KR20010041062A - Method for Producing Biotin - Google Patents

Abstract

The present invention relates to an S-acenosylmethionine synthetase gene having SEQ ID NO: 1, and a biotin biosynthetic gene having SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7 bioS1, bioS2 and / or bioS3, and possibly bioA, bioB, bioF, A recombinant product containing one or more additional biotin synthetic gene sequences selected from the group bioC, bioD, bioH, bioP, bioW, bioX, bioY or bioR. The present invention relates to an organism containing this recombinant product and its use for producing biotin, and a method for producing biotin.

Description

Method for Producing Biotin {Method for Producing Biotin}

As coenzymes, biotin (vitamin H) plays an essential role in enzyme-catalyzed carboxylation and decarboxylation reactions. Biotin is therefore an essential factor in living cells. Almost all animals and some microorganisms must take biotin from the outside because they cannot synthesize biotin themselves. Therefore, biotin is an essential vitamin for these organisms. In contrast, bacteria, yeasts and plants themselves can synthesize biotin 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).

The synthesis of biotin has been investigated in bacterial organisms, especially Gram-positive bacteria Escherichia coli and Gram-positive bacteria Bacillus sphaericus (Brown et al. Biotechnol. Genet. Eng. Rev. 9, 1991: 295-326]. Pimelyl-CoA (PmCoA) derived from fatty acid synthesis has already been considered as the first known intermediate in E. coli (Escherichia coli and Salmonella, eds. Neidhardt, FC et al. ASM Press, Washington DC, USA, 1996: 704-708, ISBN 1-55581-084-5 1996]. To date, little is known about how this biotin precursor is synthesized in this collie (Lemoine et al., Mol. Microbiol. 19, 1996: 645-647). bioC and bioH have been identified that the corresponding proteins are two genes responsible for the synthesis of Pm-CoA. The enzymatic function of the gene product, namely BioH and BioC, is not known so far (Remoin et al., Mol. Microbiol. 19, 1996: 645-647, Dsmol et al., Esccherichia 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 additional enzymatic steps. BioF first condenses Pm-CoA with alanine to form 7-keto-8-aminofellargonic acid (KAPA). KAPA is then converted to 7,8-diaminofellargonic acid (DAPA) by BioA. After the ATP-dependent carboxylation reaction, dithiobiotin (DTB) is obtained by the next step, which is promoted by BioD. DTB is converted to biotin in the last step. This step is facilitated by BioB. The chemical and enzymatic mechanisms involved in the conversion of DTB to biotin have so far been incompletely understood and elucidated.

The conversion of DTB to biotin has only been so far characterized in bacterial and plant cell extracts (WO 94/8023, EP-B-0 449 724, Sanal et al., Arch. Biochem. Biophys., Vol. 326, No. 1, 1996: 48-56 and Biochemistry 33, 1994: 3625-3631, Bald et al., Europ. J. Biochem. 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 NADPH, cysteine, thiamine, Fe ²⁺ , asparagine, serine, fructose 1,6-bisphosphate, and S-adenosylmethionine can simulate the synthesis of biotin (Osiro 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., 59, 2, 1995; 185-189, Sarsham et al. Arch. Biochem. Biophys., 326, 1, 1996: 48-56; ).

In addition to these low molecular weight factors, other proteins have been identified that simulate the synthesis of biotin from DTB in the presence of BioB. These proteins are flavodoxin and flavodoxin NADPH reductase (Burch et al., J. Biol. Chem. 270, 32, 1995: 19158-19165, Ipuku et al. Biosci. Biotechnol. Biochem., 59, 2, 1995; 185-189, Sarsham et al., Arch. Biochem. Biophys., 326, 1, 1996: 48-56). Other proteins that simulate biotin synthesis are the genes bioS1 and bioS2, which are described in German patent application No. 197.31274.8 (Priority 22.7.97).

Different results have been obtained regarding the derivation of sulfur in the biotin molecule. Investigations into biotin synthesis in whole cell extracts showed that radioactivity was introduced into biotin in the presence of ³⁵ S-labeled cysteine and into the biotin molecule when ³⁵ S-labeled methionine or S-adenosylmethionine was used. It was not possible to prove the introduction of sulfur (Ipuku et al., Biosci. Biotechnol. Biochem., 59, 2, 1995; 184-189), Birch et al., J. Biol. Chem. 270, 32, 1995: 19158-19165].

The genes encoding the described proteins, namely bioF, bioA, bioD and bioB, are encoded in this collie's bidirectional operon. This operon is located between the λ binding site and the uvrB locus at about 17 minutes on this Coli chromosome (Berlyn et al. (1996: 1715-1902)). Two additional genes (one of which bioC already has the function described in the synthesis of Pm-CoA) are additionally encoded in this operon, but so far will not impart any function to the open reading frame downstream of bioA. (WO 94/8023, Otsuka et al., J. Biol. Chem. 263, 1988: 19577-85). Highly conserved moorings for this collie protein BioF, BioA, BioD and BioB include 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), primitive bacteria such as Methanococcus janaschi and yeast such as Saccharomyces cerevisiae; Arch. Boichem. Biophys. 309, 1, 1994: 29-35) or Arabidopsis thaliana (Valette et al., CR Acad. Sci. III, Sci. Vie. 319, 2, 1996). : 99-106).

In the two Gram-positive microorganisms examined so far, the synthesis of Pm-CoA seems to proceed in a different way than in this collie. No mocha of bioH and bioC was found (Brown et al. Biotechnol. Genet. Eng. Rev. 9. 1991: 295-326).

Biotin is an optically active substance with three chiral centers. Biotin has only been produced economically by expensive multi-step chemical synthesis.

As an alternative to such chemical synthesis, many attempts have been made to construct fermentation processes for producing biotin using microorganisms. Cloning of biotin operons into multi-copy plasmids has been used successfully to enhance biotin synthesis in microorganisms transformed with these genes. The increase in biotin synthesis was also made by deselecting birA mutants to deregulate biotin gene expression (Pai C. H., J. Bacteriol. 112, 1972: 1280-1287). Combining the two trials, ie, expressing plasmid-encoded biosynthesis in a regulatory-defective strain (EP-B-0 236 429), increased productivity even further. In this regard, the biotin operon may remain under the control of its natural bidirectional promoter (EP-B-0 235 429) or its gene may be under the control of an externally regulated promoter (WO94 / 08023).

The attempts made so far to produce biotin by fermentation in this collie have not achieved economically adequate productivity.

The present invention provides an S-adenosylmethionine synthase gene having SEQ ID NO: 1 and a biotin biosynthetic gene bioS1, bioS2 and / or bioS3 having SEQ ID NO: 3, SEQ ID NO: 5 and SEQ ID NO: 7, and optionally bioA, bioB, bioF, A genetic construct containing one or more additional biotin synthetic gene sequences selected from the group bioC, bioD, bioH, bioP, bioW, bioX, bioY and bioR. The present invention also relates to the use of the gene constructs for producing organisms and biotin containing the gene constructs, and to methods of producing biotin.

Figure 1 shows the pHS1 metK gene construct obtained by cloning the metK gene encoding SAM synthase in vector pHS1.

Figure 2 shows the pHS1 metK bioS1 gene construct obtained by cloning the metK gene in plasmid pHS1 bioS1.

It is an object of the present invention to develop an industrial fermentation process for producing biotin, which synthesizes biotin as much as possible.

The inventors have found that for this purpose the S-adenosylmethionine synthase (SAM synthetase) gene having SEQ ID NO: 1 and one or more additional biotin biosynthetic genes bioS1, bioS2 or bioS3 having SEQ ID NO: 3, SEQ ID NO: 5 and SEQ ID NO: 7, and their Functional variants, analogs, or derivatives are expressed in prokaryotic or eukaryotic host organisms capable of synthesizing biotin, culturing the organism, isolating cells or purifying biotin, and immediately using the synthesized biotin to produce biotin. It has been found to be made by the method according to the invention for

The genes used in the method according to the invention, namely the SAM synthetase gene having SEQ ID NO: 1 and the biotin biosynthetic genes bioS1, bioS2 and bioS3 having SEQ ID NO: 3, SEQ ID NO: 5 and SEQ ID NO: 7, were added to the SwissProt-database. Accession No. P04384 (metK), U29581 (bioS1), P39171 (bioS2) and D90811 (bioS3). Many moieties of this Coli MetK are described in the database. These moters include other eubacteia (e.g. H. influenzae and non-subtilis) and eukaryotes (e.g. yeast: E. cerevisiae, plants: P. deltoides ), Arthropods: D. melanogaster and mammals: R. norvegicus).

The productivity of biotin biosynthesis may include one or more SAM synthetase genes having SEQ ID NO: 1 and functional variants, analogs or derivatives thereof, comprising one or more biotin synthesis genes bioS1, bioS2 or bioS3 having SEQ ID NO: 3, SEQ ID NO: 5 and SEQ ID NO: 7, and functional variants thereof. , By expression in prokaryotic or eukaryotic host organisms together with analogs or derivatives. Preferably a combination of SAM synthase gene and bioS1 is used for expression. To further increase biotin synthesis, one or more additional biotin genes selected from the group bioA, bioB, bioF, bioC, bioD, bioH, bioP, bioW, bioX, bioY and bioR are advantageously expressed simultaneously. Expression of these genes enhances biotin synthesis more than two times, preferably more than three times, compared to the control without these genes.

Genes used in the method according to the invention, ie SAM synthase gene having nucleotide sequence 1, bioS1 gene having nucleotide sequence 3, bioS2 gene having nucleotide sequence 5 and bioS3 gene having nucleotide sequence 7 (these sequences are respectively 2, SEQ ID NO: 4, SEQ ID NO: 6 and encoding the amino acid sequences provided in SEQ ID NO: 8), or allelic variants thereof can be obtained after isolation and sequencing below. It is understood that the variants are SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 and SEQ ID NO: 7, which each exhibit 30 to 100% homology, preferably 50 to 100% homology, very particularly preferably 80 to 100% homology at the amino acid level Should be. Allelic variants include, in particular, functional variants obtainable by deletion, insertion or substitution of nucleotides from the sequences set forth in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 and SEQ ID NO: 7, however, enzymatic activity is maintained.

In addition, the variants include O-acetylserine sulfohydrolase A, O-acetylserine sulfohydrolase B, β-cystionase (Flint et al., J. Biol. Chem., Vol. 271, 1996: 16053-16067), which may exhibit the enzymatic activity of bioS1, bioS2 or bioS3 in the synthesis of biotin or functional equivalents of biotin, for example Klebsiella, Candida, yeast or carcass It is to be understood that nifS from Caenorhabditis and its prokaryotic and eukaryotic murines.

Functional analogs of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 and SEQ ID NO: 7 are to be understood to be their prokaryotic or eukaryotic moieties, such as, for example, bacteria, fungi, plants, animals or human moron. It is also to be understood that the analog is single stranded DNA or RNA from a truncated sequence or from encoded and unencoded DNA sequences.

Derivatives are to be understood, for example, as promoter variants. A promoter located upstream of a given nucleotide sequence may be altered by one or more nucleotide substitutions or by insertion (s) and / or deletion (s) without impairing the functionality or activity of the promoter. In addition, the activity of promoters can be increased by changing their sequences, or promoters can be completely replaced by more active promoters, including those of different species of organisms.

Derivatives are also to be understood as variants whose nucleotide sequence has been changed in the region of -1 to -30 upstream of the initiation codon so that expression of the gene and / or protein is increased.

All Gram-negative or Gram-positive bacteria that synthesize biotin are suitable for use primarily as prokaryotic host organisms in the method according to the invention. Gram-negative bacteria that may be mentioned by way of example include Enterobacteriaceae (e.g., Escherichia, Aerobacter, Enterobacter, Citrobacter, Shigella). ), Klebsiella, Serratia, Erwinia or Salmonella, Pseudomonadaceae (e.g. Pseudomonas, Xanthomonas, Burgunderia) (Burkholderia), Gluconobacter, Nitrosomonas, Nitrobacter, Methanomonas, Comamonas, Cellulomonas, or Acetobacter ), Azotobacteraceae (e.g., Azotobacter, Azomonas, Beijerinckia or Derxia), Neisseriaceae (e.g. : Moraxella, Acinetobacter obacter, Kingella, Neisseria or Branhamella genus, Rhizobiaceae (e.g., Rhizobium or Agrobacterium genus), or Gram-negative genus Zymomonas, Chromobacterium or Flavoacterium. Gram-positive bacteria that may be mentioned by way of example are endospores-forming Gram-positive breathable or anaerobic bacteria (e.g. Bacillus, Sporolactobacilli or Clostridium genus), coryneform bacteria (e.g. Arthrobacter, Cellulomonas, Curtobacterium, Corynebacterium, Brevibacterium, Microbacterium, Kurthia genus), Actinomycetales (e.g. Mycobacterium, Rhodococcus, Streptomyces or Nocardia genus), Lactobacillaceae (e.g. Lactobacillus or Lactococcus), or Gram-positive cocci (eg, Micrococcus or Staphylococcus).

Methods according to the invention are Escherichia, Citrobacter, Serratia, Klebsiella, Salmonella, Pseudomonas, Comamonas, Acinetobacter, Azotobacter, Chromobacterium, Bacillus, Clostridium, Artrobacter Preference is given to using bacteria of the genus Corynebacterium, Brevibacterium, Lactococcus, Lactobacillus, Streptomyces, Rizovium, Agrobacterium or Staphylococcus. Esseria coli, Citobacter freundii, Serratia marcescens, Salmonella typhimurium, Pseudomonas mendocina, Pseudomonas aeruginosa aeruginosa, Pseudomonas mutabilis, Pseudomonas chlororaphis, Pseudomonas fluorescens, Comamonas acidovorans, Testamonas acidonines, testosterone, and testosterone. Abactobacter calcoaceticus, Azotobacter vinelandii, Chromobacterium violaceum, Bacillus subtilis, Bacillus spaerycus, Bacillus stearerophilus stemophilus Bacillus ), Bacillus pumilus, Bacillus rihenniformi (Bacillus licheniformis), Bacillus amyloliquefaciens, Bacillus megaterium, Bacillus cereus, Bacillus thuringiensis, Arthrobacter citruus citreus, Arthrobacter paraffineus, Corynebacterium glutamicum, Corynebacterium primorioxydans, Corynebacterium species, Brevibacterium ketoglulu Brevibacterium ketoglutamicum, Brevibacterium linens, Brevibacterium spp., Streptomyces lividans, Rizobium leguminosarum or Agrobacterium tumefacienium Particularly preferred are species and genus of Agrobacterium tumefaciens. Advantageously, bacteria are used that exhibit increased natural production of biotin.

The taxonomic position of the genus described has changed considerably in recent years and is still in a changing state where the wrong dependency name is fixed. Because of this taxonomic reorganization of the genus in bacterial taxonomy which has often been required in the past, families, genus and species not mentioned above are also suitable for the methods of the present invention.

All biotin-synthetic organisms such as fungi, yeasts, plants or plant cells are mainly suitable for use as eukaryotic host organisms in the method according to the invention. Yeasts that may be preferably mentioned are Rhodotorula, Yarrowia, Sporobolomyces, Saccharomyces or Schizosaccharomyces. Rhodotorula rubra, Rhodotorula glutinis, Rhodotorula graminis, Yarrowia lipolytica, Sporobolomises Salmonolocolor ), Sporobolomyces shibatanus or Saccharomyces cerevisiae is particularly preferred.

Primarily, all plants can be used as host organisms, and plants that play a role in animal or human nutrition, such as corn, wheat, barley, rye, potatoes, peas, beans, sunflowers, palms, millet, sesame seeds, copra Or flat. Plants such as Arabidopsis thaliana or Lavendula vera are also suitable. Particular preference is given to plant cell cultures, plant protoplasts or callus cultures.

Microorganisms such as bacteria, fungi, yeast or plant cells are advantageously used in the method according to the invention which can secrete biotin into the growth medium and optionally also exhibit an increased natural synthesis of biotin. Advantageously, these organisms may also be defective with regard to the regulation of their biotin biosynthesis. That is, this synthesis is not controlled or only adjusted to a very reduced degree. These regulatory deficiencies result in these organisms already having substantially increased biotin productivity. Such regulatory defects are known, for example, from Escherichia coli in the form of birA-defective mutant strains and should preferably be present in the cells as defects which can be induced by external influences, for example temperature-induced defects. Primarily, organisms that do not exhibit natural biotin production can be used if they are transformed with a biotin gene.

In order to further increase biotin productivity as a whole, the organisms in the method according to the invention advantageously comprise at least one additional biotin selected from the group bioA, bioB, bioF, bioC, bioD, bioH, bioP, bioW, bioX, bioY or bioR. Has a gene. Advantageously, these genes simulating biotin synthesis may also be present in the cell with SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7 and combinations thereof. Examples of genes that simulate biotin synthesis are the flaboredoxin gene and the flaboredoxin reductase gene. This additional gene or these additional genes may be present in the cell in one or more copies, such as a gene having SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7, or a combination thereof. They may be present in vectors or separate vectors, such as SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 and / or SEQ ID NO: 7, or inserted into chromosomes. SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 and / or SEQ ID NO: 7 may also be together in one vector or in separate vectors, or may be inserted into the genome.

The genetic construct according to the present invention is a SAM synthase gene of SEQ ID NO: 1 and a biotin synthesis gene of SEQ ID NO: 3, SEQ ID NO: 5 and / or SEQ ID NO: 7, and their functionalities, functionally linked to one or more regulatory signals to increase expression of the gene. It is to be understood that this is the gene sequence of the variant, analog or derivative. In addition to these novel regulatory sequences, the natural regulation of these sequences may still exist upstream of the actual structural gene, and in some cases, may be genetically altered so that natural regulation is interrupted and expression of the gene is increased. However, genetic constructs can also be assembled in a simple manner. That is, no further regulatory signal is inserted upstream of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 and / or SEQ ID NO: 7, and the native promoter with regulation is not removed. Instead, natural regulatory sequences are mutated such that regulation by biotin no longer occurs and gene expression is increased. SEQ ID NO: 1, SEQ ID NO: 3, 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. Additional advantageous regulatory elements can also be inserted at the 3 'end of the DNA sequence. The gene having SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7 may exist in one or more copies in the gene construct.

Regulatory sequences advantageous for the method according to the invention are, for example, cos-, tac-, trp-, tet-, trp-tet-, lpp-, lac-, lpp-lac- which are advantageously used in Gram-negative bacteria. , lacI ^q- , T7-, T5-, T3-, gal-, trc-, ara-, SP6-, λ-P _R -or λ-P _L -promoter. Further advantageous regulatory sequences are, for example, in the Gram-positive promoters amy and SPO2, in the yeast promoters ADC1, MFα, AC, P-60, CYC1, GAPDH or in the plant promoters CaMV / 35S, SSU, OCS, lib4 , usp, STLS1, B33 or nos, or in the ubiquitin promoter or in the phaseolin promoter.

Primarily, all natural promoters, such as the promoters described above in the method according to the invention, can be used with their regulatory sequences. Synthetic promoters can also be advantageously used.

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

For expression in the host organisms described above, the gene constructs are advantageously inserted into host-specific vectors that enable to achieve optimal expression of the gene in the host. Vectors are well known to the skilled person and can be found, for example, in the book Cloning Vector (Eds. Pouwels P. H. et al. Elsevier, Amsterdam-New York-Oxford, 1985, ISBN 0 444 904018). In addition to plasmids, vectors should also be understood to be phages, viruses, transposons, IS elements, plasmids, cosmids, or any other vector known to the skilled person, such as linear or circular DNA. These vectors can autonomously replicate or chromosome replicate in the host organism.

Expression systems include, for example, the above-described host organisms and plasmids, phage λ, or Mu or other source phages or transposons containing a vector suitable for that organism, such as a plasmid, virus or phage, for example an RNA polymerase / promoter system. And / or further advantageous regulatory sequences).

The term expression system is preferably to be understood to be a combination of Escherichia coli and its plasmids and phages and related promoters, and a combination of Bacillus and its plasmids and promoters.

Additional 3 'and / or 5'-terminal regulatory sequences are also suitable for advantageously expressing SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 and / or SEQ ID NO: 7 according to the present invention.

These regulatory sequences are intended to enable the specific expression of biotin genes and the expression of proteins. This may mean that depending on the host organism, for example, the gene is barely expressed or overexpressed after induction, or the gene is immediately expressed (expressed) and overexpressed.

In this regard, regulatory sequences or factors may preferably increase expression by positively affecting biotin gene expression. For example, regulatory elements can advantageously be enhanced at the level of transcription using strong transcriptional signals such as promoters and / or enhancers. In addition, however, translation can also be enhanced, for example by improving the stability of mRNA.

Enhancers should be understood to be DNA sequences that result in increased biotin gene expression, for example by improving the interaction between RNA polymerase and DNA.

The increase in proteins derived from SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, and SEQ ID NO: 7 (see SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, and SEQ ID NO: 8) and their enzymatic activity, for example, compared to the starting enzyme, for example UV irradiation By typical mutagenesis such as or by treatment with chemical mutagens and / or by specific mutagenesis such as site-directed mutagenesis, deletion (s), insertion (s) and / or substitution (s). It can be achieved by changing the gene sequence or the sequence of their molar animals. Apart from the described gene amplifications, increased enzyme activity can also be obtained by scavenging factors that inhibit enzyme biosynthesis and / or by synthesizing active enzymes instead of inactive enzymes.

The method according to the invention advantageously increases the conversion of DTB into biotin, thus combining the genes having SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 and SEQ ID NO: 7 and SEQ ID NO: 1 and SEQ ID NO: 5 or SEQ ID NO: 1 and SEQ ID NO: 7, Preferably the use of biotin genes having a combination of genes having SEQ ID NO: 1 and SEQ ID NO: 3 increases overall biotin productivity, which genes are introduced into the organism by vectors and / or by chromosomal cloning.

In the method according to the invention, microorganisms having SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 and / or SEQ ID NO: 7 are propagated in a medium capable of propagating this organism. This medium may be synthetic or natural medium. A medium known to the skilled person and suitable for the organism is used. To propagate microorganisms, the medium used contains a carbon source, a nitrogen source, an inorganic salt, and optionally small amounts of vitamins and trace elements.

Examples of advantageous carbon sources are monosaccharides, disaccharides or polysaccharides such as glucose, fructose, mannose, xylose, galactose, ribose, sorbose, ribulose, lactose, maltose, sucrose , Sugars such as raffinose, starch or cellulose, complex sugar sources (e.g. molasses), sugar phosphates (e.g. fructose-1,6-bisphosphate), sugar alcohols (e. Mannitol), polyols (e.g. 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) or amino acids (such as glutamic acid or aspartic acid), or may be used simultaneously as a nitrogen source Amino sugars.

Advantageous nitrogen sources are organic or inorganic nitrogen compounds or materials containing these compounds. Examples include ammonium salts (eg NH ₄ Cl or (NH ₄ ) ₂ SO ₄ ), nitrates or ureas, or complex nitrogen sources (eg corn steep liquor, brewer's yeast autolysates, soy flour, wheat gluten) , Yeast extract, meat extract, casein hydrolysate or yeast or potato protein, which can often be used simultaneously at the same time as a nitrogen source.

Examples of inorganic salts are salts of calcium, magnesium, sodium, manganese, potassium, zinc, copper and iron. Particularly mentioned anions of these salts are chloride, sulfate and phosphate ions. An important factor in 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 desired, growth factors such as vitamins or growth promoters such as riboflavin, thiamine, folic acid, nicotinic acid, patotothenate or pyridoxine, amino acids (e.g. alanine, cysteine, asparagine, aspartic acid, glutamine, serine, methionine or lysine) Substances such as carboxylic acid (eg citric acid, formic acid, pimelic acid or lactic acid) or dithiothritol are added to the nutrient medium.

Antibiotics are optionally added to the medium to stabilize the biotin gene-containing vector in the cells.

The proportion at which the nutrients are mixed depends on the nature of the fermentation and on a case by case basis. The media components can all be introduced separately at the start of fermentation, if desired, after being sterilized separately or together together as needed, or optionally added sequentially during fermentation.

Culture conditions are adjusted so that the organism grows optimally and the best possible yield is obtained. Preferred culture temperatures are from 15 ° C. to 40 ° C. Particular preference is given to temperatures of 25 ° C to 37 ° C. The pH is preferably maintained in the range of 3-9. PH values of 5 to 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 can be obtained after accumulating (accumulating) in the medium and destroying the cells.

The process according to the invention for producing biotin can be carried out continuously or batchwise or semibatch. Once the whole plants are regenerated from plant cells transformed with the biotin gene, they can be grown and propagated completely normally according to the method according to the invention.

1. Cloning of S-adenosylmethionine synthase gene (SEQ ID NO: 1):

The genome amplifies the gene encoding SAM synthase (metK) from this coli chromosome by polymerase chain reaction using two specific oligonucleotides, starting from coli DNA. The amplified DNA was purified, digested with restriction enzyme Acc65I, and inserted into a vector digested with the same enzyme capable of overexpressing this gene in this collie strain. One of the two oligonucleotides was used to provide a genetic construct with an optimized translation signal.

a) production of oligonucleotides to amplify metK gene from this coli chromosome

metK was amplified as an expression cassette consisting of a ribosomal binding site, an initiation codon of the coding sequence and a stop codon between two restriction enzyme recognition sites. Acc65I recognition sequences were selected for both restriction sites. MetK genes were amplified and cloned using the nucleotides PmetK1 (5'-GCGGTACCAGGTGATATTAAATATGGCAAAAC-3 ') and PmetK2 (5'-CGGGTACCGATTACTTCAGACCGGCAGC-3').

b) Performing PCR:

Condition:

0.5 µg of chromosomal DNA obtained from this Coli W3110 was used as a template. Oligonucleotides PmetK1 and PmetK2 were each used at a concentration of 15 mmol. The concentration of dNTP was 200 μM. 2.5 U of Pwo DNA polymerase (Boehringer Mannheim) in manufacturer reaction buffer was used as polymerase. PCR reaction volume was 100 μl.

Amplification:

DNA was denatured at 94 ° C. for 2 minutes. The oligonucleotides were then annealed at 55 ° C. for 30 seconds. Elongation occurred at 72 ° C. for 75 seconds. PCR reactions were performed over 30 cycles.

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

c) cloning of metK in the expression vector

Vector pHS1 (constitution is described in German patent application No. 197.31274.8, priority 22.7.97, Example 1, pp. 14-17) was digested with Acc65I and shrimp alkaline phosphatase (SAP) (Schoringer Mannheim) Dephosphorylation was used. After denaturation of SAP, the vector and fragment were linked in a molar ratio of 1: 3 using a Rapid DNA Ligation kit according to the manufacturer's instructions. This Coli XL-1-blue strain was transformed with the ligation mixture. Positive clones were revealed by plasmid preparation and restriction analysis. Restriction digestion and sequencing determined the exact orientation of the metK fragment in pHS1. The resulting construct was named pHS1 metK (FIG. 1). The sequence of pHS1 metK is provided in SEQ ID NO: 9. SEQ ID NO: 10 shows the amino acid sequence deduced from the metK-coding region.

2. Composition of Plasmids pHBbio14 and pHS1 bioS1

The construction of the plasmids pHBbio14 and pHS1 bioS1 has already been described (German patent application no. 197.31274.8, priority 22.7.97, examples 1, 2 and 5).

3. Composition of pHS1 metK bioS1

Plasmid pHS1 bioS1 (SEQ ID NO: 11 (German Patent Application No. 197.31274.8, Priority 22.7.97), SEQ ID NO: 12 shows the amino acid sequence inferred from the bioS1-coding region) and pHS1 using the plasmid preparation method (Schlinger) metK (SEQ ID NO: 9) was purified. Fragments carrying the metK gene were isolated by digestion with Acc65I from pHS1 metK. pHS1 bioS1 was digested with Acc65I and dephosphorylated with shrimp alkaline phosphatase (SAP) (Möllinger Mannheim). After denaturation of SAP according to the manufacturer's instructions, the vector and metK fragments were linked in a molar ratio of 1: 3 using a high speed DNA ligation kit according to the manufacturer's instructions. This Coli XL-1-blue strain was transformed with the ligation mixture. Positive clones were identified by plasmid preparation and restriction analysis. Restriction digestion and sequencing determined the exact orientation of the metK fragment in pHS1 bioS1. The resulting construct was named pHS1 metK bioS1 (FIG. 2). The sequence of pHS1 metK bioS1 is provided in SEQ ID NO: 13. SEQ ID NO: 14 shows an amino acid sequence inferred from a metK-coding region and SEQ ID NO: 15 shows an amino acid sequence inferred from a bioS1-coding region.

4. Increased biotin productivity by overexpressing metK, bioS1 and metK and bioS1 together

Natural rifampicin-resistant colonies were isolated from BM4086 (Ketner and Campbell, J. Molec. Biology, 1975, 96:13) by plating on rifampicin plates. P1 lysate was produced from one of these resistant strains. This P1 lysate was transduced with strain W3110 and then clones were selected using rifampicin. The resulting strains were transformed with plasmid pHBbio14 using the CaCl ₂ method (Maniatis et al. (Molecular Cloning, Cols Spring Harbor Laboratory Press, 1989)) and in LB containing 100 μg of ampicillin per ml. It was grown. The isolated, transformed strain (LU5560) was transformed in each case with plasmid pHS1, pHS1 metK, pHS1 bioS1 or pHS1 metK bioS1 using CaCl ₂ method, followed by 100 μg of ampicillin and 25 μg of kanamycin per mL. It was selected from the containing LB agar.

One colony from each transformant was inoculated in DYT culture containing the appropriate antibiotic in each case and incubated for 12 hours. Incubated overnight (= ONC) TB medium containing 30 g of glycerol per liter and appropriate antibiotics (Sambrook, J., Frisch, EF, Maniatis, 2nd ed. Cold Spring Harbor Laboratory Press., 1989 ISBN 0-87969-373-8]) was inoculated with 10 ml culture. When plasmids pHS1, pHS1 metK, pHS1 bioS1 and pHS1 metK bioS1 were present, 1 mM IPTG and 0.5% arabinose were added simultaneously to induce expression of metK and bioS1 genes, or a combination of both genes, respectively. After 24 hours, cells were removed from the culture supernatant by centrifugation and the biotin concentration in the supernatant was determined by competitive ELISA using streptavidin. The results of this determination are shown in Table I below.

Strain Plasmid I Plasmid II Biotin mg / L LU5580 pHBbio14 Control, no plasmid 11 LU5580 pHBbio14 pHS1 25 LU5580 pHBbio14 pHS1 bioS1 45 LU5580 pHBbio14 pHS1 metK 37 LU5580 pHBbio14 pHS1 metK bioS1 52

<110> BASF Aktiengesellschaft

<120> Verfahren zur Herstellung von Biotin

<130> 0050_48792

<140> 19806872.7

<141> 1998-02-19

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atg gca aaa cac ctt ttt acg tcc gag tcc gtc tct gaa ggg cat cct 48

Met Ala Lys His Leu Phe Thr Ser Glu Ser Val Ser Glu Gly His Pro

1 5 10 15

gac aaa att gct gac caa att tct gat gcc gtt tta gac gcg atc ctc 96

Asp Lys Ile Ala Asp Gln Ile Ser Asp Ala Val Leu Asp Ala Ile Leu

20 25 30

gaa cag gat ccg aaa gca cgc gtt gct tgc gaa acc tac gta aaa acc 144

Glu Gln Asp Pro Lys Ala Arg Val Ala Cys Glu Thr Tyr Val Lys Thr

35 40 45

ggc atg gtt tta gtt ggc ggc gaa atc acc acc agc gcc tgg gta gac 192

Gly Met Val Leu Val Gly Gly Glu Ile Thr Thr Ser Ala Trp Val Asp

50 55 60

atc gaa gag atc acc cgt aac acc gtt cgc gaa att ggc tat gtg cat 240

Ile Glu Glu Ile Thr Arg Asn Thr Val Arg Glu Ile Gly Tyr Val His

65 70 75 80

tcc gac atg ggc ttt gac gct aac tcc tgt gcg gtt ctg agc gct atc 288

Ser Asp Met Gly Phe Asp Ala Asn Ser Cys Ala Val Leu Ser Ala Ile

85 90 95

ggc aaa cag tct cct gac atc aac cag ggc gtt gac cgt gcc gat ccg 336

Gly Lys Gln Ser Pro Asp Ile Asn Gln Gly Val Asp Arg Ala Asp Pro

100 105 110

ctg gaa cag ggc gcg ggt gac cag ggt ctg atg ttt ggc tac gca act 384

Leu Glu Gln Gly Ala Gly Asp Gln Gly Leu Met Phe Gly Tyr Ala Thr

115 120 125

aat gaa acc gac gtg ctg atg cca gca cct atc acc tat gca cac cgt 432

Asn Glu Thr Asp Val Leu Met Pro Ala Pro Ile Thr Tyr Ala His Arg

130 135 140

ctg gta cag cgt cag gct gaa gtg cgt aaa aac ggc act ctg ccg tgg 480

Leu Val Gln Arg Gln Ala Glu Val Arg Lys Asn Gly Thr Leu Pro Trp

145 150 155 160

ctg cgc ccg gac gcg aaa agc cag gtg act ttt cag tat gac gac ggc 528

Leu Arg Pro Asp Ala Lys Ser Gln Val Thr Phe Gln Tyr Asp Asp Gly

165 170 175

aaa atc gtt ggt atc gat gct gtc gtg ctt tcc act cag cac tct gaa 576

Lys Ile Val Gly Ile Asp Ala Val Val Leu Ser Thr Gln His Ser Glu

180 185 190

gag atc gac cag aaa tcg ctg caa gaa gcg gta atg gaa gag atc atc 624

Glu Ile Asp Gln Lys Ser Leu Gln Glu Ala Val Met Glu Glu Ile Ile

195 200 205

aag cca att ctg ccc gct gaa tgg ctg act tct gcc acc aaa ttc ttc 672

Lys Pro Ile Leu Pro Ala Glu Trp Leu Thr Ser Ala Thr Lys Phe Phe

210 215 220

atc aac ccg acc ggt cgt ttc gtt atc ggt ggc cca atg ggt gac tgc 720

Ile Asn Pro Thr Gly Arg Phe Val Ile Gly Gly Pro Met Gly Asp Cys

225 230 235 240

ggt ctg act ggt cgt aaa att atc gtt gat acc tac ggc ggc atg gcg 768

Gly Leu Thr Gly Arg Lys Ile Ile Val Asp Thr Tyr Gly Gly Met Ala

245 250 255

cgt cac ggt ggc ggt gca ttc tct ggt aaa gat cca tca aaa gtg gac 816

Arg His Gly Gly Gly Ala Phe Ser Gly Lys Asp Pro Ser Lys Val Asp

260 265 270

cgt tcc gca gcc tac gca gca cgt tat gtc gcg aaa aac atc gtt gct 864

Arg Ser Ala Ala Tyr Ala Ala Arg Tyr Val Ala Lys Asn Ile Val Ala

275 280 285

gct ggc ctg gcc gat cgt tgt gaa att cag gtt tcc tac gca atc ggc 912

Ala Gly Leu Ala Asp Arg Cys Glu Ile Gln Val Ser Tyr Ala Ile Gly

290 295 300

gtg gct gaa ccg acc tcc atc atg gta gaa act ttc ggt act gag aaa 960

Val Ala Glu Pro Thr Ser Ile Met Val Glu Thr Phe Gly Thr Glu Lys

305 310 315 320

gtg cct tct gaa caa ctg acc ctg ctg gta cgt gag ttc ttc gac ctg 1008

Val Pro Ser Glu Gln Leu Thr Leu Leu Val Arg Glu Phe Phe Asp Leu

325 330 335

cgc cca tac ggt ctg att cag atg ctg gat ctg ctg cac ccg atc tac 1056

Arg Pro Tyr Gly Leu Ile Gln Met Leu Asp Leu Leu His Pro Ile Tyr

340 345 350

aaa gaa acc gca gca tac ggt cac ttt ggt cgt gaa cat ttc ccg tgg 1104

Lys Glu Thr Ala Ala Tyr Gly His Phe Gly Arg Glu His Phe Pro Trp

355 360 365

gaa aaa acc gac aaa gcg cag ctg ctg cgc gat gct gcc ggt ctg aag 1152

Glu Lys Thr Asp Lys Ala Gln Leu Leu Arg Asp Ala Ala Gly Leu Lys

370 375 380

taa 1155

385

<210> 2

<211> 384

<212> PRT

<213> Escherichia coli

<400> 2

Met Ala Lys His Leu Phe Thr Ser Glu Ser Val Ser Glu Gly His Pro

1 5 10 15

Asp Lys Ile Ala Asp Gln Ile Ser Asp Ala Val Leu Asp Ala Ile Leu

20 25 30

Glu Gln Asp Pro Lys Ala Arg Val Ala Cys Glu Thr Tyr Val Lys Thr

35 40 45

Gly Met Val Leu Val Gly Gly Glu Ile Thr Thr Ser Ala Trp Val Asp

50 55 60

Ile Glu Glu Ile Thr Arg Asn Thr Val Arg Glu Ile Gly Tyr Val His

65 70 75 80

Ser Asp Met Gly Phe Asp Ala Asn Ser Cys Ala Val Leu Ser Ala Ile

85 90 95

Gly Lys Gln Ser Pro Asp Ile Asn Gln Gly Val Asp Arg Ala Asp Pro

100 105 110

Leu Glu Gln Gly Ala Gly Asp Gln Gly Leu Met Phe Gly Tyr Ala Thr

115 120 125

Asn Glu Thr Asp Val Leu Met Pro Ala Pro Ile Thr Tyr Ala His Arg

130 135 140

Leu Val Gln Arg Gln Ala Glu Val Arg Lys Asn Gly Thr Leu Pro Trp

145 150 155 160

Leu Arg Pro Asp Ala Lys Ser Gln Val Thr Phe Gln Tyr Asp Asp Gly

165 170 175

Lys Ile Val Gly Ile Asp Ala Val Val Leu Ser Thr Gln His Ser Glu

180 185 190

Glu Ile Asp Gln Lys Ser Leu Gln Glu Ala Val Met Glu Glu Ile Ile

195 200 205

Lys Pro Ile Leu Pro Ala Glu Trp Leu Thr Ser Ala Thr Lys Phe Phe

210 215 220

Ile Asn Pro Thr Gly Arg Phe Val Ile Gly Gly Pro Met Gly Asp Cys

225 230 235 240

Gly Leu Thr Gly Arg Lys Ile Ile Val Asp Thr Tyr Gly Gly Met Ala

245 250 255

Arg His Gly Gly Gly Ala Phe Ser Gly Lys Asp Pro Ser Lys Val Asp

260 265 270

Arg Ser Ala Ala Tyr Ala Ala Arg Tyr Val Ala Lys Asn Ile Val Ala

275 280 285

Ala Gly Leu Ala Asp Arg Cys Glu Ile Gln Val Ser Tyr Ala Ile Gly

290 295 300

Val Ala Glu Pro Thr Ser Ile Met Val Glu Thr Phe Gly Thr Glu Lys

305 310 315 320

Val Pro Ser Glu Gln Leu Thr Leu Leu Val Arg Glu Phe Phe Asp Leu

325 330 335

Arg Pro Tyr Gly Leu Ile Gln Met Leu Asp Leu Leu His Pro Ile Tyr

340 345 350

Lys Glu Thr Ala Ala Tyr Gly His Phe Gly Arg Glu His Phe Pro Trp

355 360 365

Glu Lys Thr Asp Lys Ala Gln Leu Leu Arg Asp Ala Ala Gly Leu Lys

370 375 380

<210> 3

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<212> DNA

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<220>

<221> CDS

<222> (1) .. (1206)

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atg aac gtt ttt aat ccc gcg cag ttt cgc gcc cag ttt ccc gca cta 48

Met Asn Val Phe Asn Pro Ala Gln Phe Arg Ala Gln Phe Pro Ala Leu

1 5 10 15

cag gat gcg ggc gtc tat ctc gac agc gcc gcg acc gcg ctt aaa cct 96

Gln Asp Ala Gly Val Tyr Leu Asp Ser Ala Ala Thr Ala Leu Lys Pro

20 25 30

gaa gcc gtg gtt gaa gcc acc caa cag ttt tac agt ctg agc gcc gga 144

Glu Ala Val Val Glu Ala Thr Gln Gln Phe Tyr Ser Leu Ser Ala Gly

35 40 45

aac gtc cat cgc agc cag ttt gcc gaa gcc caa cgc ctg acc gcg cgt 192

Asn Val His Arg Ser Gln Phe Ala Glu Ala Gln Arg Leu Thr Ala Arg

50 55 60

tat gaa gct gca cga gag aaa gtg gcg caa tta ctg aat gca ccg gat 240

Tyr Glu Ala Ala Arg Glu Lys Val Ala Gln Leu Leu Asn Ala Pro Asp

65 70 75 80

gat aaa act atc gtc tgg acg cgc ggc acc act gaa tcc atc aac atg 288

Asp Lys Thr Ile Val Trp Thr Arg Gly Thr Thr Glu Ser Ile Asn Met

85 90 95

gtg gca caa tgc tat gcg cgt ccg cgt ctg caa ccg ggc gat gag att 336

Val Ala Gln Cys Tyr Ala Arg Pro Arg Leu Gln Pro Gly Asp Glu Ile

100 105 110

att gtc agc gtg gca gaa cac cac gcc aac ctc gtc ccc tgg ctg atg 384

Ile Val Ser Val Ala Glu His His Ala Asn Leu Val Pro Trp Leu Met

115 120 125

gtc gcc caa caa act gga gcc aaa gtg gtg aaa ttg ccg ctt aat gcg 432

Val Ala Gln Gln Thr Gly Ala Lys Val Val Lys Leu Pro Leu Asn Ala

130 135 140

cag cga ctg ccg gat gtc gat ttg ttg cca gaa ctg att act ccc cgt 480

Gln Arg Leu Pro Asp Val Asp Leu Leu Pro Glu Leu Ile Thr Pro Arg

145 150 155 160

agt cgg att ctg gcg ttg ggt cag atg tcg aac gtt act ggc ggt tgc 528

Ser Arg Ile Leu Ala Leu Gly Gln Met Ser Asn Val Thr Gly Gly Cys

165 170 175

ccg gat ctg gcg cga gcg att acc ttt gct cat tca gcc ggg atg gtg 576

Pro Asp Leu Ala Arg Ala Ile Thr Phe Ala His Ser Ala Gly Met Val

180 185 190

gtg atg gtt gat ggt gct cag ggg gca gtg cat ttc ccc gcg gat gtt 624

Val Met Val Asp Gly Ala Gln Gly Ala Val His Phe Pro Ala Asp Val

195 200 205

cag caa ctg gat att gat ttc tat gct ttt tca ggt cac aaa ctg tat 672

Gln Gln Leu Asp Ile Asp Phe Tyr Ala Phe Ser Gly His Lys Leu Tyr

210 215 220

ggc ccg aca ggt atc ggc gtg ctg tat ggt aaa tca gaa ctg ctg gag 720

Gly Pro Thr Gly Ile Gly Val Leu Tyr Gly Lys Ser Glu Leu Leu Glu

225 230 235 240

gcg atg tcg ccc tgg ctg ggc ggc ggc aaa atg gtt cac gaa gtg agt 768

Ala Met Ser Pro Trp Leu Gly Gly Gly Lys Met Val His Glu Val Ser

245 250 255

ttt gac ggc ttc acg act caa tct gcg ccg tgg aaa ctg gaa gct gga 816

Phe Asp Gly Phe Thr Thr Gln Ser Ala Pro Trp Lys Leu Glu Ala Gly

260 265 270

acg cca aat gtc gct ggt gtc ata gga tta agc gcg gcg ctg gaa tgg 864

Thr Pro Asn Val Ala Gly Val Ile Gly Leu Ser Ala Ala Leu Glu Trp

275 280 285

ctg gca gat tac gat atc aac cag gcc gaa agc tgg agc cgt agc tta 912

Leu Ala Asp Tyr Asp Ile Asn Gln Ala Glu Ser Trp Ser Arg Ser Leu

290 295 300

gca acg ctg gcg gaa gat gcg ctg gcg aaa cgt ccc ggc ttt cgt tca 960

Ala Thr Leu Ala Glu Asp Ala Leu Ala Lys Arg Pro Gly Phe Arg Ser

305 310 315 320

ttc cgc tgc cag gat tcc agc ctg ctg gcc ttt gat ttt gct ggc gtt 1008

Phe Arg Cys Gln Asp Ser Ser Leu Leu Ala Phe Asp Phe Ala Gly Val

325 330 335

cat cat agc gat atg gtg acg ctg ctg gcg gag tac ggt att gcc ctg 1056

His His Ser Asp Met Val Thr Leu Leu Ala Glu Tyr Gly Ile Ala Leu

340 345 350

cgg gcc ggg cag cat tgc gct cag ccg cta ctg gca gaa tta ggc gta 1104

Arg Ala Gly Gln His Cys Ala Gln Pro Leu Leu Ala Glu Leu Gly Val

355 360 365

acc ggc aca ctg cgc gcc tct ttt gcg cca tat aat aca aag agt gat 1152

Thr Gly Thr Leu Arg Ala Ser Phe Ala Pro Tyr Asn Thr Lys Ser Asp

370 375 380

gtg gat gcg ctg gtg aat gcc gtt gac cgc gcg ctg gaa tta ttg gtg 1200

Val Asp Ala Leu Val Asn Ala Val Asp Arg Ala Leu Glu Leu Leu Val

385 390 395 400

gat taa 1206

Asp

<210> 4

<211> 401

<212> PRT

<213> Escherichia coli

<400> 4

Met Asn Val Phe Asn Pro Ala Gln Phe Arg Ala Gln Phe Pro Ala Leu

1 5 10 15

Gln Asp Ala Gly Val Tyr Leu Asp Ser Ala Ala Thr Ala Leu Lys Pro

20 25 30

Glu Ala Val Val Glu Ala Thr Gln Gln Phe Tyr Ser Leu Ser Ala Gly

35 40 45

Asn Val His Arg Ser Gln Phe Ala Glu Ala Gln Arg Leu Thr Ala Arg

50 55 60

Tyr Glu Ala Ala Arg Glu Lys Val Ala Gln Leu Leu Asn Ala Pro Asp

65 70 75 80

Asp Lys Thr Ile Val Trp Thr Arg Gly Thr Thr Glu Ser Ile Asn Met

85 90 95

Val Ala Gln Cys Tyr Ala Arg Pro Arg Leu Gln Pro Gly Asp Glu Ile

100 105 110

Ile Val Ser Val Ala Glu His His Ala Asn Leu Val Pro Trp Leu Met

115 120 125

Val Ala Gln Gln Thr Gly Ala Lys Val Val Lys Leu Pro Leu Asn Ala

130 135 140

Gln Arg Leu Pro Asp Val Asp Leu Leu Pro Glu Leu Ile Thr Pro Arg

145 150 155 160

Ser Arg Ile Leu Ala Leu Gly Gln Met Ser Asn Val Thr Gly Gly Cys

165 170 175

Pro Asp Leu Ala Arg Ala Ile Thr Phe Ala His Ser Ala Gly Met Val

180 185 190

Val Met Val Asp Gly Ala Gln Gly Ala Val His Phe Pro Ala Asp Val

195 200 205

Gln Gln Leu Asp Ile Asp Phe Tyr Ala Phe Ser Gly His Lys Leu Tyr

210 215 220

Gly Pro Thr Gly Ile Gly Val Leu Tyr Gly Lys Ser Glu Leu Leu Glu

225 230 235 240

Ala Met Ser Pro Trp Leu Gly Gly Gly Lys Met Val His Glu Val Ser

245 250 255

Phe Asp Gly Phe Thr Thr Gln Ser Ala Pro Trp Lys Leu Glu Ala Gly

260 265 270

Thr Pro Asn Val Ala Gly Val Ile Gly Leu Ser Ala Ala Leu Glu Trp

275 280 285

Leu Ala Asp Tyr Asp Ile Asn Gln Ala Glu Ser Trp Ser Arg Ser Leu

290 295 300

Ala Thr Leu Ala Glu Asp Ala Leu Ala Lys Arg Pro Gly Phe Arg Ser

305 310 315 320

Phe Arg Cys Gln Asp Ser Ser Leu Leu Ala Phe Asp Phe Ala Gly Val

325 330 335

His His Ser Asp Met Val Thr Leu Leu Ala Glu Tyr Gly Ile Ala Leu

340 345 350

Arg Ala Gly Gln His Cys Ala Gln Pro Leu Leu Ala Glu Leu Gly Val

355 360 365

Thr Gly Thr Leu Arg Ala Ser Phe Ala Pro Tyr Asn Thr Lys Ser Asp

370 375 380

Val Asp Ala Leu Val Asn Ala Val Asp Arg Ala Leu Glu Leu Leu Val

385 390 395 400

Asp

<210> 5

<211> 1215

<212> DNA

<213> Escherichia coli

<220>

<221> CDS

<222> (1) .. (1215)

<400> 5

atg aaa tta ccg att tat ctc gac tac tcc gca acc acg ccg gtg gac 48

Met Lys Leu Pro Ile Tyr Leu Asp Tyr Ser Ala Thr Thr Pro Val Asp

1 5 10 15

ccg cgt gtt gcc gag aaa atg atg cag ttt atg acg atg gac gga acc 96

Pro Arg Val Ala Glu Lys Met Met Gln Phe Met Thr Met Asp Gly Thr

20 25 30

ttt ggt aac ccg gcc tcc cgt tct cac cgt ttc ggc tgg cag gct gaa 144

Phe Gly Asn Pro Ala Ser Arg Ser His Arg Phe Gly Trp Gln Ala Glu

35 40 45

gaa gcg gta gat atc gcc cgt aat cag att gcc gat ctg gtc ggc gct 192

Glu Ala Val Asp Ile Ala Arg Asn Gln Ile Ala Asp Leu Val Gly Ala

50 55 60

gat ccg cgt gaa atc gtc ttt acc tct ggt gca acc gaa tct gac aac 240

Asp Pro Arg Glu Ile Val Phe Thr Ser Gly Ala Thr Glu Ser Asp Asn

65 70 75 80

ctg gcg atc aaa ggt gca gcc aac ttt tat cag aaa aaa ggc aag cac 288

Leu Ala Ile Lys Gly Ala Ala Asn Phe Tyr Gln Lys Lys Gly Lys His

85 90 95

atc atc acc agc aaa acc gaa cac aaa gcg gta ctg gat acc tgc cgt 336

Ile Ile Thr Ser Lys Thr Glu His Lys Ala Val Leu Asp Thr Cys Arg

100 105 110

cag ctg gag cgc gaa ggt ttt gaa gtc acc tac ctg gca ccg cag cgt 384

Gln Leu Glu Arg Glu Gly Phe Glu Val Thr Tyr Leu Ala Pro Gln Arg

115 120 125

aac ggc att atc gac ctg aaa gaa ctt gaa gca gcg atg cgt gac gac 432

Asn Gly Ile Ile Asp Leu Lys Glu Leu Glu Ala Ala Met Arg Asp Asp

130 135 140

acc atc ctc gtg tcc atc atg cac gta aat aac gaa atc ggc gtg gtg 480

Thr Ile Leu Val Ser Ile Met His Val Asn Asn Glu Ile Gly Val Val

145 150 155 160

cag gat atc gcg gct atc ggc gaa atg tgc cgt gct cgt ggc att atc 528

Gln Asp Ile Ala Ala Ile Gly Glu Met Cys Arg Ala Arg Gly Ile Ile

165 170 175

tat cac gtt gat gca acc cag agc gtg ggt aaa ctg cct atc gac ctg 576

Tyr His Val Asp Ala Thr Gln Ser Val Gly Lys Leu Pro Ile Asp Leu

180 185 190

agc cag ttg aaa gtt gac ctg atg tct ttc tcc ggt cac aaa atc tat 624

Ser Gln Leu Lys Val Asp Leu Met Ser Phe Ser Gly His Lys Ile Tyr

195 200 205

ggc ccg aaa ggt atc ggt gcg ctg tat gta cgt cgt aaa ccg cgc gta 672

Gly Pro Lys Gly Ile Gly Ala Leu Tyr Val Arg Arg Lys Pro Arg Val

210 215 220

cgc atc gaa gcg caa atg cac ggc ggc ggt cac gag cgc ggt atg cgt 720

Arg Ile Glu Ala Gln Met His Gly Gly Gly His Glu Arg Gly Met Arg

225 230 235 240

tcc ggc act ctg cct gtt cac cag atc gtc gga atg ggc gag gcc tat 768

Ser Gly Thr Leu Pro Val His Gln Ile Val Gly Met Gly Glu Ala Tyr

245 250 255

cgc atc gca aaa gaa gag atg gcg acc gag atg gaa cgt ctg cgc ggc 816

Arg Ile Ala Lys Glu Glu Met Ala Thr Glu Met Glu Arg Leu Arg Gly

260 265 270

ctg cgt aac cgt ctg tgg aac ggc atc aaa gat atc gaa gaa gtt tac 864

Leu Arg Asn Arg Leu Trp Asn Gly Ile Lys Asp Ile Glu Glu Val Tyr

275 280 285

ctg aac ggt gac ctg gaa cac ggt gcg ccg aac att ctc aac gtc agc 912

Leu Asn Gly Asp Leu Glu His Gly Ala Pro Asn Ile Leu Asn Val Ser

290 295 300

ttc aac tac gtt gaa ggt gag tcg ctg att atg gcg ctg aaa gac ctc 960

Phe Asn Tyr Val Glu Gly Glu Ser Leu Ile Met Ala Leu Lys Asp Leu

305 310 315 320

gca gtt tct tca ggt tcc gcc tgt acg tca gca agc ctc gaa ccg tcc 1008

Ala Val Ser Ser Gly Ser Ala Cys Thr Ser Ala Ser Leu Glu Pro Ser

325 330 335

tac gtg ctg cgc gcg ctg ggg ctg aac gac gag ctg gca cat agc tct 1056

Tyr Val Leu Arg Ala Leu Gly Leu Asn Asp Glu Leu Ala His Ser Ser

340 345 350

atc cgt ttc tct tta ggt cgt ttt act act gaa gaa gag atc gac tac 1104

Ile Arg Phe Ser Leu Gly Arg Phe Thr Thr Glu Glu Glu Ile Asp Tyr

355 360 365

acc atc gag tta gtt cgt aaa tcc atc ggt cgt ctg cgt gac ctt tct 1152

Thr Ile Glu Leu Val Arg Lys Ser Ile Gly Arg Leu Arg Asp Leu Ser

370 375 380

ccg ctg tgg gaa atg tac aag cag ggc gtg gat ctg aac agc atc gaa 1200

Pro Leu Trp Glu Met Tyr Lys Gln Gly Val Asp Leu Asn Ser Ile Glu

385 390 395 400

tgg gct cat cat taa 1215

Trp Ala His His

405

<210> 6

<211> 404

<212> PRT

<213> Escherichia coli

<400> 6

Met Lys Leu Pro Ile Tyr Leu Asp Tyr Ser Ala Thr Thr Pro Val Asp

1 5 10 15

Pro Arg Val Ala Glu Lys Met Met Gln Phe Met Thr Met Asp Gly Thr

20 25 30

Phe Gly Asn Pro Ala Ser Arg Ser His Arg Phe Gly Trp Gln Ala Glu

35 40 45

Glu Ala Val Asp Ile Ala Arg Asn Gln Ile Ala Asp Leu Val Gly Ala

50 55 60

Asp Pro Arg Glu Ile Val Phe Thr Ser Gly Ala Thr Glu Ser Asp Asn

65 70 75 80

Leu Ala Ile Lys Gly Ala Ala Asn Phe Tyr Gln Lys Lys Gly Lys His

85 90 95

Ile Ile Thr Ser Lys Thr Glu His Lys Ala Val Leu Asp Thr Cys Arg

100 105 110

Gln Leu Glu Arg Glu Gly Phe Glu Val Thr Tyr Leu Ala Pro Gln Arg

115 120 125

Asn Gly Ile Ile Asp Leu Lys Glu Leu Glu Ala Ala Met Arg Asp Asp

130 135 140

Thr Ile Leu Val Ser Ile Met His Val Asn Asn Glu Ile Gly Val Val

145 150 155 160

Gln Asp Ile Ala Ala Ile Gly Glu Met Cys Arg Ala Arg Gly Ile Ile

165 170 175

Tyr His Val Asp Ala Thr Gln Ser Val Gly Lys Leu Pro Ile Asp Leu

180 185 190

Ser Gln Leu Lys Val Asp Leu Met Ser Phe Ser Gly His Lys Ile Tyr

195 200 205

Gly Pro Lys Gly Ile Gly Ala Leu Tyr Val Arg Arg Lys Pro Arg Val

210 215 220

Arg Ile Glu Ala Gln Met His Gly Gly Gly His Glu Arg Gly Met Arg

225 230 235 240

Ser Gly Thr Leu Pro Val His Gln Ile Val Gly Met Gly Glu Ala Tyr

245 250 255

Arg Ile Ala Lys Glu Glu Met Ala Thr Glu Met Glu Arg Leu Arg Gly

260 265 270

Leu Arg Asn Arg Leu Trp Asn Gly Ile Lys Asp Ile Glu Glu Val Tyr

275 280 285

Leu Asn Gly Asp Leu Glu His Gly Ala Pro Asn Ile Leu Asn Val Ser

290 295 300

Phe Asn Tyr Val Glu Gly Glu Ser Leu Ile Met Ala Leu Lys Asp Leu

305 310 315 320

Ala Val Ser Ser Gly Ser Ala Cys Thr Ser Ala Ser Leu Glu Pro Ser

325 330 335

Tyr Val Leu Arg Ala Leu Gly Leu Asn Asp Glu Leu Ala His Ser Ser

340 345 350

Ile Arg Phe Ser Leu Gly Arg Phe Thr Thr Glu Glu Glu Ile Asp Tyr

355 360 365

Thr Ile Glu Leu Val Arg Lys Ser Ile Gly Arg Leu Arg Asp Leu Ser

370 375 380

Pro Leu Trp Glu Met Tyr Lys Gln Gly Val Asp Leu Asn Ser Ile Glu

385 390 395 400

Trp Ala His His

<210> 7

<211> 1221

<212> DNA

<213> Escherichia coli

<220>

<221> CDS

<222> (1) .. (1221)

<400> 7

atg att ttt tcc gtc gac aaa gtg cgg gcc gac ttt ccg gtg ctt tcg 48

Met Ile Phe Ser Val Asp Lys Val Arg Ala Asp Phe Pro Val Leu Ser

1 5 10 15

cgt gag gta aac ggt ttg ccg ctg gct tat ctc gac agc gcc gcc agt 96

Arg Glu Val Asn Gly Leu Pro Leu Ala Tyr Leu Asp Ser Ala Ala Ser

20 25 30

gcg cag aaa ccg agc cag gtg att gac gcc gag gcc gag ttt tat cgt 144

Ala Gln Lys Pro Ser Gln Val Ile Asp Ala Glu Ala Glu Phe Tyr Arg

35 40 45

cat ggc tac gcg gcg gtg cat cgt ggt att cat acc tta agc gcc cag 192

His Gly Tyr Ala Ala Val His Arg Gly Ile His Thr Leu Ser Ala Gln

50 55 60

gcg acc gag aaa atg gag aac gtg cgc aag cgg gca tcg ctg ttt att 240

Ala Thr Glu Lys Met Glu Asn Val Arg Lys Arg Ala Ser Leu Phe Ile

65 70 75 80

aat gcc cgt tcg gcg gaa gag ctg gtg ttc gtc cgc ggc acg acg gaa 288

Asn Ala Arg Ser Ala Glu Glu Leu Val Phe Val Arg Gly Thr Thr Glu

85 90 95

ggg atc aat ctg gtc gcc aat agc tgg ggc aac agc aac gtg cgg gcg 336

Gly Ile Asn Leu Val Ala Asn Ser Trp Gly Asn Ser Asn Val Arg Ala

100 105 110

ggc gat aac atc atc atc agt cag atg gag cac cac gct aac att gtt 384

Gly Asp Asn Ile Ile Ile Ser Gln Met Glu His His Ala Asn Ile Val

115 120 125

ccc tgg cag atg ctt tgc gca cgc gtt ggc gca gag ctg cgt gtg atc 432

Pro Trp Gln Met Leu Cys Ala Arg Val Gly Ala Glu Leu Arg Val Ile

130 135 140

ccg ctc aat ccc gat ggt acg ttg caa ctg gag acg ctg cct acg ctg 480

Pro Leu Asn Pro Asp Gly Thr Leu Gln Leu Glu Thr Leu Pro Thr Leu

145 150 155 160

ttt gat gag aaa act cgc ctg ctg gca att act cat gtc tcc aac gtg 528

Phe Asp Glu Lys Thr Arg Leu Leu Ala Ile Thr His Val Ser Asn Val

165 170 175

ctt ggc aca gaa aat cca ctg gcg gaa atg atc acg ctt gcg cac cag 576

Leu Gly Thr Glu Asn Pro Leu Ala Glu Met Ile Thr Leu Ala His Gln

180 185 190

cat ggc gca aaa gtg ctg gtg gat ggc gct cag gcg gtg atg cat cat 624

His Gly Ala Lys Val Leu Val Asp Gly Ala Gln Ala Val Met His His

195 200 205

ccg gtg gat gtt cag gcg ctg gat tgc gac ttt tac gtg ttc tcc ggg 672

Pro Val Asp Val Gln Ala Leu Asp Cys Asp Phe Tyr Val Phe Ser Gly

210 215 220

cat aaa ctg tat ggc ccc acc gga att ggc att ctt tat gtg aaa gaa 720

His Lys Leu Tyr Gly Pro Thr Gly Ile Gly Ile Leu Tyr Val Lys Glu

225 230 235 240

gcc ttg ttg cag gag atg ccg ccg tgg gaa ggg ggc ggt tct atg atc 768

Ala Leu Leu Gln Glu Met Pro Pro Trp Glu Gly Gly Gly Ser Met Ile

245 250 255

gcc acc gtc agc ctg agt gaa ggc act acc tgg acc aaa gca cca tgg 816

Ala Thr Val Ser Leu Ser Glu Gly Thr Thr Trp Thr Lys Ala Pro Trp

260 265 270

cgg ttt gaa gcc ggt aca ccc aat acc ggg ggc atc att ggt ctt ggc 864

Arg Phe Glu Ala Gly Thr Pro Asn Thr Gly Gly Ile Ile Gly Leu Gly

275 280 285

gcg gcg ctg gag tat gtt tcg gcg ctg ggg ctt aat aac ata gcc gag 912

Ala Ala Leu Glu Tyr Val Ser Ala Leu Gly Leu Asn Asn Ile Ala Glu

290 295 300

tat gaa cag aat ctg atg cat tat gcg cta tca cag ctg gaa tct gta 960

Tyr Glu Gln Asn Leu Met His Tyr Ala Leu Ser Gln Leu Glu Ser Val

305 310 315 320

ccg gat ctc act ctc tat ggc cca caa aac agg ctt ggc gtt att gct 1008

Pro Asp Leu Thr Leu Tyr Gly Pro Gln Asn Arg Leu Gly Val Ile Ala

325 330 335

ttt aat ctc ggt aaa cac cac gcc tat gat gtt ggc agt ttt ctc gat 1056

Phe Asn Leu Gly Lys His His Ala Tyr Asp Val Gly Ser Phe Leu Asp

340 345 350

aat tac ggc att gct gtg cgt acc gga cat cac tgc gca atg cca ttg 1104

Asn Tyr Gly Ile Ala Val Arg Thr Gly His His Cys Ala Met Pro Leu

355 360 365

atg gcc tat tac aac gtc cct gcg atg tgt cgg gcg tcg ctg gcc atg 1152

Met Ala Tyr Tyr Asn Val Pro Ala Met Cys Arg Ala Ser Leu Ala Met

370 375 380

tat aac acc cat gaa gaa gtg gat cgt ctg gtg acc ggc ctg caa cgt 1200

Tyr Asn Thr His Glu Glu Val Asp Arg Leu Val Thr Gly Leu Gln Arg

385 390 395 400

att cac cgt ttg ctg gga taa 1221

Ile His Arg Leu Leu Gly

405

<210> 8

<211> 406

<212> PRT

<213> Escherichia coli

<400> 8

Met Ile Phe Ser Val Asp Lys Val Arg Ala Asp Phe Pro Val Leu Ser

1 5 10 15

Arg Glu Val Asn Gly Leu Pro Leu Ala Tyr Leu Asp Ser Ala Ala Ser

20 25 30

Ala Gln Lys Pro Ser Gln Val Ile Asp Ala Glu Ala Glu Phe Tyr Arg

35 40 45

His Gly Tyr Ala Ala Val His Arg Gly Ile His Thr Leu Ser Ala Gln

50 55 60

Ala Thr Glu Lys Met Glu Asn Val Arg Lys Arg Ala Ser Leu Phe Ile

65 70 75 80

Asn Ala Arg Ser Ala Glu Glu Leu Val Phe Val Arg Gly Thr Thr Glu

85 90 95

Gly Ile Asn Leu Val Ala Asn Ser Trp Gly Asn Ser Asn Val Arg Ala

100 105 110

Gly Asp Asn Ile Ile Ile Ser Gln Met Glu His His Ala Asn Ile Val

115 120 125

Pro Trp Gln Met Leu Cys Ala Arg Val Gly Ala Glu Leu Arg Val Ile

130 135 140

Pro Leu Asn Pro Asp Gly Thr Leu Gln Leu Glu Thr Leu Pro Thr Leu

145 150 155 160

Phe Asp Glu Lys Thr Arg Leu Leu Ala Ile Thr His Val Ser Asn Val

165 170 175

Leu Gly Thr Glu Asn Pro Leu Ala Glu Met Ile Thr Leu Ala His Gln

180 185 190

His Gly Ala Lys Val Leu Val Asp Gly Ala Gln Ala Val Met His His

195 200 205

Pro Val Asp Val Gln Ala Leu Asp Cys Asp Phe Tyr Val Phe Ser Gly

210 215 220

His Lys Leu Tyr Gly Pro Thr Gly Ile Gly Ile Leu Tyr Val Lys Glu

225 230 235 240

Ala Leu Leu Gln Glu Met Pro Pro Trp Glu Gly Gly Gly Ser Met Ile

245 250 255

Ala Thr Val Ser Leu Ser Glu Gly Thr Thr Trp Thr Lys Ala Pro Trp

260 265 270

Arg Phe Glu Ala Gly Thr Pro Asn Thr Gly Gly Ile Ile Gly Leu Gly

275 280 285

Ala Ala Leu Glu Tyr Val Ser Ala Leu Gly Leu Asn Asn Ile Ala Glu

290 295 300

Tyr Glu Gln Asn Leu Met His Tyr Ala Leu Ser Gln Leu Glu Ser Val

305 310 315 320

Pro Asp Leu Thr Leu Tyr Gly Pro Gln Asn Arg Leu Gly Val Ile Ala

325 330 335

Phe Asn Leu Gly Lys His His Ala Tyr Asp Val Gly Ser Phe Leu Asp

340 345 350

Asn Tyr Gly Ile Ala Val Arg Thr Gly His His Cys Ala Met Pro Leu

355 360 365

Met Ala Tyr Tyr Asn Val Pro Ala Met Cys Arg Ala Ser Leu Ala Met

370 375 380

Tyr Asn Thr His Glu Glu Val Asp Arg Leu Val Thr Gly Leu Gln Arg

385 390 395 400

Ile His Arg Leu Leu Gly

405

<210> 9

<211> 3720

<212> DNA

<213> Escherichia coli

<220>

<221> CDS

<222> (530) .. (1684)

<400> 9

gacgtctgtg tggaattgtg agcggataac aatttcacac agggccctcg gacaccgagg 60

agaatgtcaa gaggcgaaca cacaacgtct tggagcgcca gaggaggaac gagctaaaac 120

ggagcttttt tgccctgcgt gaccagatcc cggagttgga aaacaatgaa aaggccccca 180

aggtagttat ccttaaaaaa gccacagcat acatcctgtc cgtccaagca gaggagcaaa 240

agctcatttc tgaagaggac ttgttgcgga aacgacgaga acagttgaaa cacaaacttg 300

aacagctacg gaactcttgt gcgtaaggaa aagtaaggaa aacgattcct tctaacagaa 360

atgtcctgag caatcaccta tgaactgtcg actcgagata gcatttttat ccataagatt 420

agccgatcct aaggtttaca attgtgagcg ctcacaatta tgatagattc aattgtgagc 480

ggataacaat ttcacacacg ctagcggtac caaagaggag aaattaact atg gca aaa 538

Met ala lys

One

cac ctt ttt acg tcc gag tcc gtc tct gaa ggg cat cct gac aaa att 586

His Leu Phe Thr Ser Glu Ser Val Ser Glu Gly His Pro Asp Lys Ile

5 10 15

gct gac caa att tct gat gcc gtt tta gac gcg atc ctc gaa cag gat 634

Ala Asp Gln Ile Ser Asp Ala Val Leu Asp Ala Ile Leu Glu Gln Asp

20 25 30 35

ccg aaa gca cgc gtt gct tgc gaa acc tac gta aaa acc ggc atg gtt 682

Pro Lys Ala Arg Val Ala Cys Glu Thr Tyr Val Lys Thr Gly Met Val

40 45 50

tta gtt ggc ggc gaa atc acc acc agc gcc tgg gta gac atc gaa gag 730

Leu Val Gly Gly Glu Ile Thr Thr Ser Ala Trp Val Asp Ile Glu Glu

55 60 65

atc acc cgt aac acc gtt cgc gaa att ggc tat gtg cat tcc gac atg 778

Ile Thr Arg Asn Thr Val Arg Glu Ile Gly Tyr Val His Ser Asp Met

70 75 80

ggc ttt gac gct aac tcc tgt gcg gtt ctg agc gct atc ggc aaa cag 826

Gly Phe Asp Ala Asn Ser Cys Ala Val Leu Ser Ala Ile Gly Lys Gln

85 90 95

tct cct gac atc aac cag ggc gtt gac cgt gcc gat ccg ctg gaa cag 874

Ser Pro Asp Ile Asn Gln Gly Val Asp Arg Ala Asp Pro Leu Glu Gln

100 105 110 115

ggc gcg ggt gac cag ggt ctg atg ttt ggc tac gca act aat gaa acc 922

Gly Ala Gly Asp Gln Gly Leu Met Phe Gly Tyr Ala Thr Asn Glu Thr

120 125 130

gac gtg ctg atg cca gca cct atc acc tat gca cac cgt ctg gta cag 970

Asp Val Leu Met Pro Ala Pro Ile Thr Tyr Ala His Arg Leu Val Gln

135 140 145

cgt cag gct gaa gtg cgt aaa aac ggc act ctg ccg tgg ctg cgc ccg 1018

Arg Gln Ala Glu Val Arg Lys Asn Gly Thr Leu Pro Trp Leu Arg Pro

150 155 160

gac gcg aaa agc cag gtg act ttt cag tat gac gac ggc aaa atc gtt 1066

Asp Ala Lys Ser Gln Val Thr Phe Gln Tyr Asp Asp Gly Lys Ile Val

165 170 175

ggt atc gat gct gtc gtg ctt tcc act cag cac tct gaa gag atc gac 1114

Gly Ile Asp Ala Val Val Leu Ser Thr Gln His Ser Glu Glu Ile Asp

180 185 190 195

cag aaa tcg ctg caa gaa gcg gta atg gaa gag atc atc aag cca att 1162

Gln Lys Ser Leu Gln Glu Ala Val Met Glu Glu Ile Ile Lys Pro Ile

200 205 210

ctg ccc gct gaa tgg ctg act tct gcc acc aaa ttc ttc atc aac ccg 1210

Leu Pro Ala Glu Trp Leu Thr Ser Ala Thr Lys Phe Phe Ile Asn Pro

215 220 225

acc ggt cgt ttc gtt atc ggt ggc cca atg ggt gac tgc ggt ctg act 1258

Thr Gly Arg Phe Val Ile Gly Gly Pro Met Gly Asp Cys Gly Leu Thr

230 235 240

ggt cgt aaa att atc gtt gat acc tac ggc ggc atg gcg cgt cac ggt 1306

Gly Arg Lys Ile Ile Val Asp Thr Tyr Gly Gly Met Ala Arg His Gly

245 250 255

ggc ggt gca ttc tct ggt aaa gat cca tca aaa gtg gac cgt tcc gca 1354

Gly Gly Ala Phe Ser Gly Lys Asp Pro Ser Lys Val Asp Arg Ser Ala

260 265 270 275

gcc tac gca gca cgt tat gtc gcg aaa aac atc gtt gct gct ggc ctg 1402

Ala Tyr Ala Ala Arg Tyr Val Ala Lys Asn Ile Val Ala Ala Gly Leu

280 285 290

gcc gat cgt tgt gaa att cag gtt tcc tac gca atc ggc gtg gct gaa 1450

Ala Asp Arg Cys Glu Ile Gln Val Ser Tyr Ala Ile Gly Val Ala Glu

295 300 305

ccg acc tcc atc atg gta gaa act ttc ggt act gag aaa gtg cct tct 1498

Pro Thr Ser Ile Met Val Glu Thr Phe Gly Thr Glu Lys Val Pro Ser

310 315 320

gaa caa ctg acc ctg ctg gta cgt gag ttc ttc gac ctg cgc cca tac 1546

Glu Gln Leu Thr Leu Leu Val Arg Glu Phe Phe Asp Leu Arg Pro Tyr

325 330 335

ggt ctg att cag atg ctg gat ctg ctg cac ccg atc tac aaa gaa acc 1594

Gly Leu Ile Gln Met Leu Asp Leu Leu His Pro Ile Tyr Lys Glu Thr

340 345 350 355

gca gca tac ggt cac ttt ggt cgt gaa cat ttc ccg tgg gaa aaa acc 1642

Ala Ala Tyr Gly His Phe Gly Arg Glu His Phe Pro Trp Glu Lys Thr

360 365 370

gac aaa gcg cag ctg ctg cgc gat gct gcc ggt ctg aag taa 1684

Asp Lys Ala Gln Leu Leu Arg Asp Ala Ala Gly Leu Lys

375 380 385

tcggtaccgc ttgatatcga attcctgcag cccgggggat cccatggtac gcgtgctaga 1744

ggcatcaaat aaaacgaaag gctcagtcga aagactgggc ctttcgtttt atctgttgtt 1804

tgtcggtgaa cgctctcctg agtaggacaa atccgccgcc ctagacctag gggatatatt 1864

ccgcttcctc gctcactgac tcgctacgct cggtcgttcg actgcggcga gcggaaatgg 1924

cttacgaacg gggcggagat ttcctggaag atgccaggaa gatacttaac agggaagtga 1984

gagggccgcg gcaaagccgt ttttccatag gctccgcccc cctgacaagc atcacgaaat 2044

ctgacgctca aatcagtggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc 2104

ccctggcggc tccctcgtgc gctctcctgt tcctgccttt cggtttaccg gtgtcattcc 2164

gctgttatgg ccgcgtttgt ctcattccac gcctgacact cagttccggg taggcagttc 2224

gctccaagct ggactgtatg cacgaacccc ccgttcagtc cgaccgctgc gccttatccg 2284

gtaactatcg tcttgagtcc aacccggaaa gacatgcaaa agcaccactg gcagcagcca 2344

ctggtaattg atttagagga gttagtcttg aagtcatgcg ccggttaagg ctaaactgaa 2404

aggacaagtt ttggtgactg cgctcctcca agccagttac ctcggttcaa agagttggta 2464

gctcagagaa ccttcgaaaa accgccctgc aaggcggttt tttcgttttc agagcaagag 2524

attacgcgca gaccaaaacg atctcaagaa gatcatctta ttaatcagat aaaatatttc 2584

tagatttcag tgcaatttat ctcttcaaat gtagcacctg aagtcagccc catacgatat 2644

aagttgttac tagtgcttgg attctcacca ataaaaaacg cccggcggca accgagcgtt 2704

ctgaacaaat ccagatggag ttctgaggtc attactggat ctatcaacag gagtccaagc 2764

gagctctcga accccagagt cccgctcaga agaactcgtc aagaaggcga tagaaggcga 2824

tgcgctgcga atcgggagcg gcgataccgt aaagcacgag gaagcggtca gcccattcgc 2884

cgccaagctc ttcagcaata tcacgggtag ccaacgctat gtcctgatag cggtccgcca 2944

cacccagccg gccacagtcg atgaatccag aaaagcggcc attttccacc atgatattcg 3004

gcaagcaggc atcgccatgg gtcacgacga gatcctcgcc gtcgggcatg cgcgccttga 3064

gcctggcgaa cagttcggct ggcgcgagcc cctgatgctc ttcgtccaga tcatcctgat 3124

cgacaagacc ggcttccatc cgagtacgtg ctcgctcgat gcgatgtttc gcttggtggt 3184

cgaatgggca ggtagccgga tcaagcgtat gcagccgccg cattgcatca gccatgatgg 3244

atactttctc ggcaggagca aggtgagatg acaggagatc ctgccccggc acttcgccca 3304

atagcagcca gtcccttccc gcttcagtga caacgtcgag cacagctgcg caaggaacgc 3364

ccgtcgtggc cagccacgat agccgcgctg cctcgtcctg cagttcattc agggcaccgg 3424

acaggtcggt cttgacaaaa agaaccgggc gcccctgcgc tgacagccgg aacacggcgg 3484

catcagagca gccgattgtc tgttgtgccc agtcatagcc gaatagcctc tccacccaag 3544

cggccggaga acctgcgtgc aatccatctt gttcaatcat gcgaaacgat cctcatcctg 3604

tctcttgatc agatcttgat cccctgcgcc atcagatcct tggcggcaag aaagccatcc 3664

agtttacttt gcagggcttc ccaaccttac cagagggcgc cccagctggc aattcc 3720

<210> 10

<211> 384

<212> PRT

<213> Escherichia coli

<400> 10

Met Ala Lys His Leu Phe Thr Ser Glu Ser Val Ser Glu Gly His Pro

1 5 10 15

Asp Lys Ile Ala Asp Gln Ile Ser Asp Ala Val Leu Asp Ala Ile Leu

20 25 30

Glu Gln Asp Pro Lys Ala Arg Val Ala Cys Glu Thr Tyr Val Lys Thr

35 40 45

Gly Met Val Leu Val Gly Gly Glu Ile Thr Thr Ser Ala Trp Val Asp

50 55 60

Ile Glu Glu Ile Thr Arg Asn Thr Val Arg Glu Ile Gly Tyr Val His

65 70 75 80

Ser Asp Met Gly Phe Asp Ala Asn Ser Cys Ala Val Leu Ser Ala Ile

85 90 95

Gly Lys Gln Ser Pro Asp Ile Asn Gln Gly Val Asp Arg Ala Asp Pro

100 105 110

Leu Glu Gln Gly Ala Gly Asp Gln Gly Leu Met Phe Gly Tyr Ala Thr

115 120 125

Asn Glu Thr Asp Val Leu Met Pro Ala Pro Ile Thr Tyr Ala His Arg

130 135 140

Leu Val Gln Arg Gln Ala Glu Val Arg Lys Asn Gly Thr Leu Pro Trp

145 150 155 160

Leu Arg Pro Asp Ala Lys Ser Gln Val Thr Phe Gln Tyr Asp Asp Gly

165 170 175

Lys Ile Val Gly Ile Asp Ala Val Val Leu Ser Thr Gln His Ser Glu

180 185 190

Glu Ile Asp Gln Lys Ser Leu Gln Glu Ala Val Met Glu Glu Ile Ile

195 200 205

Lys Pro Ile Leu Pro Ala Glu Trp Leu Thr Ser Ala Thr Lys Phe Phe

210 215 220

Ile Asn Pro Thr Gly Arg Phe Val Ile Gly Gly Pro Met Gly Asp Cys

225 230 235 240

Gly Leu Thr Gly Arg Lys Ile Ile Val Asp Thr Tyr Gly Gly Met Ala

245 250 255

Arg His Gly Gly Gly Ala Phe Ser Gly Lys Asp Pro Ser Lys Val Asp

260 265 270

Arg Ser Ala Ala Tyr Ala Ala Arg Tyr Val Ala Lys Asn Ile Val Ala

275 280 285

Ala Gly Leu Ala Asp Arg Cys Glu Ile Gln Val Ser Tyr Ala Ile Gly

290 295 300

Val Ala Glu Pro Thr Ser Ile Met Val Glu Thr Phe Gly Thr Glu Lys

305 310 315 320

Val Pro Ser Glu Gln Leu Thr Leu Leu Val Arg Glu Phe Phe Asp Leu

325 330 335

Arg Pro Tyr Gly Leu Ile Gln Met Leu Asp Leu Leu His Pro Ile Tyr

340 345 350

Lys Glu Thr Ala Ala Tyr Gly His Phe Gly Arg Glu His Phe Pro Trp

355 360 365

Glu Lys Thr Asp Lys Ala Gln Leu Leu Arg Asp Ala Ala Gly Leu Lys

370 375 380

<210> 11

<211> 3794

<212> DNA

<213> Escherichia coli

<220>

<221> CDS

<222> (601) .. (1806)

<400> 11

gacgtctgtg tggaattgtg agcggataac aatttcacac agggccctcg gacaccgagg 60

agaatgtcaa gaggcgaaca cacaacgtct tggagcgcca gaggaggaac gagctaaaac 120

ggagcttttt tgccctgcgt gaccagatcc cggagttgga aaacaatgaa aaggccccca 180

aggtagttat ccttaaaaaa gccacagcat acatcctgtc cgtccaagca gaggagcaaa 240

agctcatttc tgaagaggac ttgttgcgga aacgacgaga acagttgaaa cacaaacttg 300

aacagctacg gaactcttgt gcgtaaggaa aagtaaggaa aacgattcct tctaacagaa 360

atgtcctgag caatcaccta tgaactgtcg actcgagata gcatttttat ccataagatt 420

agccgatcct aaggtttaca attgtgagcg ctcacaatta tgatagattc aattgtgagc 480

ggataacaat ttcacacacg ctagcggtac cgggcccccc ctcgaggtcg acggtatcga 540

taagcttgat atcgaattcc tgcagcccgg gggatcccat ggtacgcgtc gaggagtacc 600

atg aac gtt ttt aat ccc gcg cag ttt cgc gcc cag ttt ccc gca cta 648

Met Asn Val Phe Asn Pro Ala Gln Phe Arg Ala Gln Phe Pro Ala Leu

1 5 10 15

cag gat gcg ggc gtc tat ctc gac agc gcc gcg acc gcg ctt aaa cct 696

Gln Asp Ala Gly Val Tyr Leu Asp Ser Ala Ala Thr Ala Leu Lys Pro

20 25 30

gaa gcc gtg gtt gaa gcc acc caa cag ttt tac agt ctg agc gcc gga 744

Glu Ala Val Val Glu Ala Thr Gln Gln Phe Tyr Ser Leu Ser Ala Gly

35 40 45

aac gtc cat cgc agc cag ttt gcc gaa gcc caa cgc ctg acc gcg cgt 792

Asn Val His Arg Ser Gln Phe Ala Glu Ala Gln Arg Leu Thr Ala Arg

50 55 60

tat gaa gct gca cga gag aaa gtg gcg caa tta ctg aat gca ccg gat 840

Tyr Glu Ala Ala Arg Glu Lys Val Ala Gln Leu Leu Asn Ala Pro Asp

65 70 75 80

gat aaa act atc gtc tgg acg cgc ggc acc act gaa tcc atc aac atg 888

Asp Lys Thr Ile Val Trp Thr Arg Gly Thr Thr Glu Ser Ile Asn Met

85 90 95

gtg gca caa tgc tat gcg cgt ccg cgt ctg caa ccg ggc gat gag att 936

Val Ala Gln Cys Tyr Ala Arg Pro Arg Leu Gln Pro Gly Asp Glu Ile

100 105 110

att gtc agc gtg gca gaa cac cac gcc aac ctc gtc ccc tgg ctg atg 984

Ile Val Ser Val Ala Glu His His Ala Asn Leu Val Pro Trp Leu Met

115 120 125

gtc gcc caa caa act gga gcc aaa gtg gtg aaa ttg ccg ctt aat gcg 1032

Val Ala Gln Gln Thr Gly Ala Lys Val Val Lys Leu Pro Leu Asn Ala

130 135 140

cag cga ctg ccg gat gtc gat ttg ttg cca gaa ctg att act ccc cgt 1080

Gln Arg Leu Pro Asp Val Asp Leu Leu Pro Glu Leu Ile Thr Pro Arg

145 150 155 160

agt cgg att ctg gcg ttg ggt cag atg tcg aac gtt act ggc ggt tgc 1128

Ser Arg Ile Leu Ala Leu Gly Gln Met Ser Asn Val Thr Gly Gly Cys

165 170 175

ccg gat ctg gcg cga gcg att acc ttt gct cat tca gcc ggg atg gtg 1176

Pro Asp Leu Ala Arg Ala Ile Thr Phe Ala His Ser Ala Gly Met Val

180 185 190

gtg atg gtt gat ggt gct cag ggg gca gtg cat ttc ccc gcg gat gtt 1224

Val Met Val Asp Gly Ala Gln Gly Ala Val His Phe Pro Ala Asp Val

195 200 205

cag caa ctg gat att gat ttc tat gct ttt tca ggt cac aaa ctg tat 1272

Gln Gln Leu Asp Ile Asp Phe Tyr Ala Phe Ser Gly His Lys Leu Tyr

210 215 220

ggc ccg aca ggt atc ggc gtg ctg tat ggt aaa tca gaa ctg ctg gag 1320

Gly Pro Thr Gly Ile Gly Val Leu Tyr Gly Lys Ser Glu Leu Leu Glu

225 230 235 240

gcg atg tcg ccc tgg ctg ggc ggc ggc aaa atg gtt cac gaa gtg agt 1368

Ala Met Ser Pro Trp Leu Gly Gly Gly Lys Met Val His Glu Val Ser

245 250 255

ttt gac ggc ttc acg act caa tct gcg ccg tgg aaa ctg gaa gct gga 1416

Phe Asp Gly Phe Thr Thr Gln Ser Ala Pro Trp Lys Leu Glu Ala Gly

260 265 270

acg cca aat gtc gct ggt gtc ata gga tta agc gcg gcg ctg gaa tgg 1464

Thr Pro Asn Val Ala Gly Val Ile Gly Leu Ser Ala Ala Leu Glu Trp

275 280 285

ctg gca gat tac gat atc aac cag gcc gaa agc tgg agc cgt agc tta 1512

Leu Ala Asp Tyr Asp Ile Asn Gln Ala Glu Ser Trp Ser Arg Ser Leu

290 295 300

gca acg ctg gcg gaa gat gcg ctg gcg aaa cgt ccc ggc ttt cgt tca 1560

Ala Thr Leu Ala Glu Asp Ala Leu Ala Lys Arg Pro Gly Phe Arg Ser

305 310 315 320

ttc cgc tgc cag gat tcc agc ctg ctg gcc ttt gat ttt gct ggc gtt 1608

Phe Arg Cys Gln Asp Ser Ser Leu Leu Ala Phe Asp Phe Ala Gly Val

325 330 335

cat cat agc gat atg gtg acg ctg ctg gcg gag tac ggt att gcc ctg 1656

His His Ser Asp Met Val Thr Leu Leu Ala Glu Tyr Gly Ile Ala Leu

340 345 350

cgg gcc ggg cag cat tgc gct cag ccg cta ctg gca gaa tta ggc gta 1704

Arg Ala Gly Gln His Cys Ala Gln Pro Leu Leu Ala Glu Leu Gly Val

355 360 365

acc ggc aca ctg cgc gcc tct ttt gcg cca tat aat aca aag agt gat 1752

Thr Gly Thr Leu Arg Ala Ser Phe Ala Pro Tyr Asn Thr Lys Ser Asp

370 375 380

gtg gat gcg ctg gtg aat gcc gtt gac cgc gcg ctg gaa tta ttg gtg 1800

Val Asp Ala Leu Val Asn Ala Val Asp Arg Ala Leu Glu Leu Leu Val

385 390 395 400

gat taa acgcgtgcta gaggcatcaa ataaaacgaa aggctcagtc gaaagactgg 1856

Asp

gcctttcgtt ttatctgttg tttgtcggtg aacgctctcc tgagtaggac aaatccgccg 1916

ccctagacct aggggatata ttccgcttcc tcgctcactg actcgctacg ctcggtcgtt 1976

cgactgcggc gagcggaaat ggcttacgaa cggggcggag atttcctgga agatgccagg 2036

aagatactta acagggaagt gagagggccg cggcaaagcc gtttttccat aggctccgcc 2096

cccctgacaa gcatcacgaa atctgacgct caaatcagtg gtggcgaaac ccgacaggac 2156

tataaagata ccaggcgttt ccccctggcg gctccctcgt gcgctctcct gttcctgcct 2216

ttcggtttac cggtgtcatt ccgctgttat ggccgcgttt gtctcattcc acgcctgaca 2276

ctcagttccg ggtaggcagt tcgctccaag ctggactgta tgcacgaacc ccccgttcag 2336

tccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccgga aagacatgca 2396

aaagcaccac tggcagcagc cactggtaat tgatttagag gagttagtct tgaagtcatg 2456

cgccggttaa ggctaaactg aaaggacaag ttttggtgac tgcgctcctc caagccagtt 2516

acctcggttc aaagagttgg tagctcagag aaccttcgaa aaaccgccct gcaaggcggt 2576

tttttcgttt tcagagcaag agattacgcg cagaccaaaa cgatctcaag aagatcatct 2636

tattaatcag ataaaatatt tctagatttc agtgcaattt atctcttcaa atgtagcacc 2696

tgaagtcagc cccatacgat ataagttgtt actagtgctt ggattctcac caataaaaaa 2756

cgcccggcgg caaccgagcg ttctgaacaa atccagatgg agttctgagg tcattactgg 2816

atctatcaac aggagtccaa gcgagctctc gaaccccaga gtcccgctca gaagaactcg 2876

tcaagaaggc gatagaaggc gatgcgctgc gaatcgggag cggcgatacc gtaaagcacg 2936

aggaagcggt cagcccattc gccgccaagc tcttcagcaa tatcacgggt agccaacgct 2996

atgtcctgat agcggtccgc cacacccagc cggccacagt cgatgaatcc agaaaagcgg 3056

ccattttcca ccatgatatt cggcaagcag gcatcgccat gggtcacgac gagatcctcg 3116

ccgtcgggca tgcgcgcctt gagcctggcg aacagttcgg ctggcgcgag cccctgatgc 3176

tcttcgtcca gatcatcctg atcgacaaga ccggcttcca tccgagtacg tgctcgctcg 3236

atgcgatgtt tcgcttggtg gtcgaatggg caggtagccg gatcaagcgt atgcagccgc 3296

cgcattgcat cagccatgat ggatactttc tcggcaggag caaggtgaga tgacaggaga 3356

tcctgccccg gcacttcgcc caatagcagc cagtcccttc ccgcttcagt gacaacgtcg 3416

agcacagctg cgcaaggaac gcccgtcgtg gccagccacg atagccgcgc tgcctcgtcc 3476

tgcagttcat tcagggcacc ggacaggtcg gtcttgacaa aaagaaccgg gcgcccctgc 3536

gctgacagcc ggaacacggc ggcatcagag cagccgattg tctgttgtgc ccagtcatag 3596

ccgaatagcc tctccaccca agcggccgga gaacctgcgt gcaatccatc ttgttcaatc 3656

atgcgaaacg atcctcatcc tgtctcttga tcagatcttg atcccctgcg ccatcagatc 3716

cttggcggca agaaagccat ccagtttact ttgcagggct tcccaacctt accagagggc 3776

gccccagctg gcaattcc 3794

<210> 12

<211> 401

<212> PRT

<213> Escherichia coli

<400> 12

Met Asn Val Phe Asn Pro Ala Gln Phe Arg Ala Gln Phe Pro Ala Leu

1 5 10 15

Gln Asp Ala Gly Val Tyr Leu Asp Ser Ala Ala Thr Ala Leu Lys Pro

20 25 30

Glu Ala Val Val Glu Ala Thr Gln Gln Phe Tyr Ser Leu Ser Ala Gly

35 40 45

Asn Val His Arg Ser Gln Phe Ala Glu Ala Gln Arg Leu Thr Ala Arg

50 55 60

Tyr Glu Ala Ala Arg Glu Lys Val Ala Gln Leu Leu Asn Ala Pro Asp

65 70 75 80

Asp Lys Thr Ile Val Trp Thr Arg Gly Thr Thr Glu Ser Ile Asn Met

85 90 95

Val Ala Gln Cys Tyr Ala Arg Pro Arg Leu Gln Pro Gly Asp Glu Ile

100 105 110

Ile Val Ser Val Ala Glu His His Ala Asn Leu Val Pro Trp Leu Met

115 120 125

Val Ala Gln Gln Thr Gly Ala Lys Val Val Lys Leu Pro Leu Asn Ala

130 135 140

Gln Arg Leu Pro Asp Val Asp Leu Leu Pro Glu Leu Ile Thr Pro Arg

145 150 155 160

Ser Arg Ile Leu Ala Leu Gly Gln Met Ser Asn Val Thr Gly Gly Cys

165 170 175

Pro Asp Leu Ala Arg Ala Ile Thr Phe Ala His Ser Ala Gly Met Val

180 185 190

Val Met Val Asp Gly Ala Gln Gly Ala Val His Phe Pro Ala Asp Val

195 200 205

Gln Gln Leu Asp Ile Asp Phe Tyr Ala Phe Ser Gly His Lys Leu Tyr

210 215 220

Gly Pro Thr GlyIle Gly Val Leu Tyr Gly Lys Ser Glu Leu Leu Glu

225 230 235 240

Ala Met Ser Pro Trp Leu Gly Gly Gly Lys Met Val His Glu Val Ser

245 250 255

Phe Asp Gly Phe Thr Thr Gln Ser Ala Pro Trp Lys Leu Glu Ala Gly

260 265 270

Thr Pro Asn Val Ala Gly Val Ile Gly Leu Ser Ala Ala Leu Glu Trp

275 280 285

Leu Ala Asp Tyr Asp Ile Asn Gln Ala Glu Ser Trp Ser Arg Ser Leu

290 295 300

Ala Thr Leu Ala Glu Asp Ala Leu Ala Lys Arg Pro Gly Phe Arg Ser

305 310 315 320

Phe Arg Cys Gln Asp Ser Ser Leu Leu Ala Phe Asp Phe Ala Gly Val

325 330 335

His His Ser Asp Met Val Thr Leu Leu Ala Glu Tyr Gly Ile Ala Leu

340 345 350

Arg Ala Gly Gln His Cys Ala Gln Pro Leu Leu Ala Glu Leu Gly Val

355 360 365

Thr Gly Thr Leu Arg Ala Ser Phe Ala Pro Tyr Asn Thr Lys Ser Asp

370 375 380

Val Asp Ala Leu Val Asn Ala Val Asp Arg Ala Leu Glu Leu Leu Val

385 390 395 400

Asp

<210> 13

<211> 4975

<212> DNA

<213> Escherichia coli

<220>

<221> CDS

<222> (530) .. (1684)

<220>

<221> CDS

<222> (1782) .. (2987)

<400> 13

gacgtctgtg tggaattgtg agcggataac aatttcacac agggccctcg gacaccgagg 60

agaatgtcaa gaggcgaaca cacaacgtct tggagcgcca gaggaggaac gagctaaaac 120

ggagcttttt tgccctgcgt gaccagatcc cggagttgga aaacaatgaa aaggccccca 180

aggtagttat ccttaaaaaa gccacagcat acatcctgtc cgtccaagca gaggagcaaa 240

agctcatttc tgaagaggac ttgttgcgga aacgacgaga acagttgaaa cacaaacttg 300

aacagctacg gaactcttgt gcgtaaggaa aagtaaggaa aacgattcct tctaacagaa 360

atgtcctgag caatcaccta tgaactgtcg actcgagata gcatttttat ccataagatt 420

agccgatcct aaggtttaca attgtgagcg ctcacaatta tgatagattc aattgtgagc 480

ggataacaat ttcacacacg ctagcggtac caaagaggag aaattaact atg gca aaa 538

Met ala lys

One

cac ctt ttt acg tcc gag tcc gtc tct gaa ggg cat cct gac aaa att 586

His Leu Phe Thr Ser Glu Ser Val Ser Glu Gly His Pro Asp Lys Ile

5 10 15

gct gac caa att tct gat gcc gtt tta gac gcg atc ctc gaa cag gat 634

Ala Asp Gln Ile Ser Asp Ala Val Leu Asp Ala Ile Leu Glu Gln Asp

20 25 30 35

ccg aaa gca cgc gtt gct tgc gaa acc tac gta aaa acc ggc atg gtt 682

Pro Lys Ala Arg Val Ala Cys Glu Thr Tyr Val Lys Thr Gly Met Val

40 45 50

tta gtt ggc ggc gaa atc acc acc agc gcc tgg gta gac atc gaa gag 730

Leu Val Gly Gly Glu Ile Thr Thr Ser Ala Trp Val Asp Ile Glu Glu

55 60 65

atc acc cgt aac acc gtt cgc gaa att ggc tat gtg cat tcc gac atg 778

Ile Thr Arg Asn Thr Val Arg Glu Ile Gly Tyr Val His Ser Asp Met

70 75 80

ggc ttt gac gct aac tcc tgt gcg gtt ctg agc gct atc ggc aaa cag 826

Gly Phe Asp Ala Asn Ser Cys Ala Val Leu Ser Ala Ile Gly Lys Gln

85 90 95

tct cct gac atc aac cag ggc gtt gac cgt gcc gat ccg ctg gaa cag 874

Ser Pro Asp Ile Asn Gln Gly Val Asp Arg Ala Asp Pro Leu Glu Gln

100 105 110 115

ggc gcg ggt gac cag ggt ctg atg ttt ggc tac gca act aat gaa acc 922

Gly Ala Gly Asp Gln Gly Leu Met Phe Gly Tyr Ala Thr Asn Glu Thr

120 125 130

gac gtg ctg atg cca gca cct atc acc tat gca cac cgt ctg gta cag 970

Asp Val Leu Met Pro Ala Pro Ile Thr Tyr Ala His Arg Leu Val Gln

135 140 145

cgt cag gct gaa gtg cgt aaa aac ggc act ctg ccg tgg ctg cgc ccg 1018

Arg Gln Ala Glu Val Arg Lys Asn Gly Thr Leu Pro Trp Leu Arg Pro

150 155 160

gac gcg aaa agc cag gtg act ttt cag tat gac gac ggc aaa atc gtt 1066

Asp Ala Lys Ser Gln Val Thr Phe Gln Tyr Asp Asp Gly Lys Ile Val

165 170 175

ggt atc gat gct gtc gtg ctt tcc act cag cac tct gaa gag atc gac 1114

Gly Ile Asp Ala Val Val Leu Ser Thr Gln His Ser Glu Glu Ile Asp

180 185 190 195

cag aaa tcg ctg caa gaa gcg gta atg gaa gag atc atc aag cca att 1162

Gln Lys Ser Leu Gln Glu Ala Val Met Glu Glu Ile Ile Lys Pro Ile

200 205 210

ctg ccc gct gaa tgg ctg act tct gcc acc aaa ttc ttc atc aac ccg 1210

Leu Pro Ala Glu Trp Leu Thr Ser Ala Thr Lys Phe Phe Ile Asn Pro

215 220 225

acc ggt cgt ttc gtt atc ggt ggc cca atg ggt gac tgc ggt ctg act 1258

Thr Gly Arg Phe Val Ile Gly Gly Pro Met Gly Asp Cys Gly Leu Thr

230 235 240

ggt cgt aaa att atc gtt gat acc tac ggc ggc atg gcg cgt cac ggt 1306

Gly Arg Lys Ile Ile Val Asp Thr Tyr Gly Gly Met Ala Arg His Gly

245 250 255

ggc ggt gca ttc tct ggt aaa gat cca tca aaa gtg gac cgt tcc gca 1354

Gly Gly Ala Phe Ser Gly Lys Asp Pro Ser Lys Val Asp Arg Ser Ala

260 265 270 275

gcc tac gca gca cgt tat gtc gcg aaa aac atc gtt gct gct ggc ctg 1402

Ala Tyr Ala Ala Arg Tyr Val Ala Lys Asn Ile Val Ala Ala Gly Leu

280 285 290

gcc gat cgt tgt gaa att cag gtt tcc tac gca atc ggc gtg gct gaa 1450

Ala Asp Arg Cys Glu Ile Gln Val Ser Tyr Ala Ile Gly Val Ala Glu

295 300 305

ccg acc tcc atc atg gta gaa act ttc ggt act gag aaa gtg cct tct 1498

Pro Thr Ser Ile Met Val Glu Thr Phe Gly Thr Glu Lys Val Pro Ser

310 315 320

gaa caa ctg acc ctg ctg gta cgt gag ttc ttc gac ctg cgc cca tac 1546

Glu Gln Leu Thr Leu Leu Val Arg Glu Phe Phe Asp Leu Arg Pro Tyr

325 330 335

ggt ctg att cag atg ctg gat ctg ctg cac ccg atc tac aaa gaa acc 1594

Gly Leu Ile Gln Met Leu Asp Leu Leu His Pro Ile Tyr Lys Glu Thr

340 345 350 355

gca gca tac ggt cac ttt ggt cgt gaa cat ttc ccg tgg gaa aaa acc 1642

Ala Ala Tyr Gly His Phe Gly Arg Glu His Phe Pro Trp Glu Lys Thr

360 365 370

gac aaa gcg cag ctg ctg cgc gat gct gcc ggt ctg aag taa 1684

Asp Lys Ala Gln Leu Leu Arg Asp Ala Ala Gly Leu Lys

375 380 385

tcggtaccgg gccccccctc gaggtcgacg gtatcgataa gcttgatatc gaattcctgc 1744

agcccggggg atcccatggt acgcgtcgag gagtacc atg aac gtt ttt aat ccc 1799

Met Asn Val Phe Asn Pro

390

gcg cag ttt cgc gcc cag ttt ccc gca cta cag gat gcg ggc gtc tat 1847

Ala Gln Phe Arg Ala Gln Phe Pro Ala Leu Gln Asp Ala Gly Val Tyr

395 400 405

ctc gac agc gcc gcg acc gcg ctt aaa cct gaa gcc gtg gtt gaa gcc 1895

Leu Asp Ser Ala Ala Thr Ala Leu Lys Pro Glu Ala Val Val Glu Ala

410 415 420

acc caa cag ttt tac agt ctg agc gcc gga aac gtc cat cgc agc cag 1943

Thr Gln Gln Phe Tyr Ser Leu Ser Ala Gly Asn Val His Arg Ser Gln

425 430 435

ttt gcc gaa gcc caa cgc ctg acc gcg cgt tat gaa gct gca cga gag 1991

Phe Ala Glu Ala Gln Arg Leu Thr Ala Arg Tyr Glu Ala Ala Arg Glu

440 445 450 455

aaa gtg gcg caa tta ctg aat gca ccg gat gat aaa act atc gtc tgg 2039

Lys Val Ala Gln Leu Leu Asn Ala Pro Asp Asp Lys Thr Ile Val Trp

460 465 470

acg cgc ggc acc act gaa tcc atc aac atg gtg gca caa tgc tat gcg 2087

Thr Arg Gly Thr Thr Glu Ser Ile Asn Met Val Ala Gln Cys Tyr Ala

475 480 485

cgt ccg cgt ctg caa ccg ggc gat gag att att gtc agc gtg gca gaa 2135

Arg Pro Arg Leu Gln Pro Gly Asp Glu Ile Ile Val Ser Val Ala Glu

490 495 500

cac cac gcc aac ctc gtc ccc tgg ctg atg gtc gcc caa caa act gga 2183

His His Ala Asn Leu Val Pro Trp Leu Met Val Ala Gln Gln Thr Gly

505 510 515

gcc aaa gtg gtg aaa ttg ccg ctt aat gcg cag cga ctg ccg gat gtc 2231

Ala Lys Val Val Lys Leu Pro Leu Asn Ala Gln Arg Leu Pro Asp Val

520 525 530 535

gat ttg ttg cca gaa ctg att act ccc cgt agt cgg att ctg gcg ttg 2279

Asp Leu Leu Pro Glu Leu Ile Thr Pro Arg Ser Arg Ile Leu Ala Leu

540 545 550

ggt cag atg tcg aac gtt act ggc ggt tgc ccg gat ctg gcg cga gcg 2327

Gly Gln Met Ser Asn Val Thr Gly Gly Cys Pro Asp Leu Ala Arg Ala

555 560 565

att acc ttt gct cat tca gcc ggg atg gtg gtg atg gtt gat ggt gct 2375

Ile Thr Phe Ala His Ser Ala Gly Met Val Val Met Val Asp Gly Ala

570 575 580

cag ggg gca gtg cat ttc ccc gcg gat gtt cag caa ctg gat att gat 2423

Gln Gly Ala Val His Phe Pro Ala Asp Val Gln Gln Leu Asp Ile Asp

585 590 595

ttc tat gct ttt tca ggt cac aaa ctg tat ggc ccg aca ggt atc ggc 2471

Phe Tyr Ala Phe Ser Gly His Lys Leu Tyr Gly Pro Thr Gly Ile Gly

600 605 610 615

gtg ctg tat ggt aaa tca gaa ctg ctg gag gcg atg tcg ccc tgg ctg 2519

Val Leu Tyr Gly Lys Ser Glu Leu Leu Glu Ala Met Ser Pro Trp Leu

620 625 630

ggc ggc ggc aaa atg gtt cac gaa gtg agt ttt gac ggc ttc acg act 2567

Gly Gly Gly Lys Met Val His Glu Val Ser Phe Asp Gly Phe Thr Thr

635 640 645

caa tct gcg ccg tgg aaa ctg gaa gct gga acg cca aat gtc gct ggt 2615

Gln Ser Ala Pro Trp Lys Leu Glu Ala Gly Thr Pro Asn Val Ala Gly

650 655 660

gtc ata gga tta agc gcg gcg ctg gaa tgg ctg gca gat tac gat atc 2663

Val Ile Gly Leu Ser Ala Ala Leu Glu Trp Leu Ala Asp Tyr Asp Ile

665 670 675

aac cag gcc gaa agc tgg agc cgt agc tta gca acg ctg gcg gaa gat 2711

Asn Gln Ala Glu Ser Trp Ser Arg Ser Leu Ala Thr Leu Ala Glu Asp

680 685 690 695

gcg ctg gcg aaa cgt ccc ggc ttt cgt tca ttc cgc tgc cag gat tcc 2759

Ala Leu Ala Lys Arg Pro Gly Phe Arg Ser Phe Arg Cys Gln Asp Ser

700 705 710

agc ctg ctg gcc ttt gat ttt gct ggc gtt cat cat agc gat atg gtg 2807

Ser Leu Leu Ala Phe Asp Phe Ala Gly Val His His Ser Asp Met Val

715 720 725

acg ctg ctg gcg gag tac ggt att gcc ctg cgg gcc ggg cag cat tgc 2855

Thr Leu Leu Ala Glu Tyr Gly Ile Ala Leu Arg Ala Gly Gln His Cys

730 735 740

gct cag ccg cta ctg gca gaa tta ggc gta acc ggc aca ctg cgc gcc 2903

Ala Gln Pro Leu Leu Ala Glu Leu Gly Val Thr Gly Thr Leu Arg Ala

745 750 755

tct ttt gcg cca tat aat aca aag agt gat gtg gat gcg ctg gtg aat 2951

Ser Phe Ala Pro Tyr Asn Thr Lys Ser Asp Val Asp Ala Leu Val Asn

760 765 770 775

gcc gtt gac cgc gcg ctg gaa tta ttg gtg gat taa acgcgtgcta 2997

Ala Val Asp Arg Ala Leu Glu Leu Leu Val Asp

780 785

gaggcatcaa ataaaacgaa aggctcagtc gaaagactgg gcctttcgtt ttatctgttg 3057

tttgtcggtg aacgctctcc tgagtaggac aaatccgccg ccctagacct aggggatata 3117

ttccgcttcc tcgctcactg actcgctacg ctcggtcgtt cgactgcggc gagcggaaat 3177

ggcttacgaa cggggcggag atttcctgga agatgccagg aagatactta acagggaagt 3237

gagagggccg cggcaaagcc gtttttccat aggctccgcc cccctgacaa gcatcacgaa 3297

atctgacgct caaatcagtg gtggcgaaac ccgacaggac tataaagata ccaggcgttt 3357

ccccctggcg gctccctcgt gcgctctcct gttcctgcct ttcggtttac cggtgtcatt 3417

ccgctgttat ggccgcgttt gtctcattcc acgcctgaca ctcagttccg ggtaggcagt 3477

tcgctccaag ctggactgta tgcacgaacc ccccgttcag tccgaccgct gcgccttatc 3537

cggtaactat cgtcttgagt ccaacccgga aagacatgca aaagcaccac tggcagcagc 3597

cactggtaat tgatttagag gagttagtct tgaagtcatg cgccggttaa ggctaaactg 3657

aaaggacaag ttttggtgac tgcgctcctc caagccagtt acctcggttc aaagagttgg 3717

tagctcagag aaccttcgaa aaaccgccct gcaaggcggt tttttcgttt tcagagcaag 3777

agattacgcg cagaccaaaa cgatctcaag aagatcatct tattaatcag ataaaatatt 3837

tctagatttc agtgcaattt atctcttcaa atgtagcacc tgaagtcagc cccatacgat 3897

ataagttgtt actagtgctt ggattctcac caataaaaaa cgcccggcgg caaccgagcg 3957

ttctgaacaa atccagatgg agttctgagg tcattactgg atctatcaac aggagtccaa 4017

gcgagctctc gaaccccaga gtcccgctca gaagaactcg tcaagaaggc gatagaaggc 4077

gatgcgctgc gaatcgggag cggcgatacc gtaaagcacg aggaagcggt cagcccattc 4137

gccgccaagc tcttcagcaa tatcacgggt agccaacgct atgtcctgat agcggtccgc 4197

cacacccagc cggccacagt cgatgaatcc agaaaagcgg ccattttcca ccatgatatt 4257

cggcaagcag gcatcgccat gggtcacgac gagatcctcg ccgtcgggca tgcgcgcctt 4317

gagcctggcg aacagttcgg ctggcgcgag cccctgatgc tcttcgtcca gatcatcctg 4377

atcgacaaga ccggcttcca tccgagtacg tgctcgctcg atgcgatgtt tcgcttggtg 4437

gtcgaatggg caggtagccg gatcaagcgt atgcagccgc cgcattgcat cagccatgat 4497

ggatactttc tcggcaggag caaggtgaga tgacaggaga tcctgccccg gcacttcgcc 4557

caatagcagc cagtcccttc ccgcttcagt gacaacgtcg agcacagctg cgcaaggaac 4617

gcccgtcgtg gccagccacg atagccgcgc tgcctcgtcc tgcagttcat tcagggcacc 4677

ggacaggtcg gtcttgacaa aaagaaccgg gcgcccctgc gctgacagcc ggaacacggc 4737

ggcatcagag cagccgattg tctgttgtgc ccagtcatag ccgaatagcc tctccaccca 4797

agcggccgga gaacctgcgt gcaatccatc ttgttcaatc atgcgaaacg atcctcatcc 4857

tgtctcttga tcagatcttg atcccctgcg ccatcagatc cttggcggca agaaagccat 4917

ccagtttact ttgcagggct tcccaacctt accagagggc gccccagctg gcaattcc 4975

<210> 14

<211> 384

<212> PRT

<213> Escherichia coli

<400> 14

Met Ala Lys His Leu Phe Thr Ser Glu Ser Val Ser Glu Gly His Pro

1 5 10 15

Asp Lys Ile Ala Asp Gln Ile Ser Asp Ala Val Leu Asp Ala Ile Leu

20 25 30

Glu Gln Asp Pro Lys Ala Arg Val Ala Cys Glu Thr Tyr Val Lys Thr

35 40 45

Gly Met Val Leu Val Gly Gly Glu Ile Thr Thr Ser Ala Trp Val Asp

50 55 60

Ile Glu Glu Ile Thr Arg Asn Thr Val Arg Glu Ile Gly Tyr Val His

65 70 75 80

Ser Asp Met Gly Phe Asp Ala Asn Ser Cys Ala Val Leu Ser Ala Ile

85 90 95

Gly Lys Gln Ser Pro Asp Ile Asn Gln Gly Val Asp Arg Ala Asp Pro

100 105 110

Leu Glu Gln Gly Ala Gly Asp Gln Gly Leu Met Phe Gly Tyr Ala Thr

115 120 125

Asn Glu Thr Asp Val Leu Met Pro Ala Pro Ile Thr Tyr Ala His Arg

130 135 140

Leu Val Gln Arg Gln Ala Glu Val Arg Lys Asn Gly Thr Leu Pro Trp

145 150 155 160

Leu Arg Pro Asp Ala Lys Ser Gln Val Thr Phe Gln Tyr Asp Asp Gly

165 170 175

Lys Ile Val Gly Ile Asp Ala Val Val Leu Ser Thr Gln His Ser Glu

180 185 190

Glu Ile Asp Gln Lys Ser Leu Gln Glu Ala Val Met Glu Glu Ile Ile

195 200 205

Lys Pro Ile Leu Pro Ala Glu Trp Leu Thr Ser Ala Thr Lys Phe Phe

210 215 220

Ile Asn Pro Thr Gly Arg Phe Val Ile Gly Gly Pro Met Gly Asp Cys

225 230 235 240

Gly Leu Thr Gly Arg Lys Ile Ile Val Asp Thr Tyr Gly Gly Met Ala

245 250 255

Arg His Gly Gly Gly Ala Phe Ser Gly Lys Asp Pro Ser Lys Val Asp

260 265 270

Arg Ser Ala Ala Tyr Ala Ala Arg Tyr Val Ala Lys Asn Ile Val Ala

275 280 285

Ala Gly Leu Ala Asp Arg Cys Glu Ile Gln Val Ser Tyr Ala Ile Gly

290 295 300

Val Ala Glu Pro Thr Ser Ile Met Val Glu Thr Phe Gly Thr Glu Lys

305 310 315 320

Val Pro Ser Glu Gln Leu Thr Leu Leu Val Arg Glu Phe Phe Asp Leu

325 330 335

Arg Pro Tyr Gly Leu Ile Gln Met Leu Asp Leu Leu His Pro Ile Tyr

340 345 350

Lys Glu Thr Ala Ala Tyr Gly His Phe Gly Arg Glu His Phe Pro Trp

355 360 365

Glu Lys Thr Asp Lys Ala Gln Leu Leu Arg Asp Ala Ala Gly Leu Lys

370 375 380

<210> 15

<211> 401

<212> PRT

<213> Escherichia coli

<400> 15

Met Asn Val Phe Asn Pro Ala Gln Phe Arg Ala Gln Phe Pro Ala Leu

1 5 10 15

Gln Asp Ala Gly Val Tyr Leu Asp Ser Ala Ala Thr Ala Leu Lys Pro

20 25 30

Glu Ala Val Val Glu Ala Thr Gln Gln Phe Tyr Ser Leu Ser Ala Gly

35 40 45

Asn Val His Arg Ser Gln Phe Ala Glu Ala Gln Arg Leu Thr Ala Arg

50 55 60

Tyr Glu Ala Ala Arg Glu Lys Val Ala Gln Leu Leu Asn Ala Pro Asp

65 70 75 80

Asp Lys Thr Ile Val Trp Thr Arg Gly Thr Thr Glu Ser Ile Asn Met

85 90 95

Val Ala Gln Cys Tyr Ala Arg Pro Arg Leu Gln Pro Gly Asp Glu Ile

100 105 110

Ile Val Ser Val Ala Glu His His Ala Asn Leu Val Pro Trp Leu Met

115 120 125

Val Ala Gln Gln Thr Gly Ala Lys Val Val Lys Leu Pro Leu Asn Ala

130 135 140

Gln Arg Leu Pro Asp Val Asp Leu Leu Pro Glu Leu Ile Thr Pro Arg

145 150 155 160

Ser Arg Ile Leu Ala Leu Gly Gln Met Ser Asn Val Thr Gly Gly Cys

165 170 175

Pro Asp Leu Ala Arg Ala Ile Thr Phe Ala His Ser Ala Gly Met Val

180 185 190

Val Met Val Asp Gly Ala Gln Gly Ala Val His Phe Pro Ala Asp Val

195 200 205

Gln Gln Leu Asp Ile Asp Phe Tyr Ala Phe Ser Gly His Lys Leu Tyr

210 215 220

Gly Pro Thr Gly Ile Gly Val Leu Tyr Gly Lys Ser Glu Leu Leu Glu

225 230 235 240

Ala Met Ser Pro Trp Leu Gly Gly Gly Lys Met Val His Glu Val Ser

245 250 255

Phe Asp Gly Phe Thr Thr Gln Ser Ala Pro Trp Lys Leu Glu Ala Gly

260 265 270

Thr Pro Asn Val Ala Gly Val Ile Gly Leu Ser Ala Ala Leu Glu Trp

275 280 285

Leu Ala Asp Tyr Asp Ile Asn Gln Ala Glu Ser Trp Ser Arg Ser Leu

290 295 300

Ala Thr Leu Ala Glu Asp Ala Leu Ala Lys Arg Pro Gly Phe Arg Ser

305 310 315 320

Phe Arg Cys Gln Asp Ser Ser Leu Leu Ala Phe Asp Phe Ala Gly Val

325 330 335

His His Ser Asp Met Val Thr Leu Leu Ala Glu Tyr Gly Ile Ala Leu

340 345 350

Arg Ala Gly Gln His Cys Ala Gln Pro Leu Leu Ala Glu Leu Gly Val

355 360 365

Thr Gly Thr Leu Arg Ala Ser Phe Ala Pro Tyr Asn Thr Lys Ser Asp

370 375 380

Val Asp Ala Leu Val Asn Ala Val Asp Arg Ala Leu Glu Leu Leu Val

385 390 395 400

Asp

Claims (14)

S-adenosylmethionine synthase gene having SEQ ID NO: 1 and at least one additional biotin biosynthetic gene bioS1, bioS2 or bioS3 having SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7, and functional variants, analogs or derivatives thereof, for synthesizing biotin A method for producing biotin, which is expressed in a prokaryotic or eukaryotic host organism, cultured the organism, isolating and removing cells or purifying biotin, and immediately using the synthesized biotin. The method of claim 1 wherein the variant of the gene having SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 and SEQ ID NO: 7 exhibits 30-100% homology at the amino acid level deduced from the sequences claimed in claim 1, and the synthesis of biotin How to increase The method according to claim 1 or 2, wherein Escherichia, Citrobacter, Serratia, Klebsiella, Salmonella, Pseudomonas, and comamonas Comamonas, Acinetobacter, Azotobacter, Chromobacterium, Bacillus, Clostridium, Arthrobacter, Corynebacterium ), Brevibacterium, Lactococcus, Lactobacillus, Streptomyces, Streptomyces, Rhizobium, Agrobacterium, Staphylococcus, Staphylococcus, Staphylococcus A method using as an host organism an organism selected from the group of Rhodotorula, Sporobolomyces, Yarrowia, Schizosaccharomyces or Saccharomyces. 4. The method of claim 1, wherein the regulatory-deficient biotin mutant is used as a host organism. 5. The method of claim 1, wherein one or more copies of the genes having SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, and SEQ ID NO: 7 as claimed in claim 1, are alone in a prokaryotic or eukaryotic host organism. Or with one or more copies of one or more additional biotin genes selected from the group bioA, bioB, bioF, bioC, bioD, bioH, bioP, bioW, bioX, bioY or bioR. The method of any one of claims 1 to 5, wherein one or more copies of the genes having SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, and SEQ ID NO: 7 as claimed in claim 1 are solely expressed in prokaryotic or eukaryotic host organisms. Or in a shared vector or in a separate vector with one or more copies of one or more additional biotin genes selected from the group bioA, bioB, bioF, bioC, bioD, bioH, bioP, bioW, bioX, bioY or bioR. An S-adenosylmethionine synthetase gene having SEQ ID NO: 1 and one or more additional biotin biosynthetic genes bioS12, bioS2 or bioS3 having SEQ ID NO: 3, SEQ ID NO: 5 and SEQ ID NO: 7, and functional variants, analogs or derivatives thereof, A gene construct that is functionally linked to (or connected to) one or more regulatory signals and whose natural regulation is turned off to increase gene expression and / or protein expression. The genetic construct of claim 7 which is inserted into a vector suitable for expressing the gene in a prokaryotic or eukaryotic host organism. The gene construct of claim 7 or 8, wherein the genes having SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, and SEQ ID NO: 7, and functional variants, analogs or derivatives thereof, are present in the gene construct in several copies. 10. The biotin biosynthesis gene of any one of claims 7 to 9, wherein the gene is the S-adenosylmethionine synthase gene as claimed in claim 7, and at least one additional biotin biosynthesis gene having SEQ ID NO: 3 bioS1, bioS2 or bioS3, and functional variants, analogs or derivatives thereof, with one or more copies of one or more additional genes selected from the group bioA, bioB, bioF, bioC, bioD, bioH, bioP, bioW, bioX, bioY or bioR Gene constructs present in a gene construct or vector together. An organism comprising a construct that is a gene as claimed in claim 7. Use of the sequence as claimed in claim 1 for producing biotin. The bioS3 gene having SEQ ID NO: 7 or a functional variant, analog or derivative thereof, or the S-adenosylmethionine synthase gene bioS1, bioS2, bioA, bioB, bioF, bioC, bioD, bioH, bioP, bioW, bioX, bioY or Use for biotin production of a bioS3 gene having SEQ ID NO: 7 or a functional variant, analog or derivative thereof in combination with one or more additional genes selected from the bioR group. Use of a gene construct as claimed in any one of claims 7 to 10 for producing biotin.