[0061] wherein T at position 104 of SEQ ID NO: 2 may also be S, A at position 105 of SEQ ID NO: 2 may also be S, V at position 107 of SEQ ID NO: 2 may also be L or I, and L at position 111 of SEQ ID NO: 2 may also be I, or

[0062] ii) the peptide sequence AP(VI)YSH(X)(X)YD, as specified, for example, in the amino acid sequence SEQ ID NO: 2 from positions 172 to 181,

[0063] wherein the V at position 174 of SEQ ID NO: 2 may be I, and the amino acid L at position 178 and the I at position 179 of SEQ ID NO: 2 are non-conservative, or

[0064] iii) the peptide sequence RFL(KAS)F(RL), as specified, for example, from positions 243 to 248 in the amino acid sequence SEQ ID NO: 2,

[0065] wherein K at position 246 of SEQ ID NO: 2 may be S or A, and SEQ ID NO:

[0066] The amino acid R at position 248 of 2 may be L.

[0067] Conserved amino acid sequence motifs (peptide sequence motifs) are defined as short amino acid sequences within the protein sequence of a protein that are present in many homologous sequences from other microorganisms and indicate sequence segments that are important for protein function.

[0068] Conserved amino acid sequence motifs can also contain single amino acids replaced by one or two different amino acids in the homologous sequence. In peptides i) to iii), these amino acids are explicitly stated and preferred. Non-conserved amino acids can be any amino acid and are identified by the placeholder X and are not preferred. For example, peptide ii) contains two non-conserved amino acids. As is familiar to those skilled in the art, amino acid sequences are represented by single-letter codes.

[0069] Particularly preferably, the WT protein sequence that is then mutated to become a pantothenate kinase that is feedback-resistant to CoA comprises an amino acid sequence motif selected from the group consisting of the peptide sequences GSVAVGKST(TS)(AS)R(VLI)LQX(LI)L, for example as specified in the amino acid sequence of SEQ ID NO: 2 from positions 95 to 112, and AP(VI)YSH(X)(X)YD, for example as specified in the amino acid sequence of SEQ ID NO: 2 from positions 172 to 181. Particularly preferably, the WT protein sequence that is then mutated to become a pantothenate kinase that is feedback-resistant to CoA comprises an amino acid sequence motif GSVAVGKST(TS)(AS)R(VLI)LQX(LI)L, for example as specified in the amino acid sequence of SEQ ID NO: 2 from positions 95 to 112.

[0070] Pantothenate kinase activity assay can be performed as follows:

[0071] i) Production of the enzyme to be tested:

[0072] Enzymes produced by growth in shake flasks or in fermentation can be used in reactions as follows:

[0073] - as an aliquot from the culture broth without further treatment, or

[0074] - as an aliquot of a cell suspension, after redissociating the cells from the culture medium, for example by centrifugation, or

[0075] - in the form of an aliquot of said cell homogenate

[0076] a) after mechanical disruption of the cell suspension, or

[0077] b) by chemically permeabilizing cells (e.g., with chloroform)

[0078] or

[0079] - as a cell extract after removal of particulate components from said cell homogenate, or

[0080] - as an enzyme purified by, for example, chromatography.

[0081] The total protein concentration obtained in each case can be determined according to the manufacturer's instructions, for example by means of the commercially available Qubit 3.0 Fluorometer from Thermo Fisher Scientific, using the " Protein determination kit".

[0082] ii) Determination of pantothenate kinase enzyme activity: The procedure performed was the assay as described by Strauss and Begley (2002), J. Biol. Chem. 277: 48205-48209, wherein the pantothenate kinase reaction according to equation (1) (consuming ATP to form ADP) is coupled with a pyruvate kinase reaction according to equation (2) (consumed in the assay by reaction with the ADP formed in the pantothenate kinase reaction to form pyruvate and ATP) and a lactate dehydrogenase reaction according to equation (3) (reduction of pyruvate to form lactate with consumption of NADH).

[0083] (2) Phosphoenolpyruvate + ADP->gtpyruvate + ATP

[0084] (3) Pyruvate + NADH -> Lactate + NAD

[0085] Thus, for each ADP molecule from the pantothenate kinase reaction, one NADH molecule is consumed stoichiometrically, which allows the determination of pantothenate kinase activity in the following assay.

[0086] The solution, buffered to pH 7.6 with Tris-HCl, contained the following assay components (final concentrations in solution are given between brackets): ATP (1.5 mM), NADH (0.3 mM), phosphoenolpyruvate (0.5 mM), MgCl2x7H2O (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 from i) used depends on the purity. If culture medium, a cell suspension of re-isolated cells, a cell homogenate, or a cell extract is used, at least 0.1 mg of the enzyme fraction prepared in i) is used. In the case of purified enzyme, at least 5 μg of the purified enzyme fraction is used. The assay volume is 1 ml. The assay is performed at 25°C. The assay is contained in a 1 ml cuvette in a spectrophotometer set to a wavelength of 340 nm.

[0087] The reaction was started by adding calcium pantothenate (160 μM final concentration). The measurement time was 5 minutes. According to equations (1) to (3), the decrease in absorbance at 340 nm (NADH consumption) indicates the activity of pantothenate kinase. After a measurement time of five minutes, a ΔA of at least 0.02 at 340 nm (ΔA = A0-A 5min : The absorbance A0 at time 0 minutes decreases to the absorbance A after the measurement ends 5 minutes later. 5min ) was defined as the detection limit. If ΔA at 340 nm was less than 0.02 under the above assay conditions, this meant that the assay did not contain any active pantothenate kinase.

[0088] The feedback-resistant mutant of pantothenate kinase and the inventive use thereof are characterized in that:

[0089] I) having the enzymatic activity of an enzyme that is identified by EC 2.7.1.33 in the KEGG database and catalyzes the reaction from pantothenic acid to 4'-phosphopantothenate according to equation (1),

[0090] II) Its enzyme activity is not feedback inhibited by coenzyme A,

[0091] III) its recombinant expression in a host strain results in the production of free biotin and / or DTB,

[0092] It can be isolated from cell culture supernatant.

[0093] The WT gene of the feedback-resistant mutant of pantothenate kinase can be derived from a microbial strain that is also the host strain (homologous pantothenate kinase), or it can be an exogenous gene (heterologous pantothenate kinase) produced by synthesis (which particularly allows adaptation of the cds to expression in the host strain by so-called codon optimization) or isolated from a strain other than the host strain. Homologous genes of pantothenate kinase are preferred.

[0094] Preferred variants of pantothenate kinase (CoaA) have at least 5-fold reduced feedback inhibition by Coenzyme A compared to the corresponding wild-type enzyme. Particularly preferred CoaA variants have at least 10-fold reduced feedback inhibition by Coenzyme A compared to the corresponding wild-type enzyme. Particularly preferred CoaA variants do not have any feedback inhibition by Coenzyme A.

[0095] The factor that reduces the feedback inhibition of CoA is determined by the CoaA enzyme assay as described above, for example, by performing the enzyme assay in the absence of CoA and in the presence of different concentrations of CoA and determining the corresponding enzyme activity. This generates a reference relationship between enzyme activity and CoA concentration in the enzyme assay (called control) for the WT enzyme. The enzyme activity of the WT enzyme without any added CoA is defined as 100% activity, and the CoA concentration at which 50% activity is still measured is defined as IC 50 (The concentration of inhibitor CoA in the assay was such that 50% of the enzyme activity without added CoA was still measured.) Similarly, the IC 50 The feedback inhibition factor was reduced by changing the IC 50 Divide by the IC of WT enzyme 50 Thus, if the WT CoaA enzyme has an IC of, for example, 20 μM CoA 50 and the CoaA mutant had an IC of 100 μM 50 , then the feedback inhibition is reduced by a factor of 5. If the IC 50 For 200 μM CoA, the feedback inhibition is reduced by a factor of ten. If even at a CoA concentration lower than the IC of the WT enzyme (which in the chosen example corresponds to 400 μM CoA) 50 If no IC50 could be determined for the mutant even at a 20-fold higher concentration, the mutant was defined as not being feedback inhibited by CoA and exhibiting no feedback inhibition by CoA, as the enzyme activity was still >50% of the activity without inhibitor.

[0096] In the context of the present invention, the mutant pantothenate kinase with reduced feedback inhibition known from Rock et al., 2002 is a preferred mutant of the CoaA enzyme for generating production strains:

[0097] CoaA[R106A]: The arginine at position 106 of the Escherichia coli CoaA enzyme is replaced by alanine, which is called the fbr mutant CoaA-R106A.

[0098] CoaA[H177Q]: The histidine at position 177 of the Escherichia coli CoaA enzyme is replaced by glutamine, which is called the fbr mutant CoaA-H177Q.

[0099] CoaA[F247V]: The phenylalanine at position 247 of the Escherichia coli CoaA enzyme is replaced by valine, which is called the fbr mutant CoaA-F247V.

[0100] Suitable microbial strains as production strains include bacterial strains selected from the family Enterobacteriaceae, Corynebacteriaceae, Bacillaceae or γ-Proteobacteriaceae, and yeast (e.g., Saccharomyces cerevisiae, Yarrowia lipolytica) or fungi (e.g., Aspergillus niger). The method is characterized in that the microbial production strain is a bacterial strain, particularly preferably a strain from the family Enterobacteriaceae. The bacterial strain is preferably selected from Corynebacterium species (e.g., particularly preferably Corynebacterium glutamicum), Pseudomonas species (e.g., particularly preferably Aspergillus flavus), Bacillus species (e.g., particularly preferably Bacillus subtilis), Pantoea species (e.g., particularly preferably Pantoea ananas), Raoultella species (e.g., particularly preferably Raoultella terrestrial) and Escherichia species (e.g., particularly preferably Escherichia coli). The method is preferably characterized in that the microbial production strain is a strain of the species Escherichia coli, Raoultella terrestris or Pantoea ananas, particularly preferably Escherichia coli. In a particularly preferred embodiment, the microorganism is the Escherichia coli K12 W3110 strain, which is commercially available from the DSMZ-German Collection of Microorganisms and Cell Cultures GmbH under the strain number DSM 5911.

[0101] A production strain containing a gene construct comprising an expression unit of a feedback-resistant mutant of pantothenate kinase, and thus suitable for biotin and DTB production, characterized in that the expression unit preferably contains at least one cds selected from the group consisting of feedback-resistant mutants a), b) or c) of the coaA gene, particularly preferably selected from a) or c), particularly preferably mutant a):

[0102] a) the production strain comprises a modified coaA gene cds, wherein the codon for a conserved arginine has been mutated such that the resulting point mutant still has pantothenate kinase activity but is no longer inhibited by coenzyme A, preferably, the conserved arginine is the arginine at position 106 of the amino acid sequence of SEQ ID NO: 2, and

[0103] Arginine can be mutated to any of the 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 the arginine at position 106 of the amino acid sequence of SEQ ID NO: 2, which has been mutated to alanine (mutation R106A).

[0104] b) The production strain comprises the cds of a modified coaA gene, wherein the codon for a conserved histidine has been mutated so that the resulting point mutant still has pantothenate kinase activity but is no longer inhibited by coenzyme A. Preferably, the conserved histidine is the histidine at position 177 of the amino acid sequence of SEQ ID NO: 2, and the histidine may be mutated to any of the other 19 amino acids 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 histidine is the histidine at position 177 of the amino acid sequence of SEQ ID NO: 2, and the histidine has been mutated to glutamine (mutation H177Q).

[0105] c) The production strain comprises a modified coaA gene cds, wherein the codon for the conserved phenylalanine has been mutated so that the resulting point mutant still has pantothenate kinase activity but is no longer inhibited by coenzyme A. Preferably, the conserved phenylalanine is the phenylalanine at position 247 of the amino acid sequence of SEQ ID NO: 2, and the phenylalanine may be mutated to any of the 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 phenylalanine is the phenylalanine at position 247 of the amino acid sequence of SEQ ID NO: 2.

[0106] The phenylalanine at position 247 of the amino acid sequence of ID NO: 2 has been mutated to valine (mutation F247V).

[0107] The cds encoding the feedback-resistant mutant of pantothenate kinase is present extrachromosomally on a vector in the microbial strain (plasmid-encoded) or integrated into the genome of the microbial strain (genome-encoded). Preferably, the cds encoding the feedback-resistant mutant of pantothenate kinase is present extrachromosomally on a vector in the microbial strain (plasmid-encoded).

[0108] As described in Example 1 of the present invention, the E. coli coaAWT gene encoding the CoaA enzyme having SEQ ID NO: 2 (coaA-wt, SEQ ID NO: 1) and the cds of the three feedback-resistant point mutants described in Rock et al. 2002 (i.e., coa A-R106A encoding the CoaA enzyme having SEQ ID NO: 4 (SEQ ID NO: 3), coa A-H177Q encoding the CoaA enzyme having SEQ ID NO: 6 (SEQ ID NO: 5), and coa A-F247V encoding the CoaA enzyme having SEQ ID NO: 8 (SEQ ID NO: 7)) were cloned into the vector pKKj-SC101 also described in Example 1. The result was the vector pcoaA-wt ( Figure 2 ), pcoa A-R106A, pcoa A-H177Q and pcoa A-F247V. The gene constructs are generated according to the prior art and preferably with the aid of conventional recombinant DNA technology, as described in Example 1 and as are familiar to those skilled in the art. In pcoaA-wt, pcoa A-R106A, pcoa A-H177Q and pcoa A-F247V, the coaA cds is in each case functionally linked to the tac promoter, so that its expression is controlled by the tac promoter.

[0109] Production strains are produced in a known manner by transforming the gene constructs according to the invention into preferred microorganisms (host strains), preferably the gene constructs pcoa A-R106A, pcoa A-H177Q or pcoa A-F247V, particularly preferably the gene constructs pcoa A-R106A or pcoa A-F247V, particularly preferably the gene construct pcoa A-R106A.

[0110] Preferred host strains for producing the strains are selected from strains of Escherichia coli, Pantoea ananatis or Raoultella terrestris, particularly preferably strains of Escherichia coli K12 W3110 DSM 5911, Pantoea ananatis DSM 30080 or Raoultella terrestris DSM 2687. The strains are commercially available from DSMZ GmbH.

[0111] In a particularly preferred embodiment, the host strain is the strain Escherichia coli K12 W3110 DSM 5911.

[0112] The preferred production strain is the strain disclosed in Example 2 of the present invention:

[0113] Escherichia coli W3110x pcoa A-R106A

[0114] Escherichia coli W3110x pcoa A-H177Q

[0115] Escherichia coli W3110x pcoa A-F247V

[0116] R.terrigena x pcoa A-R106A

[0117] P.ananatis x pcoa A-R106A

[0118] A particularly preferred production strain is the strain:

[0119] Escherichia coli W3110x pcoa A-R106A

[0120] Escherichia coli W3110x pcoa A-H177Q

[0121] Escherichia coli W3110x pcoa A-F247V

[0122] Particularly preferred are production strains:

[0123] Escherichia coli W3110x pcoa A-R106A

[0124] Biotin and / or its biosynthetic precursor DTB is produced by culturing (growing) the production strain according to the present invention in a growth medium.

[0125] Compared to the fermentation production scale, shake flask growth is used to cultivate microorganisms on a laboratory scale. Although shake flask culture also involves specifying a specific culture medium and pH and cultivating under constant motion (shaking) in the presence of oxygen, more specific conditions regarding culture medium, temperature, pH, oxygen supply, and culture medium mixing can be established and adjusted in a fermentor. Smaller-scale cultures, such as in shake flasks, can also be used as precultures for inoculating larger-scale cultures (e.g., fermentors).

[0126] Production scale in fermentation typically starts with batches of 0.5 L and can go up to 100 000 L or more. Production scale in shake flask growth typically ranges from 10 ml to 1000 ml batch volumes.

[0127] In the context of the present invention, yield is defined as the amount of the product obtained by growing the production strain. Yield can be specified in terms of the absolute amount (mmol, or g or mg) of the product or in terms of the volumetric yield (concentration) of the product amount (mM, or g / L or mg / L) based on volume.

[0128] 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 were only found within the detection limit in the fermentation medium. Therefore, only biotin and DTB produced by secretion into the fermentation medium were considered.

[0129] Preferably, the method is characterized in that the biotin content in the fermentation supernatant is greater than 70%, preferably greater than 80%, particularly preferably greater than 90%, based on the biotin content in the fermentation batch. 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 contents, also referred to as the total biotin / DTB content. The extracellular fraction of biotin and DTB is determined as described in Example 3 ("Sample Preparation"). The intracellular fraction of biotin and DTB is determined as described in Example 8, with a detection limit of 0.1 mg / L for biotin and 0.1 mg / L for DTB.

[0130] Thus, the expression of a feedback-resistant mutant of pantothenate kinase in the production strain according to the present invention differs in the synthesis of biotin and DTB in the microbial cell and their transfer from the cell. This extracellular production or enrichment, i.e., the biosynthesis of biotin and DTB, followed by their transfer (export, secretion) from the production strain into the growth medium, has the major advantage that the volume in which biotin and DTB can accumulate is not limited to the small volume of the cell contents (cytoplasm). This avoids toxic effects caused by high concentrations of intracellular products.

[0131] Furthermore, extracellular production of biotin and DTB has the advantage of simplifying product isolation, as the products can be isolated directly from the fermentation supernatant without prior elaborate mechanical or chemical disruption of the cells. It is unknown how biotin and DTB are secreted from production strains. The mechanism may be passive, with diffusion of the product across the cell membrane, or one or more transporters may be involved in secretion.

[0132] As disclosed in the Examples of the present invention, fermentation or growth in shake flasks of a recombinant production strain according to the present invention, comprising a host strain transformed with a gene construct comprising a cds encoding a pantothenate kinase that is feedback-resistant to Coenzyme A, allows for the production of biotin and / or DTB, whereas neither biotin nor DTB can be produced in detectable amounts by an untransformed host strain, despite the presence of a naturally occurring biotin biosynthetic pathway. This was unexpected in the prior art.

[0133] Preferably, the process for the fermentative production of biotin and / or DTB is characterized by a fermentation volume of at least 0.5 L, particularly preferably at least 10 L production scale, particularly preferably at least 1000 L production scale, particularly preferably a fermentation volume of at least 10 000 L.

[0134] The amount of biotin or DTB can be quantified from the culture medium. This can be done by taking, for example, a 1 ml aliquot and measuring the cell density OD 600 This is accomplished by reducing the concentration of culture broth to at least 1.0 mL / ml, followed by removal of all solid components, e.g., by centrifugation at maximum speed in a benchtop centrifuge for five minutes, and quantification of the supernatant by LC-MS calibrated for biotin and DTB, as described in Example 3.

[0135] In a preferred embodiment, the yield of biotin at the end of fermentation after a fermentation time of up to 65 hours is at least 10 mg / L, particularly preferably at least 20 mg / L, and particularly preferably at least 50 mg / L, whereas the untransformed host strain does not produce any detectable biotin, with a detection limit of about 0.1 mg / L biotin or DTB. Thus, the use of the production strain according to the invention allows the hitherto unknown fermentative production of biotin without the need to optimize the biotin biosynthetic pathway for use in the food, feed, and pharmaceutical sectors.

[0136] In a further preferred embodiment, the process is characterized in that the yield of DTB at the end of the fermentation after a fermentation time of at most 65 h is at least 10 mg / L, particularly preferably at least 20 mg / L, particularly preferably at least 50 mg / L.

[0137] In a preferred embodiment, since DTB is a biosynthetic precursor of biotin, the method is characterized by producing desthiobiotin, which is then converted to biotin in a biotransformation, and then the biotin is separated from the biotransformation reaction. DTB can be used directly in the biotransformation, or it can be pre-enriched or separated, for example, to allow it to be used in a more concentrated form in the biotransformation. Particularly preferably, DTB is used in the biotransformation in an enriched or separated form, and particularly preferably in an isolated form.

[0138] The culture media used to grow production strains in shake flasks and ferment them are familiar to those skilled in the art from practicing microbial culture. They typically consist of a carbon source, a nitrogen source, and additives (e.g., vitamins, salts, and trace elements), as well as a sulfur source that optimizes cell growth and biotin / DTB production. Possible culture media supplements that increase biotin / DTB production include, for example, pimelic acid (1,5-pentanedicarboxylic acid, CAS No. 111-16-0).

[0139] Carbon source is that production strain can be used for forming those of biotin / DTB product.These include all forms of monosaccharide, comprise C6 sugar (hexose) such as glucose, mannose, fructose or galactose and C5 sugar (pentose) such as wood sugar, arabinose or ribose, and all possible disaccharides and polysaccharides formed therefrom, such as sucrose, lactose, maltose, maltodextrin, starch, and by hydrolysis from its monomer or oligomer of releasing (enzymatic or chemical).Other available carbon sources except sugar or carbohydrate are acetic acid (or acetate derived therefrom), ethanol, glycerine, citric acid (and its salt) or pyruvate (and its salt).However, gaseous carbon sources such as carbon dioxide or carbon monoxide are also conceivable.

[0140] Preferred carbon sources for growth of the production strain are glucose, fructose, sucrose, mannose, xylose and arabinose, with glucose and sucrose being particularly preferred, and glucose being particularly preferred.

[0141] Nitrogen sources are those that can be used by the production strain to form biomass. Examples that can be used for this purpose include:

[0142] - ammonia, in gaseous form or in the form of an aqueous solution as NH4OH, or a salt thereof, such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium acetate or ammonium nitrate, and / or

[0143] - known nitrates such as KNO3, NaNO3, ammonium nitrate Ca(NO3)2, Mg(NO3)2 and other nitrogen sources such as urea, and / or

[0144] - complex mixtures of amino acids, such as yeast extract, peptone, malt extract, soy peptone, caspases, corn steep liquor (in liquid form or in dry form as so-called CSD), and / or -NZ amines, and / or

[0145] -Basal nitrogen source for yeast.

[0146] In order to effectively produce biotin / DTB, it is necessary to meter in a sulfur source, either as a batch addition or as a continuous feed. Continuous metering can be performed as a pure feed solution or in a mixture with another feed component such as glucose. Preferably, the method is characterized in that it is performed in the presence of at least one compound selected from sulfate, sulfite, dithionite, thiosulfate and sulfide, with the use of the corresponding acid also being conceivable given the stability. Preferred sulfur sources are sulfate, sulfite, thiosulfate and sulfide, with sulfate and thiosulfate being particularly preferred. Particularly preferably, the method is performed in the presence of sulfate. Particularly preferred sulfates are compounds selected from sodium sulfate, ammonium sulfate and mixtures thereof.

[0147] The additional sulfur source is selected from the amino acids cysteine, methionine, oligotaurine and taurine, preferably from cysteine ​​and methionine, particularly preferably from L-cysteine ​​and D,L-methionine, particularly preferably D,L-methionine.

[0148] The growth (cultivation of microbial cells) can be carried out in so-called batch mode, that is, by inoculating growth medium (substratum) with the starter culture of production strain (microbial cells carrying one or more gene constructs), then cell growth without further feeding nutrient source to obtain biomass. Growth can also be carried out in so-called fed-batch mode (also referred to as growing in feed mode), that is, the method for obtaining biomass, wherein the initial stage of growing in batch mode is followed by the additional feeding of nutrient source (feed). Feed can be made up of carbon source, nitrogen source, sulphur source, one or more vitamins or trace elements important to production or aforesaid combination. Feed components can be measured together as a mixture, or measured separately in a separate feed portion. In addition, other culture medium components and additives that increase biological / DTB production, such as pimelic acid, can also be added to the feed. Feed can be supplied continuously or partially (discontinuously), or supplied with a combination of continuous and discontinuous feeding. Preferably, the method for producing biotin / DTB using microbial production strain fermentation according to the present invention is characterized in that the fermentation process is a fed-batch mode process.

[0149] Preferred carbon sources in the feed are glucose, sucrose and plant hydrolysates containing glucose or sucrose, as well as mixtures of the preferred carbon sources in any mixing ratio. A particularly preferred carbon source in the feed is glucose.

[0150] Preferably, the carbon source is metered into the culture such that the content of the carbon source in the fermentor is

[0151] The production stage should not exceed 10g / L.

[0152] The maximum concentration is 2g / L, especially preferred

[0153] 0.5 g / L, particularly preferably 0.1 g / L.

[0154] Preferred nitrogen sources in the feed are ammonia in gaseous form or in the form of aqueous solution, and its salts ammonium sulfate, ammonium phosphate, ammonium acetate and ammonium chloride, and further urea, KNO3, NaNO3 and ammonium nitrate, yeast extract, peptone, malt extract, soy peptone, caspases, corn steep liquor and NZ amines and yeast-based nitrogen sources, of which ammonia or ammonium salts, yeast extract, soy peptone or corn steep liquor (in liquid or dry form) are particularly preferred.

[0155] Preferred sulfur sources in the feed are sulfates, sulfites, thiosulfates and sulfides, with sulfates and thiosulfates being particularly preferred, and sulfates such as sodium sulfate and ammonium sulfate being particularly preferred.

[0156] Other culture medium supplements that can be added are salts of element phosphorus, chlorine, sodium, magnesium, nitrogen, potassium, calcium and iron, and salts of element molybdenum, boron, cobalt, manganese, zinc, copper and nickel in trace amounts (i.e., with μM concentrations). In addition, organic acids (e.g., acetate, citrate), amino acids (e.g., isoleucine) and vitamins (e.g., vitamin B1, vitamin B6) can be added to the culture medium. Other culture medium supplements include all types of fatty acids, monocarboxylic acids and dicarboxylic acids in the form of one of free fatty acids or their salts, wherein the C7 dicarboxylic acid pimelic acid is preferred.

[0157] Growth is carried out under pH and temperature conditions that promote growth and biotin / DTB production by the producing strain. The pH range is preferably 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 particularly preferred.

[0158] The preferred temperature range for growing the production strain is 20° C. to 40° C. The temperature range of 25° C. to 37° C. is particularly preferred, and 29° C. to 35° C. is particularly preferred.

[0159] The production strain can be grown optionally without oxygen supply (anaerobic culture) or with oxygen supply (aerobic culture). Aerobic culture with oxygen is preferred.

[0160] In the case of aerobic cultivation of the strain according to the invention for biotin production, an oxygen content of preferably at least 5% (v / v), particularly preferably at least 15% (v / v), and particularly preferably at least 30% (v / v) saturation is set. According to the prior art, the oxygen saturation in the culture is automatically regulated by a combination of gas supply and stirring speed.

[0161] The oxygen supply can be ensured by introducing compressed air or pure oxygen. Aerobic cultivation is preferably carried out by introducing compressed air. The compressed air supply in the aerobic cultivation is preferably in the range of 0.05 vvm to 10 vvm (vvm: the compressed air is introduced into the fermentation batch, specified in liters of compressed air per minute per liter of fermentation volume). Particularly preferably, 0.2 vvm to 8 vvm of compressed air is introduced, particularly preferably 0.4 to 6 vvm of compressed air, and particularly preferably 0.8 to 5 vvm of compressed air.

[0162] The maximum stirring speed is preferably 2500 rpm, particularly preferably a maximum of 2000 rpm, particularly preferably a maximum of 1800 rpm.

[0163] The incubation time is preferably 10 to 200 hours, particularly preferably 20 to 120 hours, and particularly preferably 30 to 100 hours.

[0164] In the method for fermentative production of biotin / DTB according to the present invention, the microbial production strain according to the claim is cultured in step i, and the fermentation supernatant is separated in step ii. This means that the fermentation batch obtained by the method contains extracellularly accumulated biotin / DTB in the fermentation supernatant. The biotin / DTB can be used directly or separated from the fermentation medium. Various analytical methods are available for identifying, quantifying, and determining the purity of biotin / DTB, including spectrophotometry, NMR, gas chromatography, HPLC, mass spectrometry, gravimetric analysis, or a combination of these analytical methods.

[0165] Description of the drawings

[0166] The accompanying drawings show plasmids used in the Examples.

[0167] Figure 1 :pKKj-SC101.

[0168] Figure 2 :pcoaA-wt.

[0169] Abbreviations used in the accompanying drawings:

[0170] TetR: a gene that confers tetracycline resistance

[0171] Ptac: tac accelerator

[0172] pSC101 ORI: origin of replication

[0173] coaA: coaA (pantothenate kinase gene) cds

[0174] EcoRI: Cutting site of restriction enzyme EcoRI

[0175] NdeI: Cutting site of restriction enzyme NdeI

[0176] PstI: cleavage site of restriction enzyme PstI

[0177] styI: cleavage site of restriction enzyme StyI

[0178] The following examples are used to further illustrate the present invention:

[0179] Example 1: Generation of coaA expression vector

[0180] Vector pKKj-SC101:

[0181] The coaA expression vector was constructed by using the vector pKKj-SC101 ( Figure 1 ), pKKj-SC101 was generated from the vector pKKj.pKKj, which is disclosed in EP 2 670 837 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 (bp 262-1947 of the DNA sequence disclosed in M77749.1) was removed from the 4.6 kb plasmid to generate the 2.9 kb expression vector pKKj.

[0182] Vector pKKj-tet: Plasmid DNA from vector pKKj was cut with BspHI and a 1.9 kb vector fragment was isolated. The tetracycline resistance gene was isolated from plasmid pACYC184 by PCR as a 1.4 kb gene fragment. The DNA sequence of pACYC184 is published in the GenBank gene database under accession number X06403.1. The 1.4 kb PCR product contains the sequence from nt1434 to nt2870 disclosed in X06403.1. 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 result was vector pKKj-tet, in which the tetracycline resistance gene has the same sequence as the following vector pKKj-SC101 ( Figure 1 ) in the same orientation.

[0183] Vector pKKj-SC101: Plasmid DNA of vector pKKj-tet was cut with StyI and NdeI, and a 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 published in the GenBank gene database under accession number K00042.1. The 1.4 kb DNA fragment contains 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 a reverse complement form relative to the orientation disclosed in K00042.1. The result was a 3.8 kb vector pKKj-SC101 ( Figure 1 ).

[0184] Vectors pcoaA, pcoa A-R106A, pcoa A-H177Q and pcoa A-F247V:

[0185] Plasmid DNA of vector pKKj-SC101 was cut with EcoRI and PstI, and the 3.8 kb linearized vector was isolated.

[0186] The cds of the coaA variants to be cloned were obtained by PCR ("Phusion TM High-fidelity DNA polymerase, ThermoScientific TM ) was generated as a 950 nt DNA fragment using primers coaa-13f (SEQ ID NO: 9) and coaa-14r (SEQ ID NO: 10) and was analyzed by agarose gel electrophoresis ( Gel extraction kit, Qiagen) for separation.

[0187] The template DNA used for the PCR reaction was genomic DNA from E. coli K12 strain W3110 of the coaA WT gene (coaA-wt, SEQ ID NO: 1) and synthetic genes of the mutants coa A-R106A (SEQ ID NO: 3), coa A-H177Q (SEQ ID NO: 5), and coa A-F247V (SEQ ID NO: 7) (generated by Eurofin Genomics).

[0188] pass The corresponding 950nt coaA DNA fragment was cloned into the 3.8kb pKKj-SC101 vector fragment using a cloning kit (NEB New England Biolabs). The results were the coaA expression vectors pcoaA-wt, pcoa A-R106A, pcoa A-H177Q, and pcoa A-F247V. Figure 2 The vector map of pcoaA-wt shown in FIG is representative of all four expression vectors.

[0189] Example 2: Production of production strains

[0190] The starting strains (host strains) for the production of the strains were the microbial strains Escherichia coli K12 W3110, strain number DSM 5911, Roultela terrigena, strain number DSM 2687, and Pantoea anananatis, strain number DSM 30080, which are commercially available from DSMZ-German Collection of Microorganisms and Cell Cultures GmbH. Escherichia coli W3110 was transformed in a known manner with the vector pcoaA-wt, pcoa A-R106A, pcoa A-H177Q, pcoa AA-F247V, and transformants were selected on LBtet plates. The LBtet plates contained 10 g / L tryptone (GIBCO TM), 5g / L yeast extract (BD Biosciences), 5g / L NaCl, 15g / L agar and 15mg / L tetracycline (Sigma-Aldrich). Each transformant was selected as a production strain. The production strains were named E. coli W3110x pcoA-wt, E. coli W3110x pcoa A-R106A, E. coli W3110x pcoa A-H177Q and E. coli W3110x pcoa A-F247V, and were used for growth or fermentation in shake flasks, and for analyzing the production of biotin and desthiobiotin. Similarly, R. terrigena and P. ananatis were transformed with the carrier pcoa A-R106A. The production strains were named R. terrigena x pcoa A-R106A and P. ananatis x pcoa A-R106A.

[0191] Example 3: Growth of production strains in shake flasks and analysis of biotin, DTB and pantothenic acid

[0192] Preculture: A preculture of E. coli W3110 was prepared in LB medium (10 g / L tryptone, 5 g / L yeast extract, 5 g / L NaCl), and corresponding precultures of the production strains E. coli W3110 x pcoA-wt, E. coli W3110 x pcoa A-R106A, E. coli W3110 x pcoa A-H177Q, and E. coli W3110 x pcoa A-F247V were prepared in LBtet medium (10 g / L tryptone, 5 g / L yeast extract, 5 g / L NaCl, 15 mg / L tetracycline). Growth was carried out overnight at 37° C. and 120 rpm.

[0193] Likewise, precultures of R. terrigena and P. ananatis were prepared in LB medium, and precultures of the production strains R. terrigena x pcoa A-R106A and P. ananatis x pcoa A-R106A were prepared in LBtet medium. Growth was carried out overnight at 30°C and 120 rpm.

[0194] Main culture: For all strains transformed with plasmids, 0.5 ml of the corresponding preculture was transferred into a 300 ml Erlenmeyer flask (baffled) containing 30 ml BS20 medium (WT strain E. coli W3110, R. terrigena, P. anananatis) or BS20 medium containing 15 mg / L tetracycline.

[0195] The composition of BS20 medium is as follows: 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 trisodium citrate x2H2O, 0.03 g / L FeSO4 x 7H2O, 0.05 g / L CaCl2 x 2H2O, 1 g / L MgSO4 x 7H2O, 20 g / L glucose, 0.1 g / L L-methionine, 5 mg / L thiamine (Sigma-Aldrich), 50 mg / L pyridoxal phosphate (Sigma-Aldrich) and 3 ml / L trace element solution.

[0196] The composition of the trace element solution: 0.15g / L Na2MoO4×2H2O, 2.5g / LH3BO3, 0.7g / L CoCl2×6H2O, 0.25g / L CuSO4×5H2O, 1.6g / L MnC12×4H2O, 0.3g / L ZnSO4×7H2O.

[0197] The main cultures of E. coli strains were incubated in an incubator shaker (Infors) at 37°C and 140 rpm for 24 h. The main cultures of R. terrigena and P. anananatis strains were incubated in an incubator shaker (Infors) at 30°C and 140 rpm for 24 h. After 24 h, 1 ml samples were taken and quantified using a 50 μl HPLC-MS / MS instrument obtained from ThermoScientific TM Genesys TM 10S UV / visible spectrophotometer was used to measure the cell density as OD 600 The values ​​of 100 μg / ml were calculated (optical density of the main culture, measured photometrically at 600 nm), and the contents of biotin, desthiobiotin and possibly pantothenic acid were determined by LC-MS (liquid chromatography coupled with mass spectrometry).

[0198] Sample preparation for quantification of biotin / DTB by LC-MS:

[0199] 1 ml of culture broth from shake flasks was centrifuged at 13 000 rpm for 5 minutes (Heraeus TM Fresco TM 21 centrifuge). The cell culture supernatant was separated and analyzed by LC-MS for the content of biotin, desthiobiotin, and pantothenic acid.

[0200] LC-MS analysis of biotin, desthiobiotin and pantothenic acid: The compounds quantitatively analyzed in the examples were quantitatively determined by using an LC-MS method (liquid chromatography coupled with mass spectrometry) calibrated for biotin, an LC-MS method calibrated for desthiobiotin and an LC-MS method calibrated for pantothenic acid. The reference substances used for calibration were commercially available (Sigma-Aldrich). An LC-MS apparatus consisting of a UHPLC 1290 Infinity II (liquid chromatography) combined with a 6470 triple quadrupole mass spectrometer (both from Agilent) was used. The columns used for liquid chromatography were from Waters TM of UPLC HSS T3 column (2.1×100 mm, particle size: 1.8 μm). Eluent used: Eluent A: 0.1% (v / v) formic acid in H2O Eluent B: 0.1% (v / v) formic acid in acetonitrile. Liquid chromatography operating conditions: 0% eluent B for 0.5 min isocratic flow, followed by a gradient flow to 15% eluent B over 1 min, then 15% eluent B for 1.5 min isocratic flow, then a gradient flow to 100% eluent B over 2 min, then 100% eluent B for 2 min isocratic flow. The flow rate was 0.4 ml / min. The analytes resolved by liquid chromatography - biotin, desthiobiotin and pantothenic acid - were quantified by mass spectrometry using so-called "multiple reaction monitoring" (MRM) according to the prior art. The detection limit for biotin, DTB and pantothenic acid was 0.1 mg / L in each case.

[0201] An introduction to the principles of mass spectrometry theory is available from Agilent (https: / / biter.agilent.com / , csbitrinryslidepresentation, 5991-5857_Agilent_MS_Theory_DE.pdf).

[0202] The signals from the mass spectrometer were quantified using the molecular weights of the analyte in question ("precursor ions") and the molecular fragments produced in the mass spectrometer ("product ions"), as listed in Table 1. The polarity "+" in Table 1 means that positively charged ions were measured.

[0203] Table 1: Product ions used for quantification by multiple reaction monitoring (MRM):

[0204]

[0205] Example 4: Utilization of Pantothenate

[0206] The strains E. coli W3110, E. coli W3110 x pcoaA-wt and E. coli W3110 x pcoa A-R106A were grown as described in Example 3, with the main cultures of the three strains being supplemented with 8 mg / L calcium pantothenate in each case. After an incubation time of 24 h at 37° C., the cell density OD was determined by LC-MS. 600 / ml and the content of pantothenic acid in the culture supernatant was determined (Table 2).

[0207] Table 2: Cell density and pantothenic acid content in the culture supernatant after 24 h growth of strains E. coli W3110, E. coli W3110 x pcoaA-wt and E. coli W3110 x pcoa A-R106A in shake flasks

[0208]

[0209] Example 5: Production of biotin and desthiobiotin in E. coli strains

[0210] Strains E. coli W3110, E. coli W3110 x pcoA-wt, E. coli W3110 x pcoa A-R106A, E. coli W3110 x pcoa A-H177Q and E. coli W3110 x pcoa A-F247V were grown as described in Example 3. After an incubation time of 24 h at 37° C., the cell density OD was determined by LC-MS. 600 / ml and the contents of biotin and desthiobiotin in the culture supernatant were determined (Table 3).

[0211] Table 3: Cell density and content of biotin and desthiobiotin in the culture supernatant after 24 h growth of strains E. coli W3110, E. coli W3110 x pcoA-wt, E. coli W3110 x pcoa A-R106A, E. coli W3110 x pcoa A-H177Q and E. coli W3110 x pcoa A-F247V in shake flasks

[0212]

[0213] Example 6: Production of biotin and desthiobiotin in R. terrigena strains

[0214] The strains R. terrigena and R. terrigena x pcoa A-R106A were grown as described in Example 3. After an incubation time of 24 h at 30° C., the cell density OD was determined by LC-MS. 600 / ml and the contents of biotin and desthiobiotin in the culture supernatant were determined (Table 4).

[0215] Table 4: Cell density and content of biotin and desthiobiotin in the culture supernatant after 24 h growth of strains R. terrigena and R. terrigena x pcoa A-R106A in shake flasks

[0216] Example 7: Production of biotin and desthiobiotin in P. ananatis strains

[0217] P. ananatis and P. ananatis A-R106A strains were grown as described in Example 3. After a 24 h incubation time at 30°C, the cell density OD was determined by LC-MS. 600 / ml and the contents of biotin and desthiobiotin in the culture supernatant were determined (Table 5).

[0218] Table 5: Cell density and content of biotin and desthiobiotin in the culture supernatant after 24 h of growth of P. ananatis and P. ananatis x pcoa A-R106A strains in shake flasks

[0219] Example 8: Extraction of biotin and DTB from cells grown in shake flasks

[0220] A shake flask growth of the production strain E. coli W3110 x pcoa A-R106A from Example 5 was used, which had a biotin content of 1.2 mg / L and a DTB content of 0.7 mg / L (Table 2). 30 ml of the shake flask growth was centrifuged at 6300 rpm for 10 minutes (Thermo Scientific TM Multifuge X1R, TX-400 rotor) and discard the supernatant. The cell pellet was resuspended in 10 ml of H2O and centrifuged at 6300 rpm for 5 minutes and the supernatant was discarded. The cell pellet was suspended in 2 ml of H2O. The cell suspension was used to prepare the cell extract. This was done by using a 10 ml HPLC-MS / MS kit from MP Biomedicals. TM FastPrep-24 TM The cell pellet, suspended in 2 ml of H2O, was broken into 2 x 1 ml aliquots ("lysis matrix B") in a 1.5 ml tube containing glass beads assembled by the manufacturer (3 x 20 seconds at an oscillation frequency of 6000 rpm, with pauses of 30 seconds in each case). The cell homogenates obtained were combined and centrifuged at 13 000 rpm for 5 minutes (Heraeus TM Fresco TM21 centrifuge) to prepare cell extracts. The cell extracts were analyzed by LC-MS for biotin and DTB content. For biotin / DTB, no biotin or DTB was detected in LC-MS analysis at 0.1 mg / L.

[0221] Example 9: Production of biotin / DTB by fermentation

[0222] Pre-culture 1:

[0223] In a 100 ml conical flask, 20 ml of LBtet medium was inoculated with the production strain E. coli W3110 x pcoa A-R106A and incubated on a shaker (150 rpm, 34° C.) for 7 hours. In a 100 ml conical flask, 20 ml of LB medium was inoculated with the WT strain E. coli W3110 and incubated on a shaker (150 rpm, 34° C.) for 7 hours.

[0224] Pre-culture 2:

[0225] In each case, the entire preculture 1 was then transferred to 100 ml of BS20 medium, the medium for preculture 2 of the strain E. coli W3110 x pcoa A-R106A was supplemented with 15 mg / L tetracycline (the composition of the BS20 medium is shown in Example 3). The cultures were shaken in Erlenmeyer flasks (1 L volume) at 34° C. at 150 rpm for 17 hours (Infors incubator shaker). After this incubation, the cell density OD 600 / ml were between 3 and 5 respectively.

[0226] Main training:

[0227] Fermentation in the " The experiments were carried out in a "Parallel Bioreactor System for Microbiology" fermentor. A culture vessel with a total volume of 1.8 L was used. The growth medium consisted of 600 ml of BS20 medium and 15 mg / L tetracycline (for strain E. coli W3110 x pcoa A-R106A) and 600 ml of BS20 medium (for strain E. coli W3110).

[0228] The pH in the fermenter was initially adjusted to 7.0 by pumping in a 25% NH4OH solution. During the fermentation, the pH was maintained at a value of 7.0 by automatic correction with 25% NH4OH or 4M H3PO4. Foam control was achieved by automatically metering 4% v / v Struktol J673 (Schill & Seilacher) in H2O.

[0229] For inoculation, 60 ml of the corresponding preculture 2 were pumped into the fermenter vessel. The initial volume was therefore 660 ml. The culture was initially stirred at 400 rpm and aerated with compressed air, sterilized through a sterile filter at an aeration rate of 2 vvm (vvm: compressed air introduced into the fermentation batch, specified in liters of compressed air per liter of fermentation volume per minute). Under these starting conditions, the oxygen probe was calibrated to 100% saturation before inoculation.

[0230] The target value for O2 saturation during fermentation was set at 15%. After the O2 saturation fell below the target value, a regulation cascade was initiated to return the O2 saturation to the target value. This involved first continuously increasing the gas supply (to a maximum of 5 vvm) and then continuously increasing the stirring speed (to a maximum of 1500 rpm). Fermentation was carried out at a temperature of 34°C.

[0231] Once the glucose content in the fermentor has dropped to approximately 2 g / L from an initial 20 g / L, a 56% (w / w) glucose solution is continuously metered in. The feed rate is adjusted so that the glucose concentration in the fermentor no longer exceeds 2 g / L. Glucose is measured using a glucose analyzer from YSI (Yellow Springs, Ohio, USA).

[0232] The fermentation time was 65 h. Samples were taken from the fermentation batch 24 h, 42 h, and 65 h after the start of fermentation, and the cell density OD was determined by LC-MS. 600 / ml and the biotin / DTB content in the culture supernatant was determined. The results for E. coli strain W3110 x pcoa A-R106A are summarized in Table 6. After 65 hours of fermentation, biotin production was 20.1 mg / L and DTB production was 28.6 mg / L. In a parallel fermentation of E. coli strain W3110, neither biotin nor DTB was detected (Table 7).

[0233] Table 6: Time course of cell density and biotin and DTB content in fermentation of E. coli W3110x pcoa A-R106A strain

[0234]

[0235] Table 7: Time course of cell density and biotin and DTB content in fermentations of E. coli strain W3110

[0236]

Claims

1. A method for producing biotin, desthiobiotin (DTB) or a mixture thereof, characterized in that: A microbial production strain recombinantly expressing at least one enzyme that is feedback-resistant to Coenzyme A and has pantothenate kinase enzymatic activity (EC 2.7.1.33 class protein, CoaA enzymatic activity) is cultured and then biotin, DTB, or a mixture thereof is isolated.

2. The method according to claim 1, wherein The microbial production strain is a bacterial strain.

3. The method according to one or both of claims 1 and 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. The method according to one or more of claims 1 to 4, characterized in that The cells are cultivated on an industrial scale by fermentation.

6. The method according to one or more of claims 1 to 5, characterized in that The gene expressing an enzyme that is feedback-resistant to Coenzyme A and has pantothenate kinase activity is a mutant gene whose Wt form encodes a CoaA enzyme that is feedback-inhibited by Coenzyme A.

7. The method according to one or more of claims 1 to 6, characterized in that The mutant gene expressing an enzyme that is feedback-resistant to coenzyme A and has the enzymatic activity of pantothenate kinase is a bacterial gene.

8. The method according to one or more of claims 1 to 7, characterized in that The mutant gene expressing an enzyme that is feedback-resistant to coenzyme A and has the enzymatic activity of pantothenate kinase is a bacterial gene from the family Enterobacteriaceae.

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

10. The method according to one or more of claims 1 to 9, characterized in that The enzyme having feedback resistance to Coenzyme A and having the enzymatic activity of 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% identical.

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

4.

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

13. The method according to one or more of claims 1 to 12, characterized in that After a fermentation time of at most 65 h, the yield of biotin at the end of the fermentation is at least 10 mg / L.

14. Method according to one or more of claims 1 to 13, characterized in that After a fermentation time of at most 65 h, the yield of DTB at the end of the fermentation was at least 10 mg / L.

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

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