JP2002504338A - Method for producing biotin - Google Patents

Abstract

  (57) [Summary] The present invention relates to an S-adenosylmethionine synthase gene having the sequence of SEQ ID NO: 1, a biotin biosynthesis gene having a sequence of SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7, bioS1, bioS2 and / or bioS3, and bioA, bioB, It relates to a recombinant product having at least one other biotin-synthesizing gene sequence selected from the group of bioF, bioC, bioD, bioH, bioP, bioW, bioX, bioY or bioR. The present invention also relates to an organism having the above-described genetically modified product, the use of the product for producing biotin, and a method for producing biotin.

Description

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to an S-adenosylmethionine synthase gene having the sequence of SEQ ID NO: 1, and biotin biosynthesis genes bioS1, bioS2 and / or bioS3 having the sequences of SEQ ID NO: 3, SEQ ID NO: 5 and SEQ ID NO: 7, respectively. And optionally bioA, bioB, bioF, bioC, bioD, bioH
, BioP, bioW, bioX, bioY or bioR at least one other biotin-synthesizing gene sequence.
The present invention further relates to an organism having the gene construct and the use of the gene construct for producing biotin, and a method for producing biotin.

As a coenzyme, biotin (vitamin H) plays an essential role in enzyme-catalyzed carboxylation and decarboxylation reactions. Therefore, biotin is an essential factor in living cells. Almost all animals and some microorganisms need to take biotin from the outside world. Because they cannot synthesize biotin on their own. Therefore, biotin is an essential vitamin for such organisms. In contrast, bacteria, yeasts and plants can themselves synthesize biotin from precursors (Brown et al. Biotechnol. Genet. Eng. Rev. 9, 1991:
295-326, DeMoll, E., Escherichia coli and Salmonella, eds.Neidhardt, F
C. et al. ASM Press, Washington DC, USA, 1996: 704-708, ISBN 1-55581-0
84-5).

[0003] Biotin synthesis has been investigated in bacterial organisms, particularly the gram-negative bacterium Escherichia coli and the gram-positive bacterium Bacillus sphaericus (Brown et al. Biotechnol. Genet. Eng. Rev. 9, 1991: 295-326)
. Pimeryl-CoA (PmCoA) obtained from fatty acid synthesis has previously been described in E. coli. co
is considered the first known intermediate in li (DeMoll, E., Escherichia
coli and Salmonella, eds.Neidherdt, FC et al. ASM Press, Washington
DC, USA, 1996: 704-708, ISBN 1-55581-084-5 1996). Until now, the biotin precursor has been E. coli. The pathways synthesized in E. coli were largely unknown (Lemoine et
al., Mol. Microbiol. 19, 1996: 645-647). bioC and bioH have been identified as two genes whose corresponding proteins are responsible for Pm-CoA synthesis.
The enzymatic function of the gene products, BioH and BioC, was hitherto unknown (Lemoine et al., Mol. Microbiol. 19, 1996: 645-647, DeMoll, E
., Escherichia coli and Salmonella, eds. Neidhardt, FC et al. ASM Pre
ss, Washington DC, USA, 1996: 704-708, ISBN 1-55581-084-5). Pm-Co A is converted to biotin in four additional enzymatic steps. First, BioF is Pm
-CoA and alanine are condensed to give 7-keto-8-aminopelargonic acid (KAPA
) Is formed. KAPA is then converted by BioA to 7,8-diaminopelargonic acid (DAPA). After the ATP-dependent carboxylation reaction, the subsequent step is catalyzed by BioD to give dethiobiotin (DTB). DTB is converted to biotin in the final step. This stage is catalyzed by BioB. DT
The chemical and enzymatic mechanisms involved in the conversion of B to biotin have hitherto only been incompletely understood and elucidated.

[0004] Conversion of DTB to biotin has hitherto only been characterized in bacterial and plant cell extracts (WO94 / 8023, EP-B-0449724, Sanyal et al.
Arch. Biochem. Biophys., Vol. 326, No. 1, 1996: 48-56 and Biochemistry 3
3, 1994: 3625-3631, Baldet et al. Europ. J. Biochem. 217, 1, 1993: 479-4
85, Mejean et al. Biochem. Biophys. Res.Commun., Vol. 217, No. 3, 1995:
1231-1237, Ohshiro et al., Biosci. Biotechnol. Biochem., 58, 9, 1994: 17
38-1741).

[0005] In vitro studies have demonstrated that low molecular weight factors such as NADPH, cysteine, thiamine, Fe ²⁺ , asparagine, serine, fructose 1-6-biphosphate and S-adenosylmethionine can induce biotin synthesis. (Ohshiro et al., Biosci. Biotechnol. Biochem., 58, 9, 1994:
1738-1741, Birch et al., J. Biol. Chem. 270, 32, 1995: 19158-19165, Ifu
ku et al., Biosci. Biotechnol. Biochem., 59, 2, 1995: 185-189, Sanyal et.
al. Arch. Biochem. Biophys. 326, 1, 1996: 48-56).

In addition to the above low molecular weight factors, other proteins have been identified that stimulate the synthesis of biotin from DTB in the presence of BioB. These proteins are flavodoxin and flavodoxin NADPH reductase (Birch et al., J. B.
iol. Chem. 270, 32, 1995: 19158-19165, Ifuku et al., Biosci. Biotechnol.
Biochem., 59, 2, 1995: 185-189, Sanyal et al., Arch. Biochem. Biophys.
326, 1, 1996: 48-56). Other proteins that induce biotin synthesis are the genes bioS1 and bioS2 described in the German application with application number 197.31274.8 (priority 22.7.97).

Various results have been obtained for the origin of sulfur in biotin molecules. Investigation of biotin synthesis in whole cell extracts showed that radioactivity was incorporated into biotin in the presence of ³⁵ S-labeled cysteine, with either ³⁵ S-labeled methionine or S-adenosyl. When using any of the methionines, the introduction of sulfur into the biotin molecule cannot be demonstrated (Ifuku et al., Biosci.
Biotechnol. Biochem. 59, 2, 1995: 184-189, Birch et al., J. Biol. Chem.
270, 32, 1995: 19158-19165).

[0008] The genes encoding the aforementioned proteins, ie, bioF, bioA, bioD and bioB are described in E. coli. E. coli, encoded on the bidirectional operon. The operon is located between the λ attachment site and the uvrB locus at about 17 minutes on the E. coli chromosome (Berlyn et al. 1996: 1715-1902). The other two genes, one of which, bioC, has the above function in the synthesis of Pm-CoA, are further encoded on the operon, while the open reading, which has been previously located downstream of bioA, It was not possible to add any function to the frame (WO94 / 8023, Otsuka et al., J. Biol. Chem. 263, 1988: 19577-85). E. FIG.
Highly conserved analogs for the E. coli proteins BioF, BioA, BioD and BioB are described in Sphaeryx, B. Subtilis, Synechocystis sp. (Brown et al. Biotechnol. Genet. Eng. Rev. 9, 19)
91: 295-326, Bower et al., J. Bacteriol. 175, 1996: 4122-4130, Kaneko et
al., DNA Res. 3, 3, 1996: 109-136), archaeobacte
ria), such as Methanococcus janaschi, and yeasts, such as Saccharomyces cerevisiae (Zhang et al., Arch. Biochem. Bi).
ophys. 309, 1, 1994: 29-35) or plants, such as Arabidopsis thaliana (A
rabidopsis thaliana) (Baldet et al., CR Acad. Sci. III, Sci. Vie. 31)
9, 2, 1996: 99-106).

[0009] In two gram-positive microorganisms that have been investigated so far, the synthesis of Pm-CoA depends on its E. coli. It appears to be proceeding in a different manner than synthesis in E. coli. It was not possible to find any homologues of bioH and bioC (Brown et al. Biotechnol. Genet. Eng. Rev. 9, 1991: 295-326).

[0010] Biotin is an optically active substance having three chiral centers. Previously, they were only economically produced by expensive multi-step chemical synthesis methods.

As an alternative to the chemical synthesis, many attempts have been made to construct fermentative methods for biotin production using microorganisms. Cloning of the biotin operon on multicopy plasmids has been successfully used to increase biotin synthesis in microorganisms transformed with the gene. A further increase in biotin synthesis was achieved by deregulating biotin gene expression by selection of virA mutants (Pai CH, J. Bacteriol. 112, 1972: 1280).
-1287). The combination of the two approaches, expression of the biosynthetic gene encoded in the plasmid in a regulatory deficient strain (EP-B-0236429), further increased the productivity. In this regard, the biotin operon can remain under the control of its native bidirectional promoter (EP-B-0236429), or it can bring the gene under the control of an externally regulated promoter (WO94 / 08023). issue).

Up to now, E.I. The approaches sought to produce biotin fermentatively in E. coli fail to achieve economically suitable productivity at all.

It is an object of the present invention to develop an industrial fermentative process for producing biotin that exhibits as high a biotin synthesis as possible.

The object is to provide an S-adenosylmethionine synthase (SAM synthase) having the sequence SEQ ID NO: 1 and at least one other biotin biosynthesis gene having the sequence SEQ ID NO: 3, SEQ ID NO: 5 and SEQ ID NO: 7 bioS1, bioS2 or bioS3, and functional variants, analogs or derivatives thereof, are expressed in a prokaryotic or eukaryotic host organism capable of biotin synthesis, and the organism is cultured. However, it has been found that the problem can be solved by the method for producing biotin according to the present invention in which the synthesized biotin is directly used after removing biomass or after purifying biotin.

[0015] The gene used in the method according to the invention, ie the S having the sequence of SEQ ID NO: 1
The AM synthase gene and the biotin biosynthesis genes bioS1, bioS2 and bioS3 having the sequences of SEQ ID NO: 3, SEQ ID NO: 5 and SEQ ID NO: 7 are Swi
In the ssProt database, accession number P04384 (metK
), U29581 (bioS1), P39171 (bioS2) and D908.
11 (bioS3). E. FIG. Several homologs of E. coli MetK are described in the database. These homologs can be found in organisms such as other eukaryotic bacteria (eg, H. influenza, and B. subtilis) and eukaryotes (eg,
For example, yeast: S. Cerevisiae, plant: P. Deltoides, arthropod: D. Melanogaster and honey bean: Norvegicus
)including.

[0016] The productivity of biotin biosynthesis is determined by combining one or more SAM synthase genes having the sequence of SEQ ID NO: 1 and functional variants, analogs or derivatives thereof to the sequences of SEQ ID NO: 3, SEQ ID NO: 5 and SEQ ID NO: 7. At least one biotin synthesis gene b having
It can be significantly increased by expression in prokaryotic or eukaryotic host organisms in combination with ioS1, bioS2 or bioS3, and functional variants, analogs or derivatives thereof. Advantageously, the SAM synthase gene and b
The combination with ioS1 is used for expression. bioA, bioB, bioF
, BioC, bioD, bioH, bioP, bioW, bioX, bioY and bioR are advantageously co-expressed with at least one other biotin gene, further increasing biotin synthesis. Expression of the gene increases biotin synthesis at least two-fold, advantageously three-fold or more, as compared to a control without the gene.

A SAM synthase having the nucleotide sequence of SEQ ID NO: 1, encoding the gene used in the method according to the invention, ie the amino acid sequence set forth in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 and SEQ ID NO: 8, respectively A bioS1 gene having the nucleotide sequence of SEQ ID NO: 3, b having the nucleotide sequence of SEQ ID NO: 5
The ioS2 gene as well as the bioS3 gene having the nucleotide sequence of SEQ ID NO: 7, or an allelic variant thereof, can be obtained after isolation and sequencing.
Variants are SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, each having 30-100% homology, preferably 50-100% homology, particularly preferably 80-100% homology at the amino acid level. And a variant of SEQ ID NO: 7. Allelic variants are obtained, in particular, by deleting, inserting or substituting nucleotides from the sequences set forth in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 and SEQ ID NO: 7, while maintaining enzymatic activity. Includes functional variants.

[0018] Furthermore, the mutants may have O-acetylserine sulfohydrolase A, O-acetylserine sulfohydrolase A, which can exhibit the bioS1, bioS2 or bioS3 enzymatic activity in biotin synthesis.
-Acetylserine sulfohydrolase B, β-cystathionase (Flint et al.
, J. Biol. Chem., Vol. 271, 1996: 16053-16067) or a functional equivalent of a gene such as nifS and, for example, Klebsiella, Candida,
It should be understood to be prokaryotic and eukaryotic homologues thereof from yeast or Caenorhabditis.

The 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 analogs, for example homologs of bacteria, fungi, plants, animals or humans Should be understood as a body. Furthermore, an analog is to be understood as a single-stranded DNA or RNA from a missing sequence or from coding and non-coding DNA sequences.

A derivative is to be understood as being, for example, a promoter variant. A promoter located upstream of the recited nucleotide sequence may be altered by one or more nucleotide substitutions, or insertions and / or deletions, without compromising the functionality or activity of the promoter. In addition, the activity of the promoter may be increased by altering its sequence, or may be completely replaced by a more active promoter, including promoters from different species of organisms.

Derivatives are also to be understood as variants in which the nucleotide sequence is changed in the region -1 to -30 upstream of the start codon to increase gene expression and / or protein expression. is there. Preference is given to a change in the Shine-Dalgarno sequence.

All Gram-negative or Gram-positive bacteria which synthesize biotin are in principle suitable for use as prokaryotic host organisms in the process according to the invention.
Examples of Gram-negative bacteria include Enterobacteriaceae, such as Escherichia, Aerobacterium, Enterobacter, Citrobacter, Shigella, Klebsiella, Serratia, Erwinia or Salmonella, Pseudomonas, such as Pseudomonas, Zantmonas, Burkholderia, Gluconobacter, Nitrosomonas, Nitrobacter, Methanomonas, Comamonas, Cellulomonas or Acetobacter, Azotobacteriaceae, such as Azotobacter, Azomonas, Bayerinchia or Derxia,
Neisseriaceae, such as Moraxella, Acinetobacter, Kingella, Neisseria or Branhamella, Rhizobium such as Rhizobium or Agrobacterium, or Gram-negative Zymomonas, Chromobacterium or Flavobacterium. Examples of Gram-positive bacteria include endospore-forming Gram-positive aerobic or anaerobic bacteria, such as Bacillus,
Spororactobacillus or Clostridium, Coryneform bacteria such as Arthrobacter, Cellulomonas, Curtobacterium, Corynebacterium, Brevibacterium, Microbacterium or Crucia, Actinomyces, For example, Mycobacterium, Rhodococcus, Streptomyces or Nocardia, Lactobacillus, such as Lactobacillus or Lactococcus, or Gram-positive cocci, such as Micrococcus or Staphylococcus.

Advantageously, the genus Escherichia, Citrobacter, Serratia, Klebsiella, Salmonella, Pseudomonas, Comamonas, Acinetobacter, Azotobacter, Chromobacterium, Bacillus, Clostridium, Arthrobacter, Corynebacterium, Brevibacterium, Lactococcus, Lactobacillus, Streptomyces, Rhizobium, Agrobacterium or Staphylococcus are used in the method according to the invention. Particular preference is given to genera and species, such as Escherichia coli, Citrobacter freundii, Serratia marcescens, Salmonella typhimurium, Pseudomonas mendocina, Pseudomonas aeruginosa, Pseudomonas mutabilis, Pseudomonas chlorosomas s. Calcoaceticus, Azotobacter vinelandii, Chromobacterium violaceum, Bacillus subtilis, Bacillus sphaericus, Bacillus stearothermophilus, Bacillus pmirus, Bacillus licheniformis, Bacillus amiloric facilens, Bacillus megalithium , Bacillus thuringiensis, Arthrobacter paraffineus, Arthrobacter paraffineus, Corynebacterium glutamicum, Corynebacterium primorioxydans, Corynebacterium sp., Brevibacterium ketoglutamicum, Brevi bacteria Brevibacterium species, Streptomyces lividans, Rhizobium leguminosarum or Agrobacterium tumefaciens. Advantageously, bacteria are used which exhibit an already enhanced natural production of biotin.

The taxonomic position of the genus listed has undergone considerable changes in recent years, and is still in flux as a tentative genus, and the species names have been corrected. Due to the taxonomic regrouping of the genus of bacterial taxonomy, which was often necessary in the past, other families, genera and species within the genus of the bacterial taxonomy according to the invention may also be used. Suitable for

All biotin-synthesizing organisms, such as fungi, yeasts, plants or plant cells, are in principle suitable for use as eukaryotic host organisms in the process according to the invention. Advantageously mentioned yeasts are of the genera Rhodotorula, Yarrowia, Sporoboromyces, Saccharomyces or Schizosaccharomyces. Particularly preferred are genera and species, Rhodotorula rubra, Rhodotorula glutinis, Rhodotorula graminis.
minis), Yaloa Ripolitica, Sporobolomyces salmonikola (Sporobo
lomyces salmonicolor) and Sporobolomyces sh
ibatanus) or Saccharomyces cerevisiae.

In principle, all plants may be used as host organisms, advantageously plants having a role in animal or human nutrition, such as corn, wheat, barley, rye, potato, pea. , Kidney bean, sunflower, palm, millet, sesame, copra or rapeseed. Plants such as Arabidopsis thaliana or Lavendula vera are also suitable. Particular preference is given to plant cell culture, plant cell culture, plant protoplast or callus culture.

Microorganisms, such as bacteria, fungi, yeasts or plant cells, which can secrete biotin into the growth medium and which optionally exhibit an already enhanced natural synthesis of biotin, are advantageously used in the process according to the invention. I do. Advantageously, these organisms may be deficient in regulating their biotin biosynthesis; that is, the synthesis is unregulated or
Or adjusted only to a very low degree. This deficiency in regulation results in organisms that already have increased biotin productivity. Such a deficiency in regulation is known, for example, from Escherichia coli in the form of a birA-deficient mutant and should advantageously be present in the cell as a deficiency which can be induced by external influences, for example a deficiency which is temperature-induced. is there. In principle, organisms that do not show any natural biotin production may also be used if they have been transformed with the biotin gene.

To increase the biotin productivity still further overall, the organisms of the method according to the invention are advantageously bioA, bioB, bioF, bioC, bio.
It should have at least one biotin gene selected from the group of D, bioH, bioP, bioW, bioX, bioY or bioR. Advantageously,
Said gene that induces biotin synthesis may be present in the cell in combination with the sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7 and combinations thereof. Examples of genes that induce biotin synthesis are the flavoredoxin gene and the flavoredoxin reductase gene. The additional genes or these additional genes are present in one or more copies in the cell, such as a gene having the sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 7, or a combination thereof. You may. These may be located in the same vector as the sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 and / or SEQ ID NO: 7, or on a separate vector or integrated on the chromosome. SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5
And / or SEQ ID NO: 7 may be inserted together on one vector, or on separate vectors, or in the genome.

[0029] The gene construct according to the present invention is for the gene sequence of SAM synthase gene SEQ ID NO: 1, the gene sequence of biotin synthesis gene SEQ ID NO: 3, SEQ ID NO: 5 and / or SEQ ID NO: 7, and for increasing gene expression It should be understood that a functional variant, analog or derivative thereof operably linked to one or more regulatory signals. In addition to these novel regulatory sequences, the native regulation of these sequences may still be present upstream of the virtual structural gene, and in some cases the native regulation may be switched off to increase gene expression It may be genetically altered. However, the genetic constructs can also be easily assembled, i.e. adding additional regulatory signals to SEQ ID NO: 1, SEQ ID NO: 3,
It is not inserted upstream of SEQ ID NO: 5 and / or SEQ ID NO: 7 and the sequence of the native promoter. Instead, the native regulatory sequence is mutated such that regulation by biotin is no longer performed and gene expression is increased. SEQ ID NO: 1, SEQ ID NO: 3,
The sequence of SEQ ID NO: 5 and / or SEQ ID NO: 7 may be under the control of one promoter or under the control of a separate promoter. Furthermore, advantageous regulatory elements may be inserted at the 3 'end of the DNA sequence.

The gene having the sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7 may be present in one or more copies in the gene construct.

The regulatory sequences which are advantageous for the method according to the invention are, for example, promoters which are advantageously used in gram-negative bacteria, such as cos-, tac-, trp-, tet-,
trp-tet-, lpp-, lac-, lpp-lac-, lacI q -, T 7-, T5-, T3-, gal-, trc-, ara-, SP6-, λ-P R -, or lambda -In the P _L -promoter. Further advantageous regulatory sequences are, for example, the gram-positive promoters amy and SPO2, the yeast promoters ADC1, MFα, AC, P-60, CYC1, GAPDH or the plant promoters CaMV / 35S, SSU, OCS, lib4, usp, STL
Present in S1, B33 or nos, or ubiquitin promoter or phaseolin promoter.

In principle, all native promoters, together with their regulatory sequences, can be used for the method according to the invention like the above-mentioned promoters. In addition, synthetic promoters can be used to advantage.

It has its own promoter, is under the control of a promoter of one sequence, or is under the control of a promoter of all the sequences SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7. BioA, bioB, bioF,
Other biotin genes selected from the group of bioC, bioD, bioH, bioP, bioW, bioX, bioY or bioR may be present in one or more copies in the genetic construct.

For expression in the above-described host organism, the gene construct is advantageously inserted into a host-specific vector capable of achieving optimal gene expression in the host. Vectors are known to those skilled in the art and are described, for example, in the book Cloning Vectors (Eds. Pouwels PH.
et al. Elsevier, Amsterdam-New York-Oxford, 1985, ISBN 0 444 904018). In addition to a plasmid, a vector is to be understood as any other vector known to the person skilled in the art, for example a phage, virus, transposon, IS element, fazmid, cosmid or linear or circular DNA. These vectors are capable of autonomous replication in the host organism or of being replicated chromosomally.

The expression systems include, by way of example, the host organisms listed above and suitable vectors for such organisms, such as plasmids, viruses or phages, such as RNA polymerase / promoter systems, phage λ, or Mu or other temperate phages or transposons and And / or in combination with a plasmid having other advantageous regulatory sequences.

The term expression system should advantageously be understood as a combination of Escherichia coli and its plasmids and phages and associated promoters, and also a combination of Bacillus with its plasmids and promoters.

Still other 3 ′ and / or 5′-end regulatory sequences are SEQ ID NO: 1,
Suitable for advantageously expressing SEQ ID NO: 3, SEQ ID NO: 5 and / or SEQ ID NO: 7.

The purpose of these regulatory sequences is to be able to achieve specific expression of the biotin gene and protein expression. Depending on the host organism, for example, this may mean that it is expressed or overexpressed after induction, or immediately expressed and / or overexpressed.

In this regard, the regulatory sequence or factor advantageously acts positively on biotin gene expression, thereby increasing expression. For example, regulatory elements can be advantageously enhanced at the transcriptional level using strong transcription signals, such as promoters and / or enhancers. However, further translations, such as mR
It can also be enhanced by improving the stability of the NA.

An enhancer is to be understood as a DNA sequence that results in an increase in biotin gene expression, for example by improving the interaction between RNA polymerase and DNA.

Increase in proteins (see SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 and SEQ ID NO: 8) obtained from the sequences of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 and SEQ ID NO: 7 and comparison with the starting enzyme Increased enzymatic activity, eg, by classical mutagenesis, eg, treatment with UV radiation or chemical mutagens and / or site-directed mutagenesis, eg, site-specific mutagenesis. It can be achieved by altering by mutation, deletion, insertion and / or substitution. Apart from the gene amplification described above, an increase in enzyme activity can also be achieved by eliminating factors that inhibit the biosynthesis of the enzyme and / or by synthesizing an active enzyme instead of an inactive enzyme.

Advantageously, the method according to the invention increases the conversion of DTB to biotin and thus increases all biotin productivity. This is a biotin gene having the sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 and SEQ ID NO: 7 introduced into the organism by its vector and / or by chromosomal cloning, as well as SEQ ID NO: 1 and SEQ ID NO: 5 or the sequence By using a combination of genes having the sequence of SEQ ID NO: 1 and SEQ ID NO: 7, advantageously a combination of genes having the sequence of SEQ ID NO: 1 and SEQ ID NO: 3.

In the method according to the invention, the microorganism having SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 and / or SEQ ID NO: 7 is grown in a medium in which the organism can grow. The medium may be a synthetic or natural medium. Media known to those skilled in the art as well as media appropriate for the organism are used. To enable the growth of microorganisms, the medium used contains a carbon source, a nitrogen source, inorganic salts and possibly trace amounts of vitamins and trace elements.

Examples of advantageous carbon sources are sugars, such as monosaccharides, disaccharides or polysaccharides, such as glucose, fructose, mannose, xylose, galactose, ribose, sorbose, ribulose, lactose, maltose, sucrose, raffinose, starch or cellulose. , A complex sugar source, such as molasses,
Sugar phosphates such as fructose-1,6-bisphosphate, sugar alcohols such as mannitol, polyhydric alcohols 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 seed oil or an amino acid which can also be used simultaneously as a nitrogen source, such as glutamic or aspartic acid.

Preferred nitrogen sources are organic or inorganic nitrogen compounds or materials containing said compounds. Examples are ammonium salts such as NH ₄ Cl or (NH ₄ ) ₂ SO ₄ , nitride or urea, or complex nitrogen sources such as corn steep liquor, brewer's yeast autolysate, soy flour, wheat gluten, yeast extract, meat extract , Casein hydrolyzate or yeast or potato proteins, which can often also be used as nitrogen salts at the same time.

Examples of inorganic salts are calcium, magnesium, sodium, manganese, potassium,
It is a salt of zinc, copper and iron. The anions of these salts which are mentioned in particular are chloride, sulfate and phosphate. An important factor for increasing the productivity in the method according to the invention is the addition of Fe ²⁺ or Fe ³⁺ salts and / or potassium salts to the production medium.

Optionally, other growth factors such as vitamins or growth promoters such as riboflavin, thiamine, folate, nicotinic acid, pantothenate or pyridoxine, amino acids such as alanine, cysteine, asparagine, aspartic acid, glutamine, serine, methionine or Lysine, carboxylic acid, such as citric acid,
A substance such as formic acid, pimelic acid or lactic acid, or dithiothreitol is added to the nutrient medium.

[0048] Optionally, an antibiotic may be added to the medium to stabilize the vector with the biotin gene in the cells.

The mixing ratio of the nutrients depends on the state of the fermentation and is specified in each individual case. All of the components of the medium may be introduced before fermentation, if necessary separately or together after sterilization, or they may be added later if necessary during the fermentation.

The medium conditions are arranged so that the organism grows optimally and the best possible yield is achieved. Preferred culture temperatures are between 15C and 40C. Temperatures between 25 ° C. and 37 ° C. are particularly advantageous. Advantageously, the pH is maintained in the range 3-9. P between 5 and 8
H values are particularly advantageous. In general, an incubation period of 8 to 240 hours, preferably 8 to 120 hours, is sufficient. Within this period, the greatest amount of biotin has accumulated and / or is available after disrupting the cells.

The process for producing biotin according to the invention can be carried out continuously or batchwise or fed-batch. Whole plants can be regenerated from plant cells transformed with the biotin gene and grown and grown completely normal by the method according to the invention.

Embodiments: Cloning of the S-adenosylmethionine synthase gene (SEQ ID NO: 1): Starting from genomic DNA of E. coli, the gene encoding SAM synthase (metK) was E. coli chromosomes were amplified by the polymerase chain reaction using two specific oligonucleotides. The DNA amplified as described above was purified, cut with the restriction enzyme Acc65I, cut with the same enzyme and E. coli. c
The gene was inserted into a vector capable of overexpressing the gene in the oli strain. One of the two oligonucleotides was used to provide a gene construct with optimal translation signals.

A. E.). Construction of oligonucleotides to amplify the metK gene from the E. coli chromosome: metK should be amplified as an expression cassette with a ribosome binding site between the two restriction enzyme recognition sites, a start codon and a stop codon of the coding sequence. Was. The Acc65I recognition sequence was chosen for both restriction sites. metK
The gene was replaced with the nucleotide PmetK1 (5'-GCGGTACCAGGGTGAT
ATTAAATATGGCAAAAAC-3 ') and PmetK2 (5'-CG
GGTACGATTACTTCAGACCGGCAGC-3 ′) and cloned.

B. ) Performing PCR: Conditions: E. coli as template 0.5 μg of chromosomal DNA from E. coli W3110 was used. Oligonucleotides PmetK1 and PmetK2 were in each case 1
Used at a concentration of 5 picomoles (pMol). The concentration of dNTP was 200 μM.
2.5 U of Pwo DNA polymerase (Boehringer Mannheim) in the manufacturer's reaction buffer was used as the polymerase. PCR reaction volume is 100 μl
Met.

Amplification: DNA was denatured at 94 ° C. for 2 minutes. The oligonucleotide was then added at 55 ° C for 3 hours.
Anneal for 0 seconds. Extension was performed at 72 ° C. for 75 seconds. 3 PCR reactions
Performed over 0 cycles.

The resulting DNA product, having a size of about 1145 bp, was purified and digested with Acc65I in a suitable buffer.

C. 2.) Cloning of metK in expression vector 2 μg of the vector pHS1 (construct described in DE 197.31274.8, Example 1 of priority 22.7.97, pages 14 to 17) were digested with Acc65I,
And dephosphorylated using shrimp alkaline phosphatase (SAP) (Boehringer Mannheim). After denaturing the SAP, the vectors and fragments were:
Ligation was performed using the Rapid DNA Ligation Kit at a molar ratio of 3 according to the manufacturer's instructions. The ligation mixture was added to E. coli. coli strain XL-1-
and transformed into blue. Positive clones were identified by plasmid preparation and restriction analysis. The correct orientation of the metK fragment in pHS1 was determined by restriction digestion and sequencing. The resulting construct was called pHS1metK (FIG. 1). The sequence of pHS1 metK is listed in SEQ ID NO: 9. SEQ ID NO: 10 shows an amino acid sequence derived from a region encoding metK.

2. Construction of plasmids pHBbio14 and pHS1 bioS1 The construction of plasmids pHBbio14 and pHSbioS1 has already been described (DE197.3274.8, Example 1, Example 2 and Example 5 with priority 22.7.97).

[0059] 3. Construction of pHS1 metK bioS1 Plasmid pHS1 bioS1 [SEQ ID NO: 11, (DE197.317.84.8, priority 22.7.97), SEQ ID NO: 12 shows the amino acid sequence derived from the region encoding bioS1] and pHS1 metK (SEQ ID NO: 9) was purified using the plasmid preparation method (Boehringer). The fragment containing the metK gene was isolated from pHS1 metK by digesting with Acc65I. pHS1 bioS1 was digested with Acc65I and dephosphorylated with shrimp alkaline phosphatase (SAP) (Boehringer Mannheim). After denaturing the SAP according to the manufacturer's instructions, the vector and the metK fragment were:
Ligation was performed using the Rapid DNA Ligation Kit at a molar ratio of 3 according to the manufacturer's instructions. The ligation mixture was added to E. coli. coli strain XL-1-
Transformed into blue. Positive clones were identified by plasmid preparation and restriction analysis. pHS1 The correct orientation of the metK fragment in bioS1 was determined by restriction digestion and sequencing. The resulting construct was called pHS1 metK bioS1 (FIG. 2). The sequence of pHS1 metK bioS1 is listed in SEQ ID NO: 13. SEQ ID NO: 14 shows an amino acid sequence derived from a region encoding metK, and SEQ ID NO: 15 shows an amino acid sequence derived from a region encoding bioS1.

[0060] 4. Increased biotin productivity by overexpressing metK, bioS1 and bioS1 in combination with bioS1 Innate rifampicillin resistant colonies were isolated from strain BM4086 (Ketner and Campbell J. Molec. Biology 1975 96:13). Isolated by plating on rifampicillin plates. P1 lysates were made from one of these resistant strains. Strain W3110 was transduced with this P1 lysate and clones were subsequently selected using rifampicillin. The resulting strain was transformed with the plasmid pHBbio14 using the CaCl ₂ method (Maniatis et al. Molecular Cloning C
ols Spring Harbor Laboratory Press 1989) and grown on LB containing 100 μg / ml ampicillin. The isolated transformed strain (LU5560) was isolated in each case from plasmids pHS1, pHS1.
metK, pHS1 bioS1 or pHS1 metK bioS1
Transformation was performed using the Cl ₂ method and then selected on LB agar containing 100 μg / ml ampicillin and 25 μg / ml kanamycin.

One colony from each transformant was inoculated in a DYT culture containing in each case the appropriate antibiotic and incubated for 12 hours. By overnight culture (= ONC), a TB medium containing 30 g / l of glycerol and an appropriate antibiotic (Sambrook, J. Fritsch, EF. Maniatis, T. 2nd ed.
10 ml of the culture was inoculated into Cold Spring Harbor Laboratory Press., 1989 ISBN 0-87969-373-8). In each case in which the plasmids pHS1, pHS1 metK, pHS1 bioS1 and pHS1 metK bioS1 are present, 1 mM IPTG and 0.5% arabinose are added simultaneously,
Expression of each of the tK and bioS1 genes, or a combination of the two genes, was induced. Twenty-four hours later, cells were separated from the culture supernatant by centrifugation, and the biotin concentration in the supernatant was determined by competition ELISA using streptavidin. The results of this measurement are shown in Table I.

[0062]

[Table 1]

[0063]

[Table 2]

[0064]

[Table 3]

[0065]

[Table 4]

[0066]

[Table 5]

[0067]

[Table 6]

[0068]

[Table 7]

[0069]

[Table 8]

[0070]

[Table 9]

[0071]

[Table 10]

[0072]

[Table 11]

[0073]

[Table 12]

[0074]

[Table 13]

[0075]

[Table 14]

[0076]

[Table 15]

[0077]

[Table 16]

[0078]

[Table 17]

[0079]

[Table 18]

[0080]

[Table 19]

[0081]

[Table 20]

[0082]

[Table 21]

[0083]

[Table 22]

[0084]

[Table 23]

[0085]

[Table 24]

[0086]

[Table 25]

[0087]

[Table 26]

[0088]

[Table 27]

[0089]

[Table 28]

[0090]

[Table 29]

[0091]

[Table 30]

[0092]

[Table 31]

[0093]

[Table 32]

[0094]

[Table 33]

[0095]

[Table 34]

[0096]

[Table 35]

[0097]

[Table 36]

[0098]

[Table 37]

[Sequence list]

[Brief description of the drawings]

FIG. 1 shows the plasmid pHS1 metK (3720 base pairs).

FIG. 2 shows the plasmid pHS1 metK bioS1 (4975 base pairs).

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI theme coat ゛ (Reference) C12P 17/18 C12R 1:19) // (C12N 1/21 (C12P 17/18 Z C12R 1:19) (C12P 17/18 C12N 15/00 ZNAA 5/00 A F term (reference) 4B024 AA01 AA05 BA80 CA01 DA05 DA06 DA07 DA08 DA09 DA10 DA12 EA04 HA01 4B064 AE55 CA19 CB30 CC24 DA01 DA10 4B065 AA26X AA26Y AB01 BA01 BA02

Claims (14)

[Claims] 1. A method for producing biotin, comprising the steps of:
The adenosylmethionine synthase gene, at least one other biotin biosynthesis gene bioS1 having the sequence SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7
, BioS2 or bioS3, and functional variants, analogs or derivatives thereof, are expressed in prokaryotic and eukaryotic host organisms capable of synthesizing biotin, the organisms are cultured, and the synthesized biotin is expressed in biomass. A method for producing biotin, which is used directly after separation or purification of biotin. 2. The mutant of the gene having the sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, and SEQ ID NO: 7 exhibits a homology of 30 to 100% at the amino acid level derived from the sequence of claim 1. The method according to claim 1, wherein the gene is a gene that enables an increase in biosynthesis of biotin. 3. The genus Escherichia, Citrobacter, Serratia, Klebsiella, Salmonella, Pseudomonas, Comamonas, Acinetobacter, Azotobacter, Chromobacterium, Bacillus, Clostridium, Arthrobacter, Corynebacterium Genus, Brevibacterium, Lactococcus, Lactobacillus, Streptomyces, Rhizobium, Agrobacterium, Staphylococcus, Rhodotorula, Sporoboromyces, Yaloia, Schizosaccharomyces or The method according to claim 1 or 2, wherein an organism selected from the group of the genus Saccharomyces is used as a host organism. 4. The method according to claim 1, wherein the deficient biotin mutant is used as a host organism. 5. The method according to claim 1, wherein at least one copy of the gene having the sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 and SEQ ID NO: 7 is used alone in a prokaryotic or eukaryotic host organism. Or bioA, bioB, bioF, bioC
, BioD, bioH, bioP, bioW, bioX, bioY or bio
The method according to any one of claims 1 to 4, wherein the method is expressed together with one or more copies of at least one other biotin gene selected from the group of R. 6. The method according to claim 1, wherein at least one copy of the gene having the sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 and SEQ ID NO: 7 is used alone in a prokaryotic or eukaryotic host organism. Or bioA, bioB, bioF, bioC, bioD, bioH, bioP on a shared vector or separate vectors
6. The method according to any one of claims 1 to 5, wherein the method is expressed together with one or more copies of at least one other biotin gene selected from the group of: bioW, bioX, bioY or bioR. 7. The S-adenosylmethionine synthase gene sequence of SEQ ID NO: 1 and at least one other biotin biosynthesis gene bioS1, bioS2 or bioS3 having the sequence of SEQ ID NO: 3, SEQ ID NO: 5 and SEQ ID NO: 7,
And has a functional variant, analog or derivative thereof, and is operably linked to one or more regulatory signals to increase gene and / or protein expression and / or its natural regulation is switched off Gene construct to be used. 8. The gene construct according to claim 7, wherein the gene construct is inserted into a vector suitable for expressing the gene in a prokaryotic or eukaryotic host organism. 9. The gene having the sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 and SEQ ID NO: 7 and a functional variant, analog or derivative thereof is present in the gene construct in multiple copies. 9. The genetic construct according to 7 or 8. 10. The S-adenosylmethionine synthase gene of SEQ ID NO: 1 according to claim 7, and at least one other biotin biosynthesis gene having the sequence of SEQ ID NO: 3, SEQ ID NO: 5 and SEQ ID NO: 7 bioS1, bioS2 Alternatively, bioS3, as well as functional variants, analogs or derivatives thereof, can be added to the gene construct or vector in bioA, bioB, bioF, bioC, bioD, bio.
10. The gene construct according to any one of claims 7 to 9, wherein the gene construct is present together with one or more copies of at least one other gene selected from the group of oH, bioP, bioW, bioX, bioY or bioR. . An organism having the gene construct according to any one of claims 7 to 10. 12. Use of the sequence according to claim 1 for the production of biotin. 13. A bioS3 gene alone or a S-adenosylmethionine synthase gene, bioS1, bioS2, bioA, b having the sequence of SEQ ID NO: 7 or a functional variant, analog or derivative thereof for biotin production.
ioB, bioF, bioC, bioD, bioH, bioP, bioW, bi
Use in combination with at least one other gene selected from the group of oX, bioY or bioR. 14. The method according to claim 7, which is for producing biotin.
Use of the gene construct according to the above item.