EP1054977A1

EP1054977A1 - Method for producing biotin

Info

Publication number: EP1054977A1
Application number: EP99908923A
Authority: EP
Inventors: Hartwig Schröder
Original assignee: BASF SE
Current assignee: BASF SE
Priority date: 1998-02-19
Filing date: 1999-02-17
Publication date: 2000-11-29
Also published as: CA2321264A1; CN1210403C; CN1291232A; JP2002504338A; DE19806872A1; KR20010041062A; WO1999042591A1; IL137310A0

Abstract

The invention relates to a genetic recombination product, containing an S-adenosyle-methionine-synthase gene, with the sequence SEQ ID no. 1 and a biotin biosynthesis gene bioS1, bioS2 and/or bioS3 with the sequences SEQ ID no. 3, SEQ ID no. 5 or SEQ ID no.7 and possibly at least one additional biotin synthesis gene sequence chosen from the group bioA, bioB, bioF, bioC, bioD, bioH, bioP, bioW, bioX, bioY or bioR. The invention also relates to organisms containing said genetic recombination product and to the use of same for producing biotin, as well as to a method for producing biotin.

Description

Process for the production of biotin

description

The invention relates to a gene construct containing an S-adenosyl-methionine synthase gene with the sequence SEQ ID No. 1 and a biotin biosynthesis gene bioSl, bioS2 and / or bioS3 with the sequences SEQ ID No.3, SEQ ID No.5 and SEQ ID No.7 and optionally at least one further biotin synthesis gene sequence selected from the group bioA, bioB, bioF, bioC, bioD, bioH, bioP, bioW, bioX, bioY or bioR. The invention further relates to organisms that contain this gene construct and the use of the gene construct for the production of biotin, and a method for the production of biotin.

As a coenzyme, biotin (vitamin H) plays an essential role in enzyme-catalyzed carboxylation and decarboxylation reactions. Biotin is therefore an essential factor in living cells.

Biotin has to be absorbed from the outside by almost all Ti.eren and egg micro organisms, since they cannot synthesize biotin themselves. It is therefore an essential vitamin for these organisms. Bacteria, yeasts and plants, on the other hand, can synthesize biotin themselves from precursors (Brown et al. Biotechnol. Genet. Eng. Rev. 9, 1991: 295-326, DeMoll, E., Escherichia coli and Salmonella, eds. Neidhardt, FC et al ASM Press, Washington DC, USA, 1996: 704-708, ISBN 1-55581-084-5).

Biotin synthesis was found in bacterial organisms, especially in the gram-negative bacterium Escherichia coli and in the gram-positive one

Bacillus sphaericus bacterium was investigated (Brown et al. Biotechnol. Genet. Eng. Rev. 9, 1991: 295-326). Pimelyl-CoA (PmCoA), which originates from fatty acid synthesis (DeMoll, E., Escherichia coli and Salmonella, eds. Neidhardt, FC et al. ASM Press, Washington DC), is regarded as the first intermediate known to date in E. coli. USA, 1996: 704-708, ISBN 1-55581-084-5 1996). The synthetic route of this biotin precursor in E. coli is so far largely unknown (Lemoine et al., Mol. Microbiol. 19, 1996: 645-647). With bioC and bioH two genes were identified, whose corresponding proteins are responsible for the synthesis of Pm-CoA. The enzymatic function of the gene products BioH and BioC is not yet known (Lemoine et al., Mol. Microbiol. 19, 1996: 645-647, DeMoll, E., Escherichia coli and Salmonella, eds. Neidhardt, FC et al. ASM Press , Washington DC, USA, 1996: 704-708, ISBN 1-55581-084-5). Pm-CoA is converted to biotin in four further enzymatic steps. Starting with the Pm-CoA, the condensation with alanine first takes place 7-Keto-8-Arrdno-Pelargonic Acid (KAPA) held by BioF. The conversion of KAPA to 7.8 diamino-pelargonic acid (DAPA) follows

BioA. The next step leads to dethiobiotin (DTB) after an ATP-dependent carboxylation reaction and is activated by BioD

5 talysed. The final step is the implementation of DTB

Biotin instead. This step is catalyzed by BioB. The chemical and enzymatic mechanism of conversion of DTB to

So far, biotin has only been incompletely understood and elucidated.

10 A characterization of the conversion of DTB to biotin has so far been carried out exclusively in bacterial or plant cell extracts (WO94 / 8023, EP-B-0 449 724, Sanyal et al. Arch. Biochem. Biophys., Vol. 326, No. 1 , 1996: 48-56 and Bio-istry 33, 1994: 3625-3631, Baldet et al. Europ. J. Biochem.

15 217, 1, 1993: 479-485, Mejean et al. Biochem. Biophys. Res.

Commun., Vol. 217, No. 3, 1995: 1231-1237, Ohshiro et al., Biosci. Biotechnol. Biochem., 58, 9, 1994: 1738-1741).

In vitro studies have shown that low molecular weight factors such as 20 NADPH, cysteine, thiamine, Fe ²⁺ , asparagine, serine, fructose

1-6-bisphosphate and S-adenosylmethionine can stimulate the synthesis of biotin (Ohshiro et al., Biosci. Biotechnol. Biochem.,

58, 9, 1994: 1738-1741, Birch et al. , J. Biol. Chem. 270, 32, 1995: 19158-19165, Ifuku et al. , Biosci. Biotechnol. Biochem.,

_25, 59, 2, 1995: 185-189, Sanyal et al. Arch. Biochem. Biophys. 326, 1, 1996: 48-56).

In addition to these low molecular weight factors, other proteins have been identified that stimulate the synthesis of biotin from DTB in the presence of BioB. It is Flavodoxin and

30 Flavodoxin NADPH reductase (Birch et al., J. Biol. Chem. 270, 32, 1995: 19158-19165, Ifuk et al., Biosci. Biotechnol. Biochem.,

59, 2, 1995: 185-189, Sanyal et al., Arch. Biochem. Biophys. 326, 1, 1996: 48-56). Other proteins that stimulate biotin synthesis are those in the German application

35 genes bioSl and bioS2 described with the application number 197.31274.8 (priority 22.7.97).

There are different results regarding the origin of the sulfur in the biotin molecule. In studies of biotin synthesis

⁴⁰ in whole cell extracts were shown to incorporate radioactivity in biotin in the presence of ³⁵ S-labeled cysteine; sulfur incorporation into the biotin molecule could not be detected either with ³⁵ S-labeled methionine or with S-adenosyl-methionine (Ifuku et al., Biosci. Biotechnol. Biochem. 59, 2, 1995: 184 -

45 189, Birch et al., J. Biol. Chem. 270, 32, 1995: 19158-19165).

The genes coding for the described proteins bioF, bioA, bioD and bioB are encoded in E. coli on a bidirectional operon. This operon is located between the λ attachment site and the uvrB gene locus at about 17 minutes on the E. coli chromosome (Berlyn et al. 1996: 1715 - 1902). On this operon, two additional genes are encoded, one of which, bioC, has already described functions in the synthesis of Pm-CoA, whereas up to now no function could be assigned to an open reading frame behind bioA (WO94 / 8023, Otsuka et al., J. Biol. Chem. 263, 1988: 19577-85). Highly conserved homologs to the 0 E. coli proteins BioF, A, D, B were found in B. sphaericus, B. subtilis, Syneccocystis sp. (Brown et al. Biotechnol. Genet. Eng. Rev. 9, 1991: 295-326, Bower et al., J. Bacteriol. 175, 1996: 4122-4130, Kaneko et al., DNA Res. 3, 3, 1996: 109-136), arka bacteria such as Methanococcus janaschi, yeasts such as Saccharomyces ₅ cerevisiae (Zhang et al., Arch. Biochem. Biophys. 309, 1, 1994: 29-35) or in plants such as Arabidopsis thaliana (Baldet et al., CR Acad. Sci. III, Sci. Vie. 319, 2, 1996: 99-106)).

o The synthesis of Pm-CoA appears to be different in the two gram-positive microorganisms examined so far than in E. coli. No homologs of bioH and bioC could be found (Brown et al. Biotechnol. Genet. Eng. Rev. 9, 1991: 295-326). 5

Biotin is an optically active substance with three chirality centers. So far, it will only be economically viable using a multi-stage, expensive chemical synthesis.

0 As an alternative to this chemical synthesis, numerous attempts have been made to set up a fermentative process for producing biotin with microorganisms. By cloning the biotin operon on multi-copy plasmids, the biotin synthesis in the microorganisms transformed with these genes could be increased. A further increase in biotin synthesis was achieved by deregulating biotin expression via the selection of birA mutants (Pai C. H., J. Bacteriol. 112, 1972: 1280-1287). The combination of both approaches, that is to say the expression of the plasmid-coded biosynthesis genes in a regulation-deficient strain (EP-B-0 236 429), brought a further increase in productivity. The biotin operon can either remain under the control of its native bidirectional promoter (EP-B-0 236 429), or its genes can be brought under the control of an externally adjustable promoter (WO94 / 08023). 5

The approaches pursued so far for the fermentative production of biotin in E. coli have not been able to achieve any economically sufficient ones Productivity can be achieved.

The object of the present invention was to develop a technical, fermentative process for the production of biotin which shows the highest possible biotin synthesis.

This object was achieved by the process according to the invention for the production of biotin, characterized in that an S-adenosyl-methionine synthase (SAM synthase) gene with the sequence SEQ ID No. 1 and at least one other biotin biosynthetic gene bioSl, bioS2 or bioS3 with the sequences SEQ ID No. 3, SEQ ID No. 5 and SEQ ID No.7 as well as their functional variants, analogs or derivatives in a prokaryotic or eukaryotic host organism that is able to synthesize, expresses, cultivates and synthesizes the biotin directly, after separating the biomass or after cleaning the biotin used, solved.

The genes used in the method according to the invention, the SAM synthase gene with the sequence SEQ ID No. 1 and the biotin biosynthetic blessings bioSl, bioS2 and bioS3 with the sequences SEQ ID No. 3, SEQ ID No. 5 and SEQ ID No.7 are listed in the SwissProt database under the "Accession" numbers P04384 (metK), U29581 (bioSl), P39171 (bioS2) and D90811 (bioS3). The database describes a number of homologues for MetK from E. coli. These homologs include organisms such as other eubacteria (eg H. infuenza, B. subtilis), as well as eukaryotes (eg yeasts: S. cererevisiae, Planta: P. deltoides, Arthropoda D. melanogaster, and Mammalia: R. norvegicus ).

By expressing one or more of the SAM synthase genes with the sequence SEQ ID No. 1, its functional variants, analogs or derivatives in combination with at least one of the biotin synthesis genes bioSl, bioS2 or bioS3 with the sequences SEQ ID No. 3, SEQ ID No. 5 and SEQ ID No.7 and their functional variants, analogs or derivatives in a prokaryotic or eukaryotic host organism, the productivity of biotin biosynthesis can be significantly increased. A combination of SAM synthase gene and biosl is preferably used for expression. For an increased biotin synthesis, at least one further bioting gene selected from the group bioA, bioB, bioF, bioC, bioD, bioH, bioP, bioW, bioX, bioY or bioR is advantageously also expressed at the same time. The expression of the genes increases the synthesis of biotin by at least a factor of 2 compared to the control without these genes, preferably by a factor greater than 3. After isolation and sequencing, the genes used in the method according to the invention, the SAM synthase gene with the nucleotide sequence SEQ ID No. 1, the bioSl gene with the nucleotide sequence SEQ ID No. 3, the bioS2 gene with the nucleotide sequence SEQ ID No. 5 and the bioS3 gene with the nucleotide sequence SEQ ID No.7, which are available for those in SEQ ID NO: 2 or SEQ ID No. 4 or SEQ ID No. 6 and SEQ ID No.8 encode specified amino acid sequences or their allelic variations. Variants include SEQ ID No. 1, SEQ ID No.3 or SEQ ID No.5, or SEQ ID No.7 variants to be understood, which have 30 to 100% homology at the amino acid level, preferably 50 to 100%, very particularly preferably 80 to 100% exhibit. Allelic variants include, in particular, functional variants which, by deletion, insertion or substitution of nucleotides from the SEQ ID NO: 1, SEQ ID No. 3, SEQ ID No. 5 and SEQ ID No.7 are available, but the enzymatic activity is retained.

Furthermore, functional variants are also equivalent to the genes, such as O-acetylserine sulfohydrolase A, O-acetylserine sulfohydrolase B, and β-cystathionase (see Flint et al., J. Biol. Chem., Vol. 271, 1996: 16053 - 16067) or nifS and its prokaryotic and eukaryotic homologues, e.g. from Klebsiella, Candida, yeast or Caenorhabditis, which are able to take over the enzymatic activity of bioSl, bioS2 or bioS3 in the biotin synthesis.

Among functional analogs of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 and SEQ ID No. 7 are, for example, their prokaryotic or eukaryotic homologs, such as bacterial, fungal, plant, animal or human homologs. Analogs are furthermore also to be understood as shortened sequences, single-stranded DNA or RNA of the coding and non-coding DNA sequence.

Derivatives are understood to mean, for example, promoter variants. The

Promoters that are upstream of the specified nucleotide sequences can be changed by one or more nucleotide exchanges, by insertion (s) and / or deletion (s), but without the functionality or effectiveness of the promoters being impaired. Furthermore, the effectiveness of the promoters can be increased by changing their sequence, or completely replaced by more effective promoters, including organisms of other species.

Derivatives are also to be understood as variants whose nucleotide sequence has been changed in the range from -1 to -30 before the start codon in such a way that gene expression and / or protein expression is increased. This is advantageously done by means of a modified Shine-Dalgarno sequence.

In principle, all gram-negative or gram-positive bacteria synthesizing biotin can be considered as prokaryotic host organisms of the method according to the invention. Examples of Gram-negative bacteria are Enterobacteriaceae such as the genera Escherichia, Aerobacter, Enterobacter, Citrobacter, Shigella, Klebsiella, Serratia, Erwinia or Salmonella, Pseudomonadaceae such as the genera Pseudomonas, Xanthomonas, Burkholderia, Gluco-nobacter, Nitrosomonasomonasrobas , Cellulomonas or Acetobacter, Azotobacteraceae such as the genera Azotobacter, Azomonas, Bei erinckia or Derxia, Neisseriaceae such as the genera Moraxella, Acinetobacter, Kingella, Neisseria or Branhamella, the Rhizobiaceae such as the genera Rhizobium or Zrobacterium or gramobacterium, or gr Called Flavobacterium. Examples of gram-positive bacteria are the endospores-forming gram-positive aerobic or anaerobic bacteria such as the genera Bacillus, Sporolactobacillus or Clostridium, the coryneform bacteria such as the genera Arthrobacter, Cellulomonas, Curtobacterium, Corynebacterium, Brevibacterium or Microbacterium - hia, the Actinomycetales such as the genera Mycobacterium, Rhodococcus, Streptomyces or Nocardia, the Lactobacillaceae such as the genera Laetobacillus or Laetococeus, the gram-positive cocci such as the genera Micrococcus or Staphylococcus.

Bacteria of the genera Escherichia, Citrobacter, Serratia, Klebsiella, Salmonella, Pseudomonas, Comamonas, Acinetobacter, Azotobacter, Chromobacterium, Bacillus, Clostridium, Arthrobacter, Corynebacterium, Brevibacterium, Laetococeus, Laetobacillrobium, Streptomycobacterium, Streptomycobacterium, Streptomycobacterium, Streptomycobacterium, Streptomycum . Genera and species such as Escherichia coli, Citro-bacter freundii, Serratia marcescens, Salmonella typhimurium, Pseudomonas mendocina, Pseudomonas aeruginosa, Pseudomonas mutabilis, Pseudomonas chlororaphis, Pseudomonas fluorescens, Comamonas aeidovoroni, Acaceobotactoroni, Acaceobotactoroni, Comaceotobacteria, Comamonasotobacteria, Comamonasotobacteria, Comamonasobacteria vinelandii, Chromobacterium violaceum, Bacillus subtilis, Bacillus sphaericus, Bacillus stearothermophilus, Bacillus pumilus, Bacillus licheniformis, Bacillus amyloliquefa- ciens, Bacillus megaterium, Bacillus cereus, Bacillus thuringien- sis, Arthrobacter citreus, Arthrobacter paraffineus, Corynebacterium glutamicum, Corynebacterium primorioxydans, Corynebacterium sp. , Brevibacterium ketoglutamicum, Brevibacterium linens, Brevibacteπum sp., Streptomyces lividans, Rhizobium leguminosarum or Agrobacterium tumefaciens. Bacteria are advantageously used which already have an increased natural biotin product own.

The taxonical position of the genera listed has undergone a major change in recent years and is still in flux because incorrect genus and species names have been corrected. Because of the taxonomic regrouping of the above-mentioned genera within the bacterial system, which has often been required in the past, families, genera and species other than those mentioned above are also suitable for the method according to the invention.

In principle, all biotin-synthesizing organisms such as fungi, yeasts, plants or plant cells can be considered as eukaryotic host organisms of the process according to the invention. The genera Rhodotorula, Yarrowia, Sporobolomyces, Saccharomyces or Schizosaccharomyces are preferred as yeasts. The genera and species Rhodotorula rubra, Rhodotorula glutinis, Rhodotorula graminis, Yarrowia lipolytica, Sporobolomyces salmonicolor, Sporobolomyces shibatanus or Saccharomyces cerevisiae are particularly preferred.

In principle, all plants can be used as the host organism; preference is given to plants which play a role in animal nutrition or in human nutrition, such as corn, wheat, barley, rye, potatoes, peas, beans, sunflowers, palm trees, millet, sesame, copra or Rapeseed. Plants such as Arabidopsis thaliana or Lavendula vera are also suitable. Plant cell cultures, protoplasts from plants or potassium cultures are particularly preferred.

Advantageously, microorganisms such as bacteria, fungi, yeast or plant cells are used in the method according to the invention, which are able to excrete biotin into the growth medium and which may already have an increased natural biotin synthesis. Advantageously, these organisms can still be defective with regard to the regulation of their biotin biosynthesis, that is to say there is no or only a very reduced regulation of the synthesis. The consequence of this regulatory defect is that these organisms already have a much higher bio-tin productivity. Such a regulatory defect is known for example from Escherichia coli as a birA defect mutant and should preferably be present in the cells in the form of a defect which can be induced by external influences, for example temperature-induced. In principle, it is also possible to use organisms which have no natural biotin production after they have been transformed with the biotin genes. In order to further increase the overall biotin productivity, the organisms in the process according to the invention should advantageously additionally selected at least one further biotin from the

Group bioA, bioB, bioF, bioC, bioD, bioH, bioP, bioW, bioX, bioY

5 or bioR included. Such genes can advantageously also be used in combination with the sequence SEQ ID No. 1, SEQ ID NO: 3, SEQ ID No.5 or SEQ ID NO.7 and their combinations, which stimulate biotin synthesis.

Genes that stimulate biotin synthesis are e.g. B. the Flavore- 0 doxin gene, the Flavoredoxin reductase gene. This additional gene or these additional genes, like the genes with the sequence SEQ ID No. 1, SEQ ID NO.3, SEQ ID No.5 or SEQ ID NO.7 or their combinations may be present in one or more copies in the cell. You can use the same vector as that

₁₅ sequence SEQ ID No. 1, SEQ ID NO.3, SEQ ID No.5 and / or SEQ ID No.7 can be localized or integrated on separate vectors or chromosomally. The sequences SEQ ID No. 1, SEQ ID No.3, SEQ ID No.5 and / or SEQ ID No.7 can be together on one vector or on separate vectors or

__ be inserted into the genome. 20th

Under the gene construct according to the invention, the gene sequences of the SAM synthase gene SEQ ID No. 1 and the biotin synthetic genes SEQ ID No.3, SEQ ID No.5 and / or SEQ ID No.7, as well as their functional

To understand ₂₅ different variants, analogs or derivatives that have been functionally linked to one or more regulation signals to increase gene expression. In addition to these new regulatory sequences, the natural regulation of these sequences may still be present before the actual structural genes and may have been genetically modified so that the natural regulation has been switched off and the expression of the genes increased. However, the gene construct can also have a simpler structure, that is to say no additional regulation signals are placed in front of the sequences SEQ ID No. 1, SEQ ID No. 3, SEQ ID No.5 and / or SEQ ID No.7 are inserted and the natural promoter with its regulation is not removed. Instead, the natural regulatory sequence is mutated in such a way that biotin regulation no longer takes place and gene expression is increased. The sequences SEQ ID No. 1, SEQ ID No.3, SEQ ID No.5 and / or SEQ ID No.7 can be separated under the regulation of a promoter or under the regulation.

⁴ ^ th promoters. Additional advantageous regulatory elements can also be inserted at the 3 'end of the DNA sequences. The genes with the sequences SEQ ID No. 1, SEQ ID No. 3, SEQ ID No.5 or SEQ ID No. 7 can be contained in one or more copies in the gene construct.

45

Advantageous regulatory sequences for the method according to the invention are, for example, in promoters such as cos, tac, trp, tet-, trp-tet-, lpp-, lac-, lpp-lac-, lad ^« * -. Contain T7-, T5-, T3-, gal-, trc-, ara-, SP6-, -P _R - or in the λ-P _L promoter, which are advantageously used in gram-negative bacteria.

Further advantageous regulation sequences are, for example, in the gram-positive promoters amy and SP02, in the yeast promoters

ADC1, MFα, AC, P-60, CYC1, GAPDH or in the plant promoters

CaMV / 35S, SSU, OCS, lib4, usp, STLS1, B33, nos or contained in the ubiquitin or phaseolin promoter.

In principle, all natural promoters with their regulatory sequences such as those mentioned above can be used for the method according to the invention. In addition, synthetic promoters can also be used advantageously.

further biotin genes can be selected from the gene construct

Group bioA, bioB, bioF, bioC, bioD, bioH, bioP, bioW, bioX, bioY or bioR can be contained in one or more copies, which can have their own promoter or under the regulation of the promoter of one of the sequences or under the regulation of the promoter of the entire sequences SEQ ID No. 1, SEQ ID No. 3, SEQ ID No.5 or SEQ ID No.7.

For expression in the above-mentioned host organism, the gene construct is advantageously inserted into a host-specific vector which enables optimal expression of the genes in the host. Vectors are well known to those skilled in the art and can be found, for example, in the book Cloning Vectors (Eds. Pouwels P.H. et al. Elsevier, Amsterdam-New York-Oxford, 1985, ISBN 0 444 904018). In addition to plasmids, vectors are also understood to mean all other vectors known to those skilled in the art, such as phages, viruses, transposons, IS elements, phasmids, cosmids, linear or circular DNA. These vectors can be replicated autonomously in the host organism or can be replicated chromosomally.

expression systems include the combination of the host organisms mentioned above by way of example and the vectors which match the organisms, such as plasmids, viruses or phages such as, for example, plasmids with the RNA polymerase / promoter system, the phages λ, Mu or other tempered phages or transposons and / or to understand further advantageous regulatory sequences.

Preferred under the term expression systems are the combination of Escherichia coli and its plasmids and phages and the associated promoters as well as Bacillus and its plasmids and

Understand promoters. For the advantageous expression of SEQ ID No. 1, SEQ ID No.3, SEQ ID No.5 and / or SEQ ID No. 7 further 3 'and / or 5' terminal regulatory sequences are also suitable.

These regulatory sequences are intended to enable the targeted expression of the biotin genes and the protein expression. Depending on the host organism, this can mean, for example, that the gene is only expressed or overexpressed after induction, or that it is expressed and / or overexpressed immediately.

The regulatory sequences or factors can preferably have a positive influence on biotin expression and thereby increase it. Thus, the regulatory elements can advantageously be strengthened at the transcription level by using strong transcription signals such as promoters and / or "enhancers". In addition, an increase in translation is also possible, for example, by improving the stability of the mRNA.

“Enhancers” are understood to mean, for example, DNA sequences which bring about increased expression of biotin via an improved interaction between RNA polymerase and DNA.

An increase in the sequence SEQ ID No. 1, SEQ ID No.3, SEQ ID No.5 and SEQ ID No.7 derived proteins (see SEQ ID No.2, SEQ ID No.4, SEQ ID No.6 and SEQ ID No.8) and their enzyme activity can be compared to the parent enzymes, for example, by changing the corresponding gene sequences or the sequences of its homologues by classic mutagenesis such as UV radiation or treatment with chemical mutants and / or by targeted mutagenesis such as site directed mutagenesis, deletion (s), insertion achieve (s) and / or substitution (s). In addition to the gene amplification described, increased enzyme activity can also be achieved by eliminating factors which repress enzyme synthesis and / or by synthesizing active instead of inactive enzymes.

The process according to the invention is used to convert DTB into biotin and thus overall biotin productivity via the biotin genes with the sequence SEQ ID no. 1, SEQ ID No.3, SEQ ID No.5 and SEQ ID No.7 and the combinations of the genes of the sequence SEQ ID No.l and SEQ ID No.5 or SEQ ID No.l and SEQ ID No.7, preferably the combination of the genes of the sequence SEQ ID No. 1 and SEQ ID No.3 is advantageously increased device.

In the method according to the invention, the microorganisms containing SEQ ID No. 1, SEQ ID No.3, SEQ ID No.5 and / or SEQ ID No.7 are grown in a medium which enables these organisms to grow. This medium can be a synthetic or a natural medium. Depending on the organism, media known to the person skilled in the art are used. For the growth of the microorganisms, the media used contain a carbon source, a nitrogen source, inorganic salts and possibly small amounts of vitamins and trace elements.

Advantageous carbon sources are, for example, sugars such as mono-, di- or polysaccharides such as glucose, fructose, mannose, xylose, galactose, ribose, sorbose, ribulose, lactose, maltose, sucrose, raffinose, starch or cellulose, complex sugar sources such as molasses, sugar phosphates such as fructose-1, 6-bisphosp-, sugar alcohols such as mannitol, polyols such as glycerol, alcohols such as methanol or ethanol, carboxylic acids such as citric acid, lactic acid or acetic acid, fats such as soybean oil or rapeseed oil, amino acids such as glutamic acid or aspartic acid or amino sugar, the can also be used simultaneously as a nitrogen source.

Advantageous nitrogen sources are organic or inorganic nitrogen compounds or materials that contain these compounds. Examples are ammonium salts such as NH ₄ CI or (H ₄ ) ₂ Sθ ₄ , nitrates, urea, or complex nitrogen sources such as corn steep liquor, beer yeast autolysate, soybean flour, wheat gluten, yeast extract, meat extract, casein hydrolyzate, yeast or potato protein, which are often also used simultaneously as a nitrogen source can serve.

Examples of inorganic salts are the salts of calcium, magnesium, sodium, manganese, potassium, zinc, copper and iron. The chlorine, sulfate and phosphate ions are particularly worth mentioning as the anion of these salts. An important factor for increasing productivity in the process according to the invention is the addition of Fe ^{2 + _} or Fe ³⁺ salts and / or potassium salts to the production medium.

If necessary, further growth factors are added to the nutrient medium, such as vitamins or growth promoters such as riboflavin, thiamine, folic acid, nicotinic acid, pantothenate or pyridoxine, amino acids such as alanine, cysteine, asparagine, aspartic acid, glutamine, serine, methonine or lysine, carboxylic acids such as ci- tronic acid, formic acid, pimelic acid or lactic acid, or substances such as dithiothreitol. Antibiotics can optionally be added to the medium to stabilize the vectors with the biotin genes in the cells.

The mixing ratio of the nutrients mentioned depends on the type of fermentation and is determined in each individual case. The medium components can all be introduced at the start of the fermentation, after they have been sterilized separately if necessary or sterilized together, or after the fermentation has been added as required.

The breeding conditions are determined in such a way that the organisms grow optimally and that the best possible yields are achieved. Preferred cultivation temperatures are 15 ° C to 40 ° C. Temperatures between 25 ° C and 37 ° C are particularly advantageous. the pH is preferably held in a range from 3 to 9. PH values between 5 and 8 are particularly advantageous. In general, an incubation period of 8 to 240 hours, preferably 8 to 120 hours, is sufficient. Within this time the maximum amount of biotin accumulates in the medium and / or is available after the cells have been disrupted.

The method according to the invention for the production of biotin can be carried out continuously or batch or fed-batch. If whole plants are regenerated from the plant cells transformed with the biotin genes, they can be grown and propagated in the normal way using the method according to the invention.

Examples:

1. Cloning of the S-adenosyl methionine synthase gene (SEQ ID No. 1):

The gene coding for SAM synthase (metK) was amplified from the chromosome of E. coli by a polymerase chain reaction using two specific oligonucleotides based on genomic E. coli DNA. The DNA amplified in this way was purified, digested with the restriction enzyme Acc65I and inserted into a vector cut with the same enzyme, which enables the gene to be overexpressed in E. coli strains. One of the two oligonucleotides provided the gene construct with optimized translation signals

a. ) Development of oligonucleotides for the amplification of the metK gene from the E. coli chromosome: metK is to be amplified as an expression cassette consisting of a ribosomal binding site, the start codon of the coding sequence and the stop codon between two recognition sites for restriction enzymes. The recognition sequence of Acc65l was chosen for both restriction sites. The metK gene was amplified and cloned using the oligonucleotides PmetKl (5'-GCGGTACCAGGTGATATTAAATATG-GCAAAAC-3 ') and PmetK2 (5' -CGGGTACCGATTACTTCAGACCGGCAGC-3 ').

b. ) Execution of the PCR:

Conditions:

0.5 μg of chromosomal DNA from E. coli W3110 was used as the template. The oligonucleotides PmetKl and PmetK2 were used in a concentration of 15 pmol each. The concentration of dNTP's was 200 μM. 2.5 U Pwo DNA polymerase (Boehringer Mannheim) was used as the polymerase in the manufacturer's reaction buffer. The volume of the PCR reaction was 100 μl.

Amplification conditions:

The DNA was denatured at 94 ° C. for 2 min. The oligonucleotides were then attached at 55 ° C. for 30 seconds. The elongation was carried out for 75 seconds at 72 ° C. The PCR reaction was carried out over 30 cycles.

The DNA product with a size of approx. 1145 bp was purified and digested with Acc65l in a suitable buffer.

c. ) Cloning of metK in expression vector

2 μg of the vector pHSl (construction was described in DE 197.31274.8, priority 22.7.97, examples 1. page 14 to 17) were digested with Acc65I and dephosphorylated by Shrimp Alkaline Phosphatase (SAP) (Boehringer Mannheim). After the SAP had been denatured, the vector and fragment were ligated in a molar ratio of 1: 3 using the Rapid DNA ligation kit according to the manufacturer's instructions. The transformation of the ligation approach took place in the strain E. coli XL-1-blue. Positive clones were identified by plasmid preparation and restriction analysis. The correct orientation of the MetK fragment in pHSl was determined by restriction digestion and sequencing. The construct obtained was called pHS1 metK (FIG. 1). The sequence of pHSl metK can be found in SEQ ID No.9. SEQ ID No.10 shows the deduced amino acid sequence of the coding region for metK. 2. Construction of the plasmids pHBbiol4 and pHSl bioSl

The construction of the plasmids pHBbiol4 and pHSl bioSl has already been described (DE 197.31274.8, priority 22.7.97, examples 5 1, 2 and 5).

3. Construction of pHSl metK bioSl

The plasmids pHSl bioSl [SEQ ID No.ll, (DE 197.31274.8, priority

10 22.7.97), SEQ ID No.12 shows the derived amino acid sequence of the coding region for bioSl] and pHSl metK SEQ ID No.9 were purified by a plasmid preparation method (Boehringer). The fragment carrying metK gene was isolated from pHSl metK by Acc65l digestion. pHSl bioSl was replaced by Acc65I

15 daut, and dephosphorylated by Shrimp Alkaline Phosphatase (SAP) (Boehringer Mannheim). After the SAP had been denatured according to the manufacturer's instructions, the vector and the metK fragment were ligated in a molar ratio of 1: 3 using the Rapid DNA ligation kit according to the manufacturer's instructions. The transformation

20 of the ligation mixture was carried out in the strain E. coli XL-1-blue. Positive clones were identified by plasmid preparation and restriction analysis. The correct orientation of the metK fragment in pHSl bioSl was determined by restriction digestion and sequencing. The construct obtained was pHSl metK bioSl (Figure 2)

25 called. The sequence of pHSl metK bioSl can be found in SEQ ID No.13. SEQ ID No.14 shows the deduced amino acid sequence of the coding region for metK, SEQ ID No.15 shows the deduced amino acid sequence of the coding region for bioSl

30 4. Increasing biotin productivity through overexpression of metK, bioSl and metK in combination with bioSl.

The BM4086 strain (Ketner and Campbell J. Molec. Biology 1975 96:13) was spontaneously analyzed by plating on rifampycin plates.

35 fampycin-resistant colonies isolated. A PI lysate was generated from one of these resistant strains. The W3110 strain was transduced with this PI lysate and then clones were selected by rifampycin. The strain obtained was transformed with the plasmid pHBbiol4 by the CaCl ₂ method (Maniatis et al. Molecular

40 Cloning Cols Spring Harbor Laboratory Press 1989) and drawn on LB ampicillin 100 μg / ml. The isolated, transformed strain (LU5560) was transformed with the plasmid pHSl, pHSl metK, pHSl bioSl or pHSl metK bioSl according to the CaCl ₂ method and on LB agar with ampicillin 100 μg / ml and kanamycin 25 μg / ml

45 selected. One colony each of the respective transformants was inoculated with the appropriate antibiotics in a DYT culture and incubated for 12 h. The overnight culture (= ÜNK) was used for a 10 ml culture in TB medium (Sambrook, J. Fritsch, E F. Maniatis, T. 2nd ed. Cold Spring Harbor Laboratory Press., 1989

ISBN 0-87969-373-8), which contains 30g / l glycerol, with the appropriate antibiotics. In the presence of the plasmids pHSl, pHSl metK, pHSlbioSl and pHSl metK bioSl, ImM IPTG and 0.5% arabinose were added simultaneously to induce gene expression of metK and bioSl or the combination of both genes. After 24 hours, the cells were separated from the culture supernatant by centrifugation and the biotin concentration was determined by a competitive ELISA with streptavidin in the supernatant. The results of this determination can be found in Table I.

Table I: Determination of the biotin concentration

Claims

claims

1. A process for the preparation of biotin, characterized in that an S-adenosyl-methionine synthase gene with the sequence SEQ ID No. 1 and at least one other biotin biosynthetic gene bioSl, bioS2 or bioS3 with the sequences SEQ ID No. 3, SEQ ID No. 5 or SEQ ID No. 7 and their functional variants, analogs or derivatives in a prokaryotic or eukaryotic host organism which is able to synthesize, expresses, cultivates and uses the synthesized biotin directly after separating off the biomass or after purifying the biotin.

2. The method according to claim 1, characterized in that it is in the variants of the genes with the sequences SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 5 and SEQ ID No. 7 are genes which have a homology of 30 to 100% at the amino acid level derived from the sequences according to claim 1 and enable increased biotin synthesis.

3. The method according to claim 1 or 2, characterized in that an organism selected from the group of the genera Escherichia, Citrobacter, Serratia, Klebsiella, Salmonella, Pseudomonas, Comamonas, Acinetobacter, Azotobacter, Chromobacterium, Bacillus, Chlostridium, Arthrobacter, Corynebacterium as host organism , Brevibacterium, Laetococeus, Laetobacillus, Streptomyces, Rhizobium, Agrobacterium, Staphylococcus, Rhodotorula, Sprorobolomyces, Yarrowia, Schizosaccharomyces or Saccharomyces is used.

4. Process according to claims 1 to 3, characterized in that a regulation-defective biotin mutant is used as the host organism.

5. The method according to claims 1 to 4, characterized in that the expression of at least one copy of the genes with the sequences SEQ ID No .1, SEQ ID No. 3, SEQ ID No. 5 and SEQ ID No. 7 according to claim 1 alone or with one or more copies of at least one further bioting gene selected from the group bioA, bioB, bioF, bioC, bioD, bioH, bioP, bioW, bioX, bioY or bioR in a prokaryotic or eukaryotic host organism.

Sign. ^'■ >. Process according to claims 1 to 5, characterized in that the expression of at least one copy of the genes with the sequences SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 5 and SEQ ID No. 7 according to claim 1 alone or with one or more copies of at least one further bioting gene selected from the group bioA, bioB, bioF, bioC, bioD, bioH, bioP, bioW, bioX, bioY or bioR in a prokaryotic or eukaryotic host organism on a common or done on separate vectors.

7. Gene construct containing an S-adenosyl-methionine synthase gene with SEQ ID No. 1 and at least one other biotin bio-synthesis gene bioSl, bioS2 or bioS3 with the sequences SEQ ID No. 3, SEQ ID No. 5 and SEQ ID No. 7 and their functional variants, analogs or derivatives, which is functionally linked to one or more regulation signals to increase gene expression and / or protein expression and / or whose natural regulation has been switched off.

8. A gene construct according to claim 7, characterized in that it has been inserted in a vector which is suitable for the expression of the gene in a prokaryotic or eukaryotic host organism.

9. gene construct according to claim 7 or 8, characterized in that the genes with the sequences SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 5 and SEQ ID No. 7 and their functional variants, analogs or derivatives are present in multiple copies in the gene construct.

10. Gene construct according to claims 7 to 9, characterized in that the S-adenosyl-methionine synthase gene SEQ ID No. 1 and at least one other biotin biosynthetic gene bioSl, bioS2 or bioS3 with the sequences SEQ ID No. 3, SEQ ID No. 5 and SEQ ID No. 7 and their functional variants, analogs or derivatives according to claim 7 together with one or more copies of at least one further gene selected from the group bioA, bioB, bioF, bioC, bioD, bioH, bioP, bioW, bioX, bioY or bioR in the gene construct or Vector is present.

11. organisms containing a gene construct according to claims 7 to 10.

12. Use of the sequences according to claim 1 for the production of biotin.

13. Use of the bioS3 gene with the sequence SEQ ID No. 7 of its functional variants, analogs or derivatives alone or in combination with at least one further gene selected from the group S-adenosyl methionine synthase genes, bioSl, bioS2, bioA, bioB, bioF, bioC, bioD, bioH, bioP, bioW, bioX , bioY or bioR for the production of biotin.

14. Use of a gene construct according to claims 7 to 10 for the production of biotin.