CA2321264A1 - Method for producing biotin - Google Patents
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- CA2321264A1 CA2321264A1 CA002321264A CA2321264A CA2321264A1 CA 2321264 A1 CA2321264 A1 CA 2321264A1 CA 002321264 A CA002321264 A CA 002321264A CA 2321264 A CA2321264 A CA 2321264A CA 2321264 A1 CA2321264 A1 CA 2321264A1
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
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- C12N9/1085—Transferases (2.) transferring alkyl or aryl groups other than methyl groups (2.5)
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/13—Transferases (2.) transferring sulfur containing groups (2.8)
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
- C12P17/18—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing at least two hetero rings condensed among themselves or condensed with a common carbocyclic ring system, e.g. rifamycin
- C12P17/185—Heterocyclic compounds containing sulfur atoms as ring hetero atoms in the condensed system
- C12P17/186—Heterocyclic compounds containing sulfur atoms as ring hetero atoms in the condensed system containing a 2-oxo-thieno[3,4-d]imidazol nucleus, e.g. Biotin
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Abstract
The invention relates to a genetic recombination product, containing an S-adenosyle-methionine-synthase gene, with the sequence SEQ ID no. 1 and a biotin biosynthesis gene bioS1, bioS2 and/or bioS3 with the sequences SEQ ID
no. 3, SEQ ID no. 5 or SEQ ID no.7 and possibly at least one additional biotin synthesis gene sequence chosen from the group bioA, bioB, bioF, bioC, bioD, bioH, bioP, bioW, bioX, bioY or bioR. The invention also relates to organisms containing said genetic recombination product and to the use of same for producing biotin, as well as to a method for producing biotin.
no. 3, SEQ ID no. 5 or SEQ ID no.7 and possibly at least one additional biotin synthesis gene sequence chosen from the group bioA, bioB, bioF, bioC, bioD, bioH, bioP, bioW, bioX, bioY or bioR. The invention also relates to organisms containing said genetic recombination product and to the use of same for producing biotin, as well as to a method for producing biotin.
Description
METHOD FOR PRODUCING BIOTIN
The invention relates to a gene construct which contains an S-adenosylmethionine synthase gene, having the sequence SEQ ID
No. 1, and a biotin biosynthesis gene bioSl, bioS2 and/or bioS3, having the sequences SEQ ID No.3, SEQ ID No.5 and SEQ ID No.7, respectively, and, where appropriate, at least one further biotin synthesis gene sequence selected from the group bioA, bioB, bioF, bioC, bioD, bioH, bioP, biota, bioX, bioY or bioR. The invention furthermore relates to organisms which contain this gene construct and to the use of the gene construct for preparing biotin, and also to a process for preparing biotin.
As a coenzyme, biotin (Vitamin H) plays an essential role in enzyme-catalyzed carboxylation and decarboxylation reactions.
Biotin is consequently an essential factor in living cells.
Almost all animals and some microorganisms have to take up biotin from the exterior since they are unable to synthesize biotin themselves. Biotin is therefore an essential vitamin for these organisms. By contrast, bacteria, yeasts and plants are able themselves to synthesize biotin from precursors (Brown et al.
Biotechnol. Genet. Eng. Rev. 9, 1991: 295 - 326, DeMoll, E., Escherichia coli and Salmonella, eds. Neidhardt, F. C. et al. ASM
Press, Washington DC, USA, 1996: 704 - 708, ISBN 1-55581-084-5).
The synthesis of biotin has been investigated in bacterial organisms, especially in the Gram-negative bacterium Escherichia coli and in the Gram-positive bacterium Bacillus sphaericus (Brown et al. Biotechnol. Genet. Eng. Rev. 9, 1991: 295 - 326).
Pimelyl-CoA (PmCoA), which is derived from fatty acid synthesis, has previously been regarded as the first known intermediate in E. coli (DeMoll, E., Escherichia coli and Salmonella, eds.
Neidhardt, F. C. et al. ASM Press, Washington DC, USA, 1996: 704 - 708, ISBN 1-55581-084-5 1996). Up to now, the route by which this biotin precursor is synthesized in E. coli has to a large extent been unknown (Lemoine et al., Mol. Microbiol. 19, 1996 645 - 647). bioC and bioH have been identified as being two genes whose corresponding proteins are responsible for the synthesis of Pm-CoA. The enzymic functions of the gene products, i.e. BioH and BioC, have hitherto been unknown (Lemoine et al., Mol. Microbiol.
19, 1996: 645 - 647, DeMOll, E., Escherichia coli and Salmonella, eds. Neidhardt, F. C. et al. ASM Press, Washington DC, USA, 1996:
704 - 708, ISBN 1-55581-084-5). Pm-CoA is converted into biotin in four further enzymic steps. BioF first of all condenses the Pm-CoA with alanine to form 7-keto-8-aminopelargonic acid (KAPA).
The KAPA is then converted into 7,8-diaminopelargonic acid (DAPA) 0050/48792 CA 02321264 2000-os-i4 a 2 by BioA. Following an ATP-dependent carboxylation reaction, the next step leads to dethiobiotin (DTB) and is catalyzed by BioD.
The DTB is converted into biotin in the last step. This step is catalyzed by BioB. The chemical and enzymic mechanisms involved in the conversion of DTB into biotin are so far only incompletely understood and clarified.
The conversion of DTB into biotin has so far only been characterized in bacterial and plant cell extracts (W094/8023, EP-B-0 449 724, Sanyal et al. Arch. Biochem. Biophys., Vol. 326, No. 1, 1996: 48 - 56 and Biochemistry 33, 1994: 3625 - 3631, Baldet 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 demonstrated that low molecular weight factors such as NADPH, cysteine, thiamine, Fe2+, asparagine, serine, fructose 1-6-bisphosphate and S-adenosylmethionine are able to stimulate the synthesis of biotin (Ohshiro et al., Biosci. Biotechnol: Biochem., 58, 9, 1994: 1738 - 1741, Birch et al., J. Biol. Chem. 270, 32, 1995: 19158 - 19165, Ifuku et al., Biosci. Biotechnol. Biochem., 59, 2, 1995: 185 - 189, Sanyal et al. Arch. Biochem. Biophys. 326, 1, 1996: 48 - 56).
In addition to these low molecular weight factors, other proteins have been identified which stimulate the synthesis of biotin from DTB in the presence of BioB. These proteins are flavodoxin and flavodoxin NADPH reductase (Birch et al., J. Biol. Chem. 270, 32, 1995: 19158 - 19165, Ifuk et al., Biosci. Biotechnol. Biochem., 59, 2, 1995: 185 - 189, Sanyal et al., Arch. Biochem. Biophys.
326, 1, 1996: 48 - 56). Other proteins which stimulate biotin synthesis are the genes bioS1 and bioS2, which are described in the German application having the application number 197.31274.8 (Priority 22.7.97).
Differing results have been obtained with regard to the origin of the sulfur in the biotin molecule. Investigations into the synthesis of biotin in whole cell extracts showed that radioactivity was incorporated into biotin in the presence of 35S-labeled cysteine; it was not possible to demonstrate incorporation of sulfur into the biotin molecule when either 3sS-labeled methionine or S-adenosylmethionine was used (Ifuku et al., Biosci. Biotechnol. Biochem. 59, 2, 1995: 184 - 189, Birch et al., J. Biol. Chem.270, 32, 1995: 19158 - 19165).
0050/48792 CA 02321264 2000-os-i4 The genes which encode the described proteins, i.e. bioF, bioA, bioD, and bioB, are encoded in E. coli on a bidirectional operon.
This operon is located between the ~, attachment site and the uvrB
gene locus at approx. 17 minutes on the E. coli chromosome (Berlyn et al. 1996: 1715 - 1902). A further two genes, one of which, i.e. bioC, already possesses described functions in the synthesis of Pm-CoA, are additionally encoded on this operon, whereas it has not so far been possible to assign any function to an open reading frame which is located downstream of bioA
(W094/8023, Otsuka et al., J. Biol. Chem. 263, 1988: 19577 - 85).
Highly conserved homologues to the E. coli proteins BioF, BioA, BioD and BioB have been found in B. sphaericus, B. subtilis, Syneccocystis sp. (Brown et al. Biotechnol. Genet. Eng. Rev. 9, 1991: 295 - 326, Bower et al., J. Bacteriol. 175, 1996: 4122 -4130, Kaneko et al., DNA Res. 3, 3, 1996: 109 - 136), archaeobacteria such as Methanococcus janaschi, and yeasts such as Saccharomyces cerevisiae (Zhang et al., Arch. Biochem.
Biophys. 309, 1, 1994: 29 - 35) or in plants such as Arabidopsis thaliana (Baldet et al., C. R. Acad. Sci. III, Sci. Vie. 319, 2, 1996: 99 - 106).
In the two Gram-positive microorganisms which have so far been investigated, the synthesis of Pm-CoA appears to proceed in a different manner from that in E. coli. It was not possible to find any homologues of bioH and bioC (Brown et al. Biotechnol.
Genet. Eng. Rev. 9, 1991: 295 - 326).
Biotin is an optically active substance which has three centers of chirality. It has hitherto only been prepared economically by way of an expensive, multi-step chemical synthesis.
As an alternative to this chemical synthesis, a large number of attempts have been made to construct a fermentative process for preparing biotin using microorganisms. Cloning the biotin operon onto multi-copy-plasmids has been successfully used to increase biotin synthesis in microorganisms which have been transformed with these genes. A further increase in biotin synthesis was achieved by deregulating biotin gene expression by means of selecting birA mutants (Pai C. H., J. Bacteriol. 112, 1972: 1280 ' 1287). Combination of the two approaches, that is expressing the plasmid-encoded biosynthesis genes in a regulation-deficient strain (EP-B-0 236 429), increased productivity still further. In this context, the biotin operon can either remain under the control of its native bidirectional promoter (EP-B-0 236 429) or else its genes can be brought under the control of a promoter which can be regulated externally (w094/08023).
0050/48792 CA 02321264 2000-os-i4 The approaches which have so far been pursued for producing biotin fermentatively in E. coli have not achieved any economically adequate productivity.
It is an object of the present invention to develop an industrial fermentative process for producing biotin which exhibits as high a biotin synthesis as possible.
We have found that this object is achieved by the process according to the invention for producing biotin, in which process an S-adenosylmethionine synthase (SAM synthase) gene, having the sequence SEQ ID No. 1, and at least one further biotin biosynthesis gene bioSl, bioS2 or bioS3, having the sequences SEQ
ID No. 3, SEQ ID No. 5 and SEQ ID No.7, and also their functional variants, analogues or derivatives, are expressed in a prokaryotic or eukaryotic host organism which is able to synthesize biotin, this organism is cultured and the synthesized biotin is used directly after separating off the biomass or after purifying the biotin.
The genes used in the process according to the invention, i.e.
the SAM synthase gene having the sequence SEQ ID No. 1 and the biotin biosynthesis genes bioSl, bioS2 and bioS3 having the sequences SEQ ID No. 3, SEQ ID No. 5 and SEQ ID No.7 are kept in the SwissProt-data base under accession numbers P04384 (metK), U29581 (bioSl), P39171 (bioS2) and D90811 (bioS3). A number of homologues to E. coli MetK are described in the data base. These homologues include organisms such as other eubacteria (e.g. H.
influenzae, and B. subtilis), and also eukaryotes (e. g. yeasts:
S. cerevisiae, Planta: P. deltoides, Arthropoda: D. melanogaster, and Mammalia: R. norvegicus).
The productivity of the biotin biosynthesis can be increased markedly by expressing one or more of the SAM synthase gene, having the sequence SEQ ID No. l, and its functional variants, analogues or derivatives in combination with at least one of the biotin synthesis genes bioSl, bioS2 or bioS3, having the sequences SEQ ID No. 3, SEQ ID No. 5 and SEQ ID No.7, and also their functional variants, analogues or derivatives, in a prokaryotic or eukaryotic host organism. A combination of the SAM
synthase gene and biosl is preferably used for the expression. At least one further biotin gene selected from the group bioA, bioB, bioF, bioC, bioD, bioH, bioP, biota, bioX, bioY and bioR is advantageously expressed at the same time in order to increase the biotin synthesis still further. Expression of the genes increases the synthesis of biotin by at least a factor of 2 as compared with the control without these genes, preferably by a 0050/48792 CA 02321264 2000-os-i4 factor which is greater than 3.
The genes used in the process according to the invention, i.e.
the SAM synthase gene having the nucleotide sequence SEQ ID No.
5 1, the bioSl gene having the nucleotide sequence SEQ ID No. 3, the bioS2 gene having the nucleotide sequence SEQ ID No. 5 and the bioS3 gene having the nucleotide sequence SEQ ID No.7, which sequences encode the amino acid sequences given in SEQ ID N0: 2, SEQ ID No. 4, SEQ ID No. 6 and SEQ ID No.8, respectively, or their allelic variations, can be obtained following isolation and sequencing. Variants are to be understood as being SEQ ID No. 1, SEQ ID No.3, SEQ ID No.5 and SEQ ID No.7 variants, respectively, which exhibit from 30 to 100% homology at the amino acid level, preferably from 50 to 100% homology, very particularly preferably from 80 to 100% homology. Allelic variants comprise, in particular, functional variants which can be obtained by the deletion, insertion or substitution of nucleotides from the sequences depicted in SEQ ID N0: 1, SEQ ID No. 3, SEQ ID No. 5 and SEQ ID No.7, with, however, the enzymic activity being retained.
In addition, variants are also to be understood as being functional equivalents of the genes, such as O-acetylserine sulfohydrolase A, O-acetylserine sulfohydrolase B, ~-cystathionase (see Flint et al., J. Biol. Chem., Vol. 271, 1996: 16053 - 16067) or nifS and its prokaryotic and eukaryotic homologues, for example from Klebsiella, Candida, yeasts or Caenorhabditis, which are able to assume the enzymic activity of bioSl, bioS2 or bioS3 in the synthesis of biotin.
Functional analogues of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID N0: 5 and SEQ ID No. 7 are to be understood as being, for example, their prokaryotic or eukaryotic homologues, such as bacterial, fungal, plant, animal or human homologues. In addition, analogues are also to be understood as being truncated sequences, or single-stranded DNA or RNA from coding and non-coding DNA
sequences.
Derivatives are to be understood, for example, as being promoter variants. The promoters, which are placed upstream of the given nucleotide sequences, can be altered by means of one or more nucleotide substitutions, or by means of (an) insertions) and/or deletions) without, however, the functionality or activity of the promoters being impaired. In addition, the activities of the promoters can be increased by means of altering their sequences, or the promoters can be completely replaced by more active 0050/48792 CA 02321264 2000-os-i4 promoters, including those from organisms of a different species.
Derivatives are also to be understood as being variants whose nucleotide sequences have been altered in the region from -1 to -30 upstream of the start codon such that expression of the gene and/or expression of a protein is increased. This is advantageously effected by altering the Shine-Dalgarno sequence.
All Gram-negative or Gram-positive bacteria which synthesize biotin are, in principal, suitable for use as prokaryotic host organisms in the process according to the invention.
Gram-negative bacteria which may be mentioned by way of example are Enterobacteriaceae such as the genera Escherichia, Aerobacter, Enterobacter, Citrobacter, Shigella, Klebsiella, Serratia, Erwinia or Salmonella, Pseudomonadaceae such as the genera Pseudomonas, Xanthomonas, Burkholderia, Gluconobacter, Nitrosomonas, Nitrobacter, Methanomonas, Comamonas, Cellulomonas or Acetobacter, Azotobacteraceae such as the genera Azotobacter, Azomonas, Beijerinckia or Derxia, Neisseriaceae such as the genera Moraxella, Acinetobacter, Kingella, Neisseria or Branhamella, the Rhizobiaceae such as the genera Rhizobium or Agrobacterium, or the Gram-negative genera Zymomonas, Chromobacterium or Flavobacterium. Gram-positive bacteria which may be mentioned by way of example are the endospore-forming Gram-positive aerobic or anaerobic bacteria such as the genera Bacillus, Sporolactobacillus or Clostridium, the coryneform bacteria such as the genera Arthrobacter, Cellulomonas, Curtobacterium, Corynebacterium, Brevibacterium, Microbacterium or Kurthia, the Actinomycetales such as the genera Mycobacterium, Rhodococcus, Streptomyces or Nocardia, the Lactobacillaceae such as the genera Lactobacillus or Lactococcus, or the Gram-positive cocci such as the genera Micrococcus or Staphylococcus.
Preference is given to using bacteria of the genera Escherichia, Citrobacter, Serratia, Klebsiella, Salmonella, Pseudomonas, Comamonas, Acinetobacter, Azotobacter, Chromobacterium, Bacillus, Clostridium, Arthrobacter, Corynebacterium, Brevibacterium, Lactococcus, Lactobacillus, Streptomyces, Rhizobium, Agrobacterium or Staphylococcus in the process according to the invention. Particular preference is given to genera and species such as Escherichia coli, Citrobacter freundii, Serratia marcescens, Salmonella typhimurium, Pseudomonas mendocina, Pseudomonas aeruginosa, Pseudomonas mutabilis, Pseudomonas chlororaphis, Pseudomonas fluorescens, Comamonas acidovorans, Comamonas testosterone, Acinetobacter calcoaceticus, Azotobacter vinelandii, Chromobacterium violaceum, Bacillus subtilis, Bacillus sphaericus, Bacillus stearothermophilus, Bacillus 0050/48792 CA 02321264 2000-os-i4 pumilus, Bacillus licheniformis, Bacillus amyloliquefaciens, Bacillus megaterium, Bacillus cereus, Bacillus thuringiensis, Arthrobacter citreus, Arthrobacter paraffineus, Corynebacterium glutamicum, Corynebacterium primorioxydans, Corynebacterium sp., Brevibacterium ketoglutamicum, Brevibacterium linens, Brevibacterium sp., Streptomyces lividans, Rhizobium leguminosarum or Agrobacterium tumefaciens. Advantageously, use is made of bacteria which already exhibit an elevated natural production of biotin.
The taxonomic position of the listed genera has been subject to considerable change in recent years and is still in a state of flux as false genera and species names are corrected. Because of these taxonomic regroupings, which have been frequently required in the past, of the said genera within bacterial systematics, families, genera and species other than those mentioned above are also suitable for the process according to the invention.
All biotin-synthesizing organisms, such as fungi, yeasts, plants or plant cells, are, in principal, suitable for use as eukaryotic host organisms in the process according to the invention. Yeasts which may preferably be mentioned are the genera Rhodotorula, Yarrowia, Sporobolomyces, Saccharomyces or Schizosaccharomyces.
Particular preference is given to the genera and species Rhodotorula rubra, Rhodotorula glutinis, Rhodotorula graminis, Yarrowia lipolytica, Sporobolomyces salmonicolor, Sporobolomyces shibatanus or Saccharomyces cerevisiae.
In principal, all plants can be used as the host organism, with preference being given to plants which play a role in animal nutrition or human nutrition, such as corn, wheat, barley, rye, potatoes, peas, beans, sunflowers, palms, millet, sesame, copra or rape. 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, yeasts or plant cells which are able to secrete biotin into the growth medium, and which, where appropriate, already additionally exhibit an increased natural synthesis of biotin, are advantageously used in the process according to the invention. Advantageously, these organisms can also be defective with regard to the regulation of their biotin biosynthesis: i.e. this synthesis is either not regulated or only regulated to a very reduced extent. This regulatory defect results in these organisms already possessing a substantially increased biotin productivity. Such a regulatory defect is known, for example, from Escherichia coli in the form 0050/48792 CA 02321264 2000-os-i4 of birA-defect mutants and should preferably be present in the cells as a defect which can be induced by external influences, for example as a defect which is temperature-inducible. In principal, organisms which do not exhibit any natural biotin production can also be used, once they have been transformed with the biotin genes.
In order to increase biotin productivity as a whole still further, the organisms in the process according to the invention should advantageously also harbor at least one further biotin gene selected from the group bioA, bioB, bioF, bivC, bioD, bioH, bioP, biota, bioX, bioY or bioR. Advantageously, those genes which stimulate biotin synthesis can also be present in the cell in combination with the sequences SEQ ID No. 1 , SEQ ID No. 3, SEQ
ID No.5 or SEQ ID No.7 and their combinations. Examples of genes which stimulate biotin synthesis are the flavoredoxin gene and the flavoredoxin reductase gene. This additional gene, or these additional genes, can be present in the cell in one or more copies, like the genes having the sequences SEQ ID No. 1 , SEQ ID
No.3, SEQ ID No.S or SEQ ID No.7 or their combinations. They can be located on the same vector as the sequences SEQ ID No. 1, SEQ
ID No.3, SEQ ID No.5 and/or SEQ ID No.7, or on separate vectors, or else integrated chromosomally. The sequences SEQ ID No. 1, SEQ
ID No.3, SEQ ID No.5 and/or SEQ ID No.7 can also be together on a vector or on separate vectors or be inserted into the genome.
The gene construct according to the invention is to be understood as being the gene sequences of the SAM synthase gene SEQ ID No. 1 and of the biotin synthesis genes SEQ ID No.3, SEQ ID No.5 and/or SEQ ID No.7, and also their functional variants, analogues or derivatives, which were linked functionally to one or more regulatory signals for the purpose of increasing expression of the genes. In addition to these new regulatory sequences, the natural regulation of these sequences can still be present upstream of the actual structural genes and, where appropriate, can have been genetically altered such that the natural regulation has been switched off and expression of genes has been increased. However, the gene construct can also be assembled in a simpler manner, i.e. no additional regulatory signals are inserted upstream of the sequences SEQ ID No. 1, SEQ ID No. 3, SEQ ID No.5 and/or SEQ ID No.7 and the natural promoter, with its regulation, is not removed. Instead, the natural regulatory sequence is mutated such that regulation by biotin no longer takes place and gene expression is increased. The sequences SEQ
ID No. 1, SEQ ID No.3, SEQ ID No.S and/or SEQ ID No.7 can be under the regulation of one promoter or under the regulation of separate promoters. Additional, advantageous regulatory elements can also be inserted at the 3' end of the DNA sequences. The 0050/48792 CA 02321264 2000-os-i4 genes having the sequences SEQ ID No. 1, SEQ ID No. 3, SEQ ID
No.5 or SEQ ID No. 7 can be present in the gene construct in one or more copies.
Advantageous regulatory sequences for the process according to the invention are present, for example, in promoters such as the cos-, tac-, trp-, tet-, trp-tet-, lpp-, lac-, lpp-lac-, lacIq--T7-, T5-, T3-, gal-, trc-, ara-, SP6-, ~,-PR- or ~.-PL-promoters, which are advantageously used in Gram-negative bacteria. Further advantageous regulatory sequences are present, for example, in the Gram-positive promoters amy and SP02, in the yeast promoters ADC1, MFa , 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 the phaseolin promoter.
In principal, all natural promoters, together with their regulatory sequences, can be used, like the abovementioned promoters, for the process according to the invention. In addition, synthetic promoters can also advantageously be used.
Other biotin genes selected from the group bioA, bioB, bioF, bioC, bioD, bioH, bioP, biota, bioX, bioY or bioR, which genes can have their own promoter or else can be under the regulation of the promoter of one of the sequences, or under the regulation of the promoter of all the sequences, SEQ ID No. 1, SEQ ID No. 3, SEQ ID No.S or SEQ ID No.7, can be present in the gene construct in one or more copies.
For expression in the abovementioned host organism, the gene construct is advantageously inserted into a host-specific vector which makes it possible to achieve optimum expression of the genes in the host. Vectors are well known to the skilled person and can be identified, for example, from the book Cloning Vectors (Eds. Pouwels P. H. et al. Elsevier, Amsterdam-New York-Oxford, 1985, ISBN 0 444 904018). In addition to plasmids, the vectors are also to be understood as being all other vectors known to the skilled person, such as phages, viruses, transposons, IS
elements, phasmids, cosmids or linear or circular DNA. These vectors can be replicated autonomously in the host organism or replicated chromosomally.
Expression systems are to be understood as being the combination of the host organisms which are mentioned above by way of example and the vectors which are appropriate for the organisms, such as plasmids, viruses or phages, for example plasmids containing the RNA polymerase/promoter system, phages ~,, or Mu or other temperate phages or transposons and/or further advantageous regulatory 0050/48792 CA 02321264 2000-os-i4 . , ,.
sequences.
The term expression systems is preferably to be understood as being the combination of Escherichia coli and its plasmids and 5 phages and the affiliated promoters, and also Bacillus and its plasmids and promoters.
Further 3' and/or 5'-terminal regulatory sequences are also 10 suitable for advantageously expressing SEQ ID No. l, SEQ ID No.3, SEQ ID No.5 and/or SEQ ID No. 7 in accordance with the invention.
These regulatory sequences are intended to make it possible to achieve specific expression of the biotin genes and expression of the protein. Depending on the host organism, this can, for example, mean that the gene is only expressed or overexpressed after induction or that it is expressed and/or overexpressed immediately.
In this context, the regulatory sequences or factors can preferably influence biotin gene expression positively and thereby increase it. For example, the regulatory elements can advantageously be reinforced at the transcriptional level by means of using strong transcription signals such as promoters and/or enhancers. In addition, however, it is also possible to reinforce translation by, for example, improving the stability of the mRNA.
Enhancers are to be understood as being, for example, DNA
sequences which bring about increased biotin gene expression by means of improving the interaction between the RNA polymerase and the DNA.
~ increase in the proteins (see SEQ ID No.2, SEQ ID No.4, SEQ ID
No.6 and 5EQ ID No.8) which are derived from the sequences SEQ ID
No. 1, SEQ ID No.3, SEQ ID No.5 and SEQ ID No.7, and in their enzyme activity, as compared with the starting enzymes, can be achieved, for example, by altering the corresponding gene sequences, or the sequences of their homologues, by means of classical mutagenesis, such as W irradiation, or by treating with chemical mutagens and/or by means of specific mutagenesis such as site-directed mutagenesis, deletion(s), insertions) and/or substitution(s). An increased enzyme activity, apart from the described gene amplification, can also be achieved by eliminating factors which repress enzyme biosynthesis and/or by synthesizing active enzymes instead of inactive enzymes.
0050/48792 CA 02321264 2000-os-i4 The process according to the invention advantageously increases the conversion of DTB into biotin, and consequently overall biotin productivity, by means of using the biotin genes having the sequences SEQ ID No. 1, SEQ ID No.3, SEQ ID No.5 and SEQ ID
No.7, and the combination of the genes having the sequences SEQ
ID No.1 and SEQ ID No.5 or SEQ ID No.1 and SEQ ID No.7, preferably the combination of the genes having the sequences SEQ
ID No.1 and SEQ ID No.3, which genes are introduced into the organisms by way of their vectors and/or by means of chromosomal cloning.
In the process according to the invention, the microorganisms harboring SEQ ID No.l, SEQ ID No.3, SEQ ID No.S and/or SEQ ID
No.7 are propagated in a medium which enables these organisms to grow. This medium can be a synthetic medium or a natural medium.
Use is made of media which are known to the skilled person and which are appropriate for the organism. In order to permit growth of the microorganisms, the media employed contain a carbon source, a nitrogen source, inorganic salts and, where appropriate, small quantities of vitamins and trace elements.
Examples of advantageous carbon sources are sugars, such as monosaccharides, disaccharides or polysaccharides, such as glucose, fructose, mannose, xylose, galactose, ribose, sorbose, ribulose, lactose, maltose, sucrose, raff inose, starch or cellulose, complex sugar sources such as molasses, sugar phosphates, such as fructose-1,6-bisphosphate, sugar alcohols, such as mannitol, polyols, such as glycerol, alcohols, such as methanol or ethanol, carboxylic acids, such as citric acid, lactic acid of acetic acid, fats, such as soy-bean oil or rapeseed oil, or amino acids, such as glutamic acid or aspartic acid, or amino sugars, which can simultaneously be used as a nitrogen source.
Advantageous nitrogen sources are organic or inorganic nitrogen compounds or materials which contain these compounds. Examples are ammonium salts, such as NH4C1 or (NH4)ZS04, nitrates or urea, or complex nitrogen sources such as corn steep liquor, brewer's yeast autolysate, soy-bean flour, wheat gluten, yeast extract, meat extract, casein hydrolysate or yeast or potato protein, which can frequently also be used simultaneously as a nitrogen source.
Examples of inorganic salts are the salts of calcium, magnesium, sodium, manganese, potassium, zinc, copper and iron. Anions of these salts which are to be mentioned in particular are the chloride, sulfate and phosphate ions. An important factor for 0050/48792 CA 02321264 2000-os-i4 increasing productivity in the process according to the invention is the addition of Fe2+ or Fe3+ salts and/or potassium salts to the production medium.
Where appropriate, further growth factors, such as vitamins or growth promoters, such as riboflavin, thiamine, folic acid, nicotinic acid, pantothenate or pyridoxine, amino acids, such as alanine, cysteine, asparagine, aspartic acid, glutamine, serine, methionine or lysine, carboxylic acids, such as citric acid, formic acid, pimelic acid or lactic acid, or substances such as dithiothreitol, are added to the nutrient medium.
Antibiotics can, where appropriate, be added to the medium in order to stabilize the biotin gene-containing vectors in the Cells.
The ratios in which the said nutrients are mixed depends on the nature of the fermentation and is laid down in each individual case. The medium components can all be initially introduced at the beginning of fermentation, after they have been, if necessary, sterilized separately or sterilized together, or else be added subsequently, as required, during fermentation.
The culture conditions are so arranged that the organisms grow optimally and that the best possible yields are achieved.
Preferred culture temperatures are from 15 °C to 40 °C.
Temperatures of between 25 °C and 37 °C are particularly advantageous. The pH is preferably kept in a range of from 3 to 9. pH values of between 5 and 8 are particularly advantageous. In general, a period of incubation of from 8 to 240 hours, preferably of from 8 to 120 hours, is sufficient. Within this time, the maximum quantity of biotin accumulates in the medium and/or is available after the cells have been disrupted.
The process according to the invention for producing biotin can be carried out continuously or batch-wise or fed-batch-wise. If whole plants are regenerated from the plant cells which have been transformed with the biotin genes, they can, according to the process according to the invention, be grown and propagated perfectly normally.
Examples:
1' Cloning of the S-adenosylmethionine synthase gene (SEQ ID
No.1):
Starting from genomic E. coli DNA, the gene which encodes SAM
syuthase (metK) was amplified from the E. coli chromosome by ' means of a polymerase chain reaction using two specific oligonucleotides. The DNA which had been amplified in this way was purified, digested with the restriction enzyme Acc65I and inserted into a vector which had been cut with the same enzyme and which enables the gene to be overexpressed in E. coli strains. One of the two oligonucleotides was used to provide the gene construct with optimized translation signals.
a.) Generation of oligonucleotides for amplifying the metK gene from the E. coli chromosome:
metK was to be amplified as an expression cassette which was composed of a ribosome binding site, the start codon of the coding sequence and the stop codon between two restriction enzyme recognition sites. The Acc65I recognition sequence was chosen for both the restriction sites. The metK gene was amplified and cloned using the nucleotides PmetK1 (5'-GCGGTACCAGGTGATATTAAATATGGCAAAAC-3') and PmetK2 (5'-CGGGTACCGATTACTTCAGACCGGCAGC-3').
b.) Implementation of the PCR:
Conditions:
0.5 ~g chromosomal DNA from E. coli W3110 was used as a template.
The oligonucleotides PmetKl and PmetK2 were employed at a concentration of in each case 15 pMol. The concentration of the dNTPs was 200 ~M. 2.5 U of Pwo DNA polymerase (Boehringer Mannheim) in the manufacturer's reaction buffer were employed as the polymerase. The PCR reaction volume was 100 ~.1.
~Plifications:
The DNA was denatured at 94 °C for 2 min. The oligonucleotides were then annealed at 55 °C for 30 seconds. The elongation took place at 72 °C for 75 seconds. The PCR reaction was carried out over 30 cycles.
The resulting DNA product, which had a size of approximately 1145 bp, was purified and digested with Acc65I in a suitable buf fer .
0050/48792 CA 02321264 2000-os-i4 c.) Cloning of metK in an expression vector 2 ~,g of the vector pHS1 (construction was described in DE
197.31274.8, priority 22.7.97, Example 1, pages 14 to 17) were digested with Acc65I and dephosphorylated using shrimp alkaline phosphatase (SAP) (Boehringer Mannheim). After the SAP had been denatured, vector and fragment were ligated, in a molar ratio of 1:3, using the Rapid DNA Ligation kit in accordance with the manufacturer's instructions. The ligation mixture was transformed into strain E. coli XL-1-blue. Positive clones were identified by plasmid preparation and restriction analysis. The correct orientation of the metK fragment in pHS1 was determined by restriction digestion and sequencing. The resulting construct was designated pHSl metK (Figure 1). The sequence of pHS1 metK is given in SEQ ID No.9. SEQ ID No.lO shows the amino acid sequence which is deduced from the metK-encoding region.
2. Construction of plasmids pHBbiol4 and pHS1 bioS1 The construction of plasmids pHBbiol4 and pHS1 bioSl has already been described (DE 197.31274.8, Priority 22.7.97, Examples 1, 2 and 5).
The invention relates to a gene construct which contains an S-adenosylmethionine synthase gene, having the sequence SEQ ID
No. 1, and a biotin biosynthesis gene bioSl, bioS2 and/or bioS3, having the sequences SEQ ID No.3, SEQ ID No.5 and SEQ ID No.7, respectively, and, where appropriate, at least one further biotin synthesis gene sequence selected from the group bioA, bioB, bioF, bioC, bioD, bioH, bioP, biota, bioX, bioY or bioR. The invention furthermore relates to organisms which contain this gene construct and to the use of the gene construct for preparing biotin, and also to a process for preparing biotin.
As a coenzyme, biotin (Vitamin H) plays an essential role in enzyme-catalyzed carboxylation and decarboxylation reactions.
Biotin is consequently an essential factor in living cells.
Almost all animals and some microorganisms have to take up biotin from the exterior since they are unable to synthesize biotin themselves. Biotin is therefore an essential vitamin for these organisms. By contrast, bacteria, yeasts and plants are able themselves to synthesize biotin from precursors (Brown et al.
Biotechnol. Genet. Eng. Rev. 9, 1991: 295 - 326, DeMoll, E., Escherichia coli and Salmonella, eds. Neidhardt, F. C. et al. ASM
Press, Washington DC, USA, 1996: 704 - 708, ISBN 1-55581-084-5).
The synthesis of biotin has been investigated in bacterial organisms, especially in the Gram-negative bacterium Escherichia coli and in the Gram-positive bacterium Bacillus sphaericus (Brown et al. Biotechnol. Genet. Eng. Rev. 9, 1991: 295 - 326).
Pimelyl-CoA (PmCoA), which is derived from fatty acid synthesis, has previously been regarded as the first known intermediate in E. coli (DeMoll, E., Escherichia coli and Salmonella, eds.
Neidhardt, F. C. et al. ASM Press, Washington DC, USA, 1996: 704 - 708, ISBN 1-55581-084-5 1996). Up to now, the route by which this biotin precursor is synthesized in E. coli has to a large extent been unknown (Lemoine et al., Mol. Microbiol. 19, 1996 645 - 647). bioC and bioH have been identified as being two genes whose corresponding proteins are responsible for the synthesis of Pm-CoA. The enzymic functions of the gene products, i.e. BioH and BioC, have hitherto been unknown (Lemoine et al., Mol. Microbiol.
19, 1996: 645 - 647, DeMOll, E., Escherichia coli and Salmonella, eds. Neidhardt, F. C. et al. ASM Press, Washington DC, USA, 1996:
704 - 708, ISBN 1-55581-084-5). Pm-CoA is converted into biotin in four further enzymic steps. BioF first of all condenses the Pm-CoA with alanine to form 7-keto-8-aminopelargonic acid (KAPA).
The KAPA is then converted into 7,8-diaminopelargonic acid (DAPA) 0050/48792 CA 02321264 2000-os-i4 a 2 by BioA. Following an ATP-dependent carboxylation reaction, the next step leads to dethiobiotin (DTB) and is catalyzed by BioD.
The DTB is converted into biotin in the last step. This step is catalyzed by BioB. The chemical and enzymic mechanisms involved in the conversion of DTB into biotin are so far only incompletely understood and clarified.
The conversion of DTB into biotin has so far only been characterized in bacterial and plant cell extracts (W094/8023, EP-B-0 449 724, Sanyal et al. Arch. Biochem. Biophys., Vol. 326, No. 1, 1996: 48 - 56 and Biochemistry 33, 1994: 3625 - 3631, Baldet 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 demonstrated that low molecular weight factors such as NADPH, cysteine, thiamine, Fe2+, asparagine, serine, fructose 1-6-bisphosphate and S-adenosylmethionine are able to stimulate the synthesis of biotin (Ohshiro et al., Biosci. Biotechnol: Biochem., 58, 9, 1994: 1738 - 1741, Birch et al., J. Biol. Chem. 270, 32, 1995: 19158 - 19165, Ifuku et al., Biosci. Biotechnol. Biochem., 59, 2, 1995: 185 - 189, Sanyal et al. Arch. Biochem. Biophys. 326, 1, 1996: 48 - 56).
In addition to these low molecular weight factors, other proteins have been identified which stimulate the synthesis of biotin from DTB in the presence of BioB. These proteins are flavodoxin and flavodoxin NADPH reductase (Birch et al., J. Biol. Chem. 270, 32, 1995: 19158 - 19165, Ifuk et al., Biosci. Biotechnol. Biochem., 59, 2, 1995: 185 - 189, Sanyal et al., Arch. Biochem. Biophys.
326, 1, 1996: 48 - 56). Other proteins which stimulate biotin synthesis are the genes bioS1 and bioS2, which are described in the German application having the application number 197.31274.8 (Priority 22.7.97).
Differing results have been obtained with regard to the origin of the sulfur in the biotin molecule. Investigations into the synthesis of biotin in whole cell extracts showed that radioactivity was incorporated into biotin in the presence of 35S-labeled cysteine; it was not possible to demonstrate incorporation of sulfur into the biotin molecule when either 3sS-labeled methionine or S-adenosylmethionine was used (Ifuku et al., Biosci. Biotechnol. Biochem. 59, 2, 1995: 184 - 189, Birch et al., J. Biol. Chem.270, 32, 1995: 19158 - 19165).
0050/48792 CA 02321264 2000-os-i4 The genes which encode the described proteins, i.e. bioF, bioA, bioD, and bioB, are encoded in E. coli on a bidirectional operon.
This operon is located between the ~, attachment site and the uvrB
gene locus at approx. 17 minutes on the E. coli chromosome (Berlyn et al. 1996: 1715 - 1902). A further two genes, one of which, i.e. bioC, already possesses described functions in the synthesis of Pm-CoA, are additionally encoded on this operon, whereas it has not so far been possible to assign any function to an open reading frame which is located downstream of bioA
(W094/8023, Otsuka et al., J. Biol. Chem. 263, 1988: 19577 - 85).
Highly conserved homologues to the E. coli proteins BioF, BioA, BioD and BioB have been found in B. sphaericus, B. subtilis, Syneccocystis sp. (Brown et al. Biotechnol. Genet. Eng. Rev. 9, 1991: 295 - 326, Bower et al., J. Bacteriol. 175, 1996: 4122 -4130, Kaneko et al., DNA Res. 3, 3, 1996: 109 - 136), archaeobacteria such as Methanococcus janaschi, and yeasts such as Saccharomyces cerevisiae (Zhang et al., Arch. Biochem.
Biophys. 309, 1, 1994: 29 - 35) or in plants such as Arabidopsis thaliana (Baldet et al., C. R. Acad. Sci. III, Sci. Vie. 319, 2, 1996: 99 - 106).
In the two Gram-positive microorganisms which have so far been investigated, the synthesis of Pm-CoA appears to proceed in a different manner from that in E. coli. It was not possible to find any homologues of bioH and bioC (Brown et al. Biotechnol.
Genet. Eng. Rev. 9, 1991: 295 - 326).
Biotin is an optically active substance which has three centers of chirality. It has hitherto only been prepared economically by way of an expensive, multi-step chemical synthesis.
As an alternative to this chemical synthesis, a large number of attempts have been made to construct a fermentative process for preparing biotin using microorganisms. Cloning the biotin operon onto multi-copy-plasmids has been successfully used to increase biotin synthesis in microorganisms which have been transformed with these genes. A further increase in biotin synthesis was achieved by deregulating biotin gene expression by means of selecting birA mutants (Pai C. H., J. Bacteriol. 112, 1972: 1280 ' 1287). Combination of the two approaches, that is expressing the plasmid-encoded biosynthesis genes in a regulation-deficient strain (EP-B-0 236 429), increased productivity still further. In this context, the biotin operon can either remain under the control of its native bidirectional promoter (EP-B-0 236 429) or else its genes can be brought under the control of a promoter which can be regulated externally (w094/08023).
0050/48792 CA 02321264 2000-os-i4 The approaches which have so far been pursued for producing biotin fermentatively in E. coli have not achieved any economically adequate productivity.
It is an object of the present invention to develop an industrial fermentative process for producing biotin which exhibits as high a biotin synthesis as possible.
We have found that this object is achieved by the process according to the invention for producing biotin, in which process an S-adenosylmethionine synthase (SAM synthase) gene, having the sequence SEQ ID No. 1, and at least one further biotin biosynthesis gene bioSl, bioS2 or bioS3, having the sequences SEQ
ID No. 3, SEQ ID No. 5 and SEQ ID No.7, and also their functional variants, analogues or derivatives, are expressed in a prokaryotic or eukaryotic host organism which is able to synthesize biotin, this organism is cultured and the synthesized biotin is used directly after separating off the biomass or after purifying the biotin.
The genes used in the process according to the invention, i.e.
the SAM synthase gene having the sequence SEQ ID No. 1 and the biotin biosynthesis genes bioSl, bioS2 and bioS3 having the sequences SEQ ID No. 3, SEQ ID No. 5 and SEQ ID No.7 are kept in the SwissProt-data base under accession numbers P04384 (metK), U29581 (bioSl), P39171 (bioS2) and D90811 (bioS3). A number of homologues to E. coli MetK are described in the data base. These homologues include organisms such as other eubacteria (e.g. H.
influenzae, and B. subtilis), and also eukaryotes (e. g. yeasts:
S. cerevisiae, Planta: P. deltoides, Arthropoda: D. melanogaster, and Mammalia: R. norvegicus).
The productivity of the biotin biosynthesis can be increased markedly by expressing one or more of the SAM synthase gene, having the sequence SEQ ID No. l, and its functional variants, analogues or derivatives in combination with at least one of the biotin synthesis genes bioSl, bioS2 or bioS3, having the sequences SEQ ID No. 3, SEQ ID No. 5 and SEQ ID No.7, and also their functional variants, analogues or derivatives, in a prokaryotic or eukaryotic host organism. A combination of the SAM
synthase gene and biosl is preferably used for the expression. At least one further biotin gene selected from the group bioA, bioB, bioF, bioC, bioD, bioH, bioP, biota, bioX, bioY and bioR is advantageously expressed at the same time in order to increase the biotin synthesis still further. Expression of the genes increases the synthesis of biotin by at least a factor of 2 as compared with the control without these genes, preferably by a 0050/48792 CA 02321264 2000-os-i4 factor which is greater than 3.
The genes used in the process according to the invention, i.e.
the SAM synthase gene having the nucleotide sequence SEQ ID No.
5 1, the bioSl gene having the nucleotide sequence SEQ ID No. 3, the bioS2 gene having the nucleotide sequence SEQ ID No. 5 and the bioS3 gene having the nucleotide sequence SEQ ID No.7, which sequences encode the amino acid sequences given in SEQ ID N0: 2, SEQ ID No. 4, SEQ ID No. 6 and SEQ ID No.8, respectively, or their allelic variations, can be obtained following isolation and sequencing. Variants are to be understood as being SEQ ID No. 1, SEQ ID No.3, SEQ ID No.5 and SEQ ID No.7 variants, respectively, which exhibit from 30 to 100% homology at the amino acid level, preferably from 50 to 100% homology, very particularly preferably from 80 to 100% homology. Allelic variants comprise, in particular, functional variants which can be obtained by the deletion, insertion or substitution of nucleotides from the sequences depicted in SEQ ID N0: 1, SEQ ID No. 3, SEQ ID No. 5 and SEQ ID No.7, with, however, the enzymic activity being retained.
In addition, variants are also to be understood as being functional equivalents of the genes, such as O-acetylserine sulfohydrolase A, O-acetylserine sulfohydrolase B, ~-cystathionase (see Flint et al., J. Biol. Chem., Vol. 271, 1996: 16053 - 16067) or nifS and its prokaryotic and eukaryotic homologues, for example from Klebsiella, Candida, yeasts or Caenorhabditis, which are able to assume the enzymic activity of bioSl, bioS2 or bioS3 in the synthesis of biotin.
Functional analogues of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID N0: 5 and SEQ ID No. 7 are to be understood as being, for example, their prokaryotic or eukaryotic homologues, such as bacterial, fungal, plant, animal or human homologues. In addition, analogues are also to be understood as being truncated sequences, or single-stranded DNA or RNA from coding and non-coding DNA
sequences.
Derivatives are to be understood, for example, as being promoter variants. The promoters, which are placed upstream of the given nucleotide sequences, can be altered by means of one or more nucleotide substitutions, or by means of (an) insertions) and/or deletions) without, however, the functionality or activity of the promoters being impaired. In addition, the activities of the promoters can be increased by means of altering their sequences, or the promoters can be completely replaced by more active 0050/48792 CA 02321264 2000-os-i4 promoters, including those from organisms of a different species.
Derivatives are also to be understood as being variants whose nucleotide sequences have been altered in the region from -1 to -30 upstream of the start codon such that expression of the gene and/or expression of a protein is increased. This is advantageously effected by altering the Shine-Dalgarno sequence.
All Gram-negative or Gram-positive bacteria which synthesize biotin are, in principal, suitable for use as prokaryotic host organisms in the process according to the invention.
Gram-negative bacteria which may be mentioned by way of example are Enterobacteriaceae such as the genera Escherichia, Aerobacter, Enterobacter, Citrobacter, Shigella, Klebsiella, Serratia, Erwinia or Salmonella, Pseudomonadaceae such as the genera Pseudomonas, Xanthomonas, Burkholderia, Gluconobacter, Nitrosomonas, Nitrobacter, Methanomonas, Comamonas, Cellulomonas or Acetobacter, Azotobacteraceae such as the genera Azotobacter, Azomonas, Beijerinckia or Derxia, Neisseriaceae such as the genera Moraxella, Acinetobacter, Kingella, Neisseria or Branhamella, the Rhizobiaceae such as the genera Rhizobium or Agrobacterium, or the Gram-negative genera Zymomonas, Chromobacterium or Flavobacterium. Gram-positive bacteria which may be mentioned by way of example are the endospore-forming Gram-positive aerobic or anaerobic bacteria such as the genera Bacillus, Sporolactobacillus or Clostridium, the coryneform bacteria such as the genera Arthrobacter, Cellulomonas, Curtobacterium, Corynebacterium, Brevibacterium, Microbacterium or Kurthia, the Actinomycetales such as the genera Mycobacterium, Rhodococcus, Streptomyces or Nocardia, the Lactobacillaceae such as the genera Lactobacillus or Lactococcus, or the Gram-positive cocci such as the genera Micrococcus or Staphylococcus.
Preference is given to using bacteria of the genera Escherichia, Citrobacter, Serratia, Klebsiella, Salmonella, Pseudomonas, Comamonas, Acinetobacter, Azotobacter, Chromobacterium, Bacillus, Clostridium, Arthrobacter, Corynebacterium, Brevibacterium, Lactococcus, Lactobacillus, Streptomyces, Rhizobium, Agrobacterium or Staphylococcus in the process according to the invention. Particular preference is given to genera and species such as Escherichia coli, Citrobacter freundii, Serratia marcescens, Salmonella typhimurium, Pseudomonas mendocina, Pseudomonas aeruginosa, Pseudomonas mutabilis, Pseudomonas chlororaphis, Pseudomonas fluorescens, Comamonas acidovorans, Comamonas testosterone, Acinetobacter calcoaceticus, Azotobacter vinelandii, Chromobacterium violaceum, Bacillus subtilis, Bacillus sphaericus, Bacillus stearothermophilus, Bacillus 0050/48792 CA 02321264 2000-os-i4 pumilus, Bacillus licheniformis, Bacillus amyloliquefaciens, Bacillus megaterium, Bacillus cereus, Bacillus thuringiensis, Arthrobacter citreus, Arthrobacter paraffineus, Corynebacterium glutamicum, Corynebacterium primorioxydans, Corynebacterium sp., Brevibacterium ketoglutamicum, Brevibacterium linens, Brevibacterium sp., Streptomyces lividans, Rhizobium leguminosarum or Agrobacterium tumefaciens. Advantageously, use is made of bacteria which already exhibit an elevated natural production of biotin.
The taxonomic position of the listed genera has been subject to considerable change in recent years and is still in a state of flux as false genera and species names are corrected. Because of these taxonomic regroupings, which have been frequently required in the past, of the said genera within bacterial systematics, families, genera and species other than those mentioned above are also suitable for the process according to the invention.
All biotin-synthesizing organisms, such as fungi, yeasts, plants or plant cells, are, in principal, suitable for use as eukaryotic host organisms in the process according to the invention. Yeasts which may preferably be mentioned are the genera Rhodotorula, Yarrowia, Sporobolomyces, Saccharomyces or Schizosaccharomyces.
Particular preference is given to the genera and species Rhodotorula rubra, Rhodotorula glutinis, Rhodotorula graminis, Yarrowia lipolytica, Sporobolomyces salmonicolor, Sporobolomyces shibatanus or Saccharomyces cerevisiae.
In principal, all plants can be used as the host organism, with preference being given to plants which play a role in animal nutrition or human nutrition, such as corn, wheat, barley, rye, potatoes, peas, beans, sunflowers, palms, millet, sesame, copra or rape. 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, yeasts or plant cells which are able to secrete biotin into the growth medium, and which, where appropriate, already additionally exhibit an increased natural synthesis of biotin, are advantageously used in the process according to the invention. Advantageously, these organisms can also be defective with regard to the regulation of their biotin biosynthesis: i.e. this synthesis is either not regulated or only regulated to a very reduced extent. This regulatory defect results in these organisms already possessing a substantially increased biotin productivity. Such a regulatory defect is known, for example, from Escherichia coli in the form 0050/48792 CA 02321264 2000-os-i4 of birA-defect mutants and should preferably be present in the cells as a defect which can be induced by external influences, for example as a defect which is temperature-inducible. In principal, organisms which do not exhibit any natural biotin production can also be used, once they have been transformed with the biotin genes.
In order to increase biotin productivity as a whole still further, the organisms in the process according to the invention should advantageously also harbor at least one further biotin gene selected from the group bioA, bioB, bioF, bivC, bioD, bioH, bioP, biota, bioX, bioY or bioR. Advantageously, those genes which stimulate biotin synthesis can also be present in the cell in combination with the sequences SEQ ID No. 1 , SEQ ID No. 3, SEQ
ID No.5 or SEQ ID No.7 and their combinations. Examples of genes which stimulate biotin synthesis are the flavoredoxin gene and the flavoredoxin reductase gene. This additional gene, or these additional genes, can be present in the cell in one or more copies, like the genes having the sequences SEQ ID No. 1 , SEQ ID
No.3, SEQ ID No.S or SEQ ID No.7 or their combinations. They can be located on the same vector as the sequences SEQ ID No. 1, SEQ
ID No.3, SEQ ID No.5 and/or SEQ ID No.7, or on separate vectors, or else integrated chromosomally. The sequences SEQ ID No. 1, SEQ
ID No.3, SEQ ID No.5 and/or SEQ ID No.7 can also be together on a vector or on separate vectors or be inserted into the genome.
The gene construct according to the invention is to be understood as being the gene sequences of the SAM synthase gene SEQ ID No. 1 and of the biotin synthesis genes SEQ ID No.3, SEQ ID No.5 and/or SEQ ID No.7, and also their functional variants, analogues or derivatives, which were linked functionally to one or more regulatory signals for the purpose of increasing expression of the genes. In addition to these new regulatory sequences, the natural regulation of these sequences can still be present upstream of the actual structural genes and, where appropriate, can have been genetically altered such that the natural regulation has been switched off and expression of genes has been increased. However, the gene construct can also be assembled in a simpler manner, i.e. no additional regulatory signals are inserted upstream of the sequences SEQ ID No. 1, SEQ ID No. 3, SEQ ID No.5 and/or SEQ ID No.7 and the natural promoter, with its regulation, is not removed. Instead, the natural regulatory sequence is mutated such that regulation by biotin no longer takes place and gene expression is increased. The sequences SEQ
ID No. 1, SEQ ID No.3, SEQ ID No.S and/or SEQ ID No.7 can be under the regulation of one promoter or under the regulation of separate promoters. Additional, advantageous regulatory elements can also be inserted at the 3' end of the DNA sequences. The 0050/48792 CA 02321264 2000-os-i4 genes having the sequences SEQ ID No. 1, SEQ ID No. 3, SEQ ID
No.5 or SEQ ID No. 7 can be present in the gene construct in one or more copies.
Advantageous regulatory sequences for the process according to the invention are present, for example, in promoters such as the cos-, tac-, trp-, tet-, trp-tet-, lpp-, lac-, lpp-lac-, lacIq--T7-, T5-, T3-, gal-, trc-, ara-, SP6-, ~,-PR- or ~.-PL-promoters, which are advantageously used in Gram-negative bacteria. Further advantageous regulatory sequences are present, for example, in the Gram-positive promoters amy and SP02, in the yeast promoters ADC1, MFa , 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 the phaseolin promoter.
In principal, all natural promoters, together with their regulatory sequences, can be used, like the abovementioned promoters, for the process according to the invention. In addition, synthetic promoters can also advantageously be used.
Other biotin genes selected from the group bioA, bioB, bioF, bioC, bioD, bioH, bioP, biota, bioX, bioY or bioR, which genes can have their own promoter or else can be under the regulation of the promoter of one of the sequences, or under the regulation of the promoter of all the sequences, SEQ ID No. 1, SEQ ID No. 3, SEQ ID No.S or SEQ ID No.7, can be present in the gene construct in one or more copies.
For expression in the abovementioned host organism, the gene construct is advantageously inserted into a host-specific vector which makes it possible to achieve optimum expression of the genes in the host. Vectors are well known to the skilled person and can be identified, for example, from the book Cloning Vectors (Eds. Pouwels P. H. et al. Elsevier, Amsterdam-New York-Oxford, 1985, ISBN 0 444 904018). In addition to plasmids, the vectors are also to be understood as being all other vectors known to the skilled person, such as phages, viruses, transposons, IS
elements, phasmids, cosmids or linear or circular DNA. These vectors can be replicated autonomously in the host organism or replicated chromosomally.
Expression systems are to be understood as being the combination of the host organisms which are mentioned above by way of example and the vectors which are appropriate for the organisms, such as plasmids, viruses or phages, for example plasmids containing the RNA polymerase/promoter system, phages ~,, or Mu or other temperate phages or transposons and/or further advantageous regulatory 0050/48792 CA 02321264 2000-os-i4 . , ,.
sequences.
The term expression systems is preferably to be understood as being the combination of Escherichia coli and its plasmids and 5 phages and the affiliated promoters, and also Bacillus and its plasmids and promoters.
Further 3' and/or 5'-terminal regulatory sequences are also 10 suitable for advantageously expressing SEQ ID No. l, SEQ ID No.3, SEQ ID No.5 and/or SEQ ID No. 7 in accordance with the invention.
These regulatory sequences are intended to make it possible to achieve specific expression of the biotin genes and expression of the protein. Depending on the host organism, this can, for example, mean that the gene is only expressed or overexpressed after induction or that it is expressed and/or overexpressed immediately.
In this context, the regulatory sequences or factors can preferably influence biotin gene expression positively and thereby increase it. For example, the regulatory elements can advantageously be reinforced at the transcriptional level by means of using strong transcription signals such as promoters and/or enhancers. In addition, however, it is also possible to reinforce translation by, for example, improving the stability of the mRNA.
Enhancers are to be understood as being, for example, DNA
sequences which bring about increased biotin gene expression by means of improving the interaction between the RNA polymerase and the DNA.
~ increase in the proteins (see SEQ ID No.2, SEQ ID No.4, SEQ ID
No.6 and 5EQ ID No.8) which are derived from the sequences SEQ ID
No. 1, SEQ ID No.3, SEQ ID No.5 and SEQ ID No.7, and in their enzyme activity, as compared with the starting enzymes, can be achieved, for example, by altering the corresponding gene sequences, or the sequences of their homologues, by means of classical mutagenesis, such as W irradiation, or by treating with chemical mutagens and/or by means of specific mutagenesis such as site-directed mutagenesis, deletion(s), insertions) and/or substitution(s). An increased enzyme activity, apart from the described gene amplification, can also be achieved by eliminating factors which repress enzyme biosynthesis and/or by synthesizing active enzymes instead of inactive enzymes.
0050/48792 CA 02321264 2000-os-i4 The process according to the invention advantageously increases the conversion of DTB into biotin, and consequently overall biotin productivity, by means of using the biotin genes having the sequences SEQ ID No. 1, SEQ ID No.3, SEQ ID No.5 and SEQ ID
No.7, and the combination of the genes having the sequences SEQ
ID No.1 and SEQ ID No.5 or SEQ ID No.1 and SEQ ID No.7, preferably the combination of the genes having the sequences SEQ
ID No.1 and SEQ ID No.3, which genes are introduced into the organisms by way of their vectors and/or by means of chromosomal cloning.
In the process according to the invention, the microorganisms harboring SEQ ID No.l, SEQ ID No.3, SEQ ID No.S and/or SEQ ID
No.7 are propagated in a medium which enables these organisms to grow. This medium can be a synthetic medium or a natural medium.
Use is made of media which are known to the skilled person and which are appropriate for the organism. In order to permit growth of the microorganisms, the media employed contain a carbon source, a nitrogen source, inorganic salts and, where appropriate, small quantities of vitamins and trace elements.
Examples of advantageous carbon sources are sugars, such as monosaccharides, disaccharides or polysaccharides, such as glucose, fructose, mannose, xylose, galactose, ribose, sorbose, ribulose, lactose, maltose, sucrose, raff inose, starch or cellulose, complex sugar sources such as molasses, sugar phosphates, such as fructose-1,6-bisphosphate, sugar alcohols, such as mannitol, polyols, such as glycerol, alcohols, such as methanol or ethanol, carboxylic acids, such as citric acid, lactic acid of acetic acid, fats, such as soy-bean oil or rapeseed oil, or amino acids, such as glutamic acid or aspartic acid, or amino sugars, which can simultaneously be used as a nitrogen source.
Advantageous nitrogen sources are organic or inorganic nitrogen compounds or materials which contain these compounds. Examples are ammonium salts, such as NH4C1 or (NH4)ZS04, nitrates or urea, or complex nitrogen sources such as corn steep liquor, brewer's yeast autolysate, soy-bean flour, wheat gluten, yeast extract, meat extract, casein hydrolysate or yeast or potato protein, which can frequently also be used simultaneously as a nitrogen source.
Examples of inorganic salts are the salts of calcium, magnesium, sodium, manganese, potassium, zinc, copper and iron. Anions of these salts which are to be mentioned in particular are the chloride, sulfate and phosphate ions. An important factor for 0050/48792 CA 02321264 2000-os-i4 increasing productivity in the process according to the invention is the addition of Fe2+ or Fe3+ salts and/or potassium salts to the production medium.
Where appropriate, further growth factors, such as vitamins or growth promoters, such as riboflavin, thiamine, folic acid, nicotinic acid, pantothenate or pyridoxine, amino acids, such as alanine, cysteine, asparagine, aspartic acid, glutamine, serine, methionine or lysine, carboxylic acids, such as citric acid, formic acid, pimelic acid or lactic acid, or substances such as dithiothreitol, are added to the nutrient medium.
Antibiotics can, where appropriate, be added to the medium in order to stabilize the biotin gene-containing vectors in the Cells.
The ratios in which the said nutrients are mixed depends on the nature of the fermentation and is laid down in each individual case. The medium components can all be initially introduced at the beginning of fermentation, after they have been, if necessary, sterilized separately or sterilized together, or else be added subsequently, as required, during fermentation.
The culture conditions are so arranged that the organisms grow optimally and that the best possible yields are achieved.
Preferred culture temperatures are from 15 °C to 40 °C.
Temperatures of between 25 °C and 37 °C are particularly advantageous. The pH is preferably kept in a range of from 3 to 9. pH values of between 5 and 8 are particularly advantageous. In general, a period of incubation of from 8 to 240 hours, preferably of from 8 to 120 hours, is sufficient. Within this time, the maximum quantity of biotin accumulates in the medium and/or is available after the cells have been disrupted.
The process according to the invention for producing biotin can be carried out continuously or batch-wise or fed-batch-wise. If whole plants are regenerated from the plant cells which have been transformed with the biotin genes, they can, according to the process according to the invention, be grown and propagated perfectly normally.
Examples:
1' Cloning of the S-adenosylmethionine synthase gene (SEQ ID
No.1):
Starting from genomic E. coli DNA, the gene which encodes SAM
syuthase (metK) was amplified from the E. coli chromosome by ' means of a polymerase chain reaction using two specific oligonucleotides. The DNA which had been amplified in this way was purified, digested with the restriction enzyme Acc65I and inserted into a vector which had been cut with the same enzyme and which enables the gene to be overexpressed in E. coli strains. One of the two oligonucleotides was used to provide the gene construct with optimized translation signals.
a.) Generation of oligonucleotides for amplifying the metK gene from the E. coli chromosome:
metK was to be amplified as an expression cassette which was composed of a ribosome binding site, the start codon of the coding sequence and the stop codon between two restriction enzyme recognition sites. The Acc65I recognition sequence was chosen for both the restriction sites. The metK gene was amplified and cloned using the nucleotides PmetK1 (5'-GCGGTACCAGGTGATATTAAATATGGCAAAAC-3') and PmetK2 (5'-CGGGTACCGATTACTTCAGACCGGCAGC-3').
b.) Implementation of the PCR:
Conditions:
0.5 ~g chromosomal DNA from E. coli W3110 was used as a template.
The oligonucleotides PmetKl and PmetK2 were employed at a concentration of in each case 15 pMol. The concentration of the dNTPs was 200 ~M. 2.5 U of Pwo DNA polymerase (Boehringer Mannheim) in the manufacturer's reaction buffer were employed as the polymerase. The PCR reaction volume was 100 ~.1.
~Plifications:
The DNA was denatured at 94 °C for 2 min. The oligonucleotides were then annealed at 55 °C for 30 seconds. The elongation took place at 72 °C for 75 seconds. The PCR reaction was carried out over 30 cycles.
The resulting DNA product, which had a size of approximately 1145 bp, was purified and digested with Acc65I in a suitable buf fer .
0050/48792 CA 02321264 2000-os-i4 c.) Cloning of metK in an expression vector 2 ~,g of the vector pHS1 (construction was described in DE
197.31274.8, priority 22.7.97, Example 1, pages 14 to 17) were digested with Acc65I and dephosphorylated using shrimp alkaline phosphatase (SAP) (Boehringer Mannheim). After the SAP had been denatured, vector and fragment were ligated, in a molar ratio of 1:3, using the Rapid DNA Ligation kit in accordance with the manufacturer's instructions. The ligation mixture was transformed into strain E. coli XL-1-blue. Positive clones were identified by plasmid preparation and restriction analysis. The correct orientation of the metK fragment in pHS1 was determined by restriction digestion and sequencing. The resulting construct was designated pHSl metK (Figure 1). The sequence of pHS1 metK is given in SEQ ID No.9. SEQ ID No.lO shows the amino acid sequence which is deduced from the metK-encoding region.
2. Construction of plasmids pHBbiol4 and pHS1 bioS1 The construction of plasmids pHBbiol4 and pHS1 bioSl has already been described (DE 197.31274.8, Priority 22.7.97, Examples 1, 2 and 5).
3. Construction of pHSl metK bioS1 The plasmids pHS1 bioS1 [SEQ ID No.ll, (DE 197.31274.8, Priority 22.7.97), SEQ ID No.l2 shows the amino acid sequence which is deduced from the bioS1-encoding region] and pHSl metK (SEQ ID
No.9) were purified using a plasmid preparation method (Boehringer). The fragment carrying the metK gene was isolated from pHS1 metK by digesting with Acc65I. pHS1 bioS1 was digested with Acc65I and dephosphorylated with shrimp alkaline phosphatase (SAP) (Boehringer Mannheim). After the SAP had been denatured in accordance with the manufacturer's instructions, the vector and the metK fragment were ligated, in a molar ratio of 1:3, using the Rapid DNA Ligation Kit in accordance with the manufacturer's instructions. The ligation mixture was transformed into strain E.
coli XL-1-blue. Positive clones were identified by plasmid preparation and restriction analysis. The correct orientation of the metK fragment in pHS1 bioS1 was determined by means of restriction digestion and sequencing. The resulting construct was designated pHS1 metK bioS1 (Figure 2). The sequence of pHSi metK
bioSi is given in SEQ ID No. l3. SEQ ID No. l4 shows the amino acid sequence which was deduced from the metK-encoding region; SEQ ID
0050/48792 CA 02321264 2000-os-i4 No.l5 shows the amino acid sequence which was deduced from the bioS1-encoding region.
No.9) were purified using a plasmid preparation method (Boehringer). The fragment carrying the metK gene was isolated from pHS1 metK by digesting with Acc65I. pHS1 bioS1 was digested with Acc65I and dephosphorylated with shrimp alkaline phosphatase (SAP) (Boehringer Mannheim). After the SAP had been denatured in accordance with the manufacturer's instructions, the vector and the metK fragment were ligated, in a molar ratio of 1:3, using the Rapid DNA Ligation Kit in accordance with the manufacturer's instructions. The ligation mixture was transformed into strain E.
coli XL-1-blue. Positive clones were identified by plasmid preparation and restriction analysis. The correct orientation of the metK fragment in pHS1 bioS1 was determined by means of restriction digestion and sequencing. The resulting construct was designated pHS1 metK bioS1 (Figure 2). The sequence of pHSi metK
bioSi is given in SEQ ID No. l3. SEQ ID No. l4 shows the amino acid sequence which was deduced from the metK-encoding region; SEQ ID
0050/48792 CA 02321264 2000-os-i4 No.l5 shows the amino acid sequence which was deduced from the bioS1-encoding region.
4. Increasing biotin productivity by overexpressing metK, bioSl and metK in combination with bio5l.
Spontaneously rifampicin-resistant colonies were isolated from strain BM4086 (Ketner and Campbell J. Molec. Biology 1975 96:13) 10 by plating on rifampicin plates. A P1 lysate was generated from one of these resistant strains. The strain W3110 was transduced with this P1 lysate and clones were subsequently selected using rifampicin. The resulting strain was transformed with plasmid pHBbiol4 using the CaClz method (Maniatis et al. Molecular Cloning 15 Cols Spring Harbour Laboratory Press 1989) and grown on LB
containing 100 ~tg of ampicillin/ml. The isolated, transformed strain (LU5560) was in each case transformed with plasmid pHSl, pHS1 metK, pHS1 bioS1 or pHS1 metK bioSl using the CaCl2 method and then selected on LB agar containing 100 ~,g of ampicillin/ml and 25 ~,g of kanamycin/ml.
One colony from each of the transformants was in each case inoculated into a DYT culture containing the appropriate antibiotics and incubated for 12 h. The overnight culture (= ONC) was used to inoculate a 10 ml culture in TB medium (Sambrook, J.
Fritsch, E F. Maniatis, T. 2nd ed. Cold Spring Harbor Laboratory Press., 1989 ISBN 0-87969-373-8), which contained g of glycerol/1 and the appropriate antibiotics. In the cases where plasmids pHSl, pHS1 metK, pHSlbioSl and pHS1 metK bioS1 30 were present, imM IPTG and 0.5~ arabinose were added simultaneously in order to induce expression of the metK and bioS1 genes or, respectively, the combination of the two genes.
After 24 h, the cells were separated off from the culture supernatant by centrifugation and the biotin concentration in the Supernatant was determined by means of a competitive ELISA
employing streptavidin. The results of this determination are shown in Table I.
Table I: Determination of the biotin concentration Strain Plasmid I Plasmid II Biotin mg/1 LU5580 pHBbiol4 Control, without ~ 11 plasmid LU5580 pHBbiol4 pHSl 25 LU5580 pHBbiol4 pHS1 bioSl 45 LU5580 pHBbiol4 pHSl metK 37 LU5580 pHBbiol4 pHSl metK bioSi 52 0050/48792 CA 02321264 2000-os-i4 SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT:
(A) NAME: BASF Aktiengesellschaft (B) STREET: Karl Bosch Strasse (C) CITY: Ludwigshafen (D) FEDERAL STATE: Rheinland-Pfalz (E) COUNTRY: Germany (F) POSTAL CODE: 67056 (ii) TITLE OF APPLICATION: Process for preparing biotin (iii) NUMBER OF SEQUENCES: 15 (iv) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.25 (EPO) (2) INFORMATION FOR SEQ ID No: 1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1155 Base pairs (B) TYPE: Nucleic acid (C) STRANDEDNESS: Single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iii) ANTISENSE: NO
(vi) ORIGINAL SOURCE:
(B) STRAIN: Escherichia coli (vii) IMMEDIATE SOURCE:
(B) CLONE: metK
(ix) FEATURES:
(A) NAME/KEY: CDS
(B) LOCATION: 1..1155 (xi) SEQUENCE DESCRIPTION: SEQ ID No: 1:
0050/48792 CA 02321264 2000-os-i4 Met Ala Lys His Leu Phe Thr Ser Glu Ser Val Ser Glu Gly His Pro Asp Lys Ile Ala Asp Gln Ile Ser Asp Ala Val Leu Asp Ala Ile Leu Glu Gln Asp Pro Lys Ala Arg Val Ala Cys Glu Thr Tyr Val Lys Thr Gly Met Val Leu Val Gly Gly Glu Ile Thr Thr Ser Ala Trp Val Asp Ile Glu Glu Ile Thr Arg Asn Thr Val Arg Glu Ile Gly Tyr Val His Ser Asp Met Gly Phe Asp Ala Asn Ser Cys Ala Val Leu Ser Ala Ile Gly Lys Gln Ser Pro Asp Ile Asn Gln Gly Val Asp Arg Ala Asp Pro Leu Glu Gln Gly Ala Gly Asp Gln Gly Leu Met Phe Gly Tyr Ala Thr Asn Glu Thr Asp Val Leu Met Pro Ala Pro Ile Thr Tyr Ala His Arg Leu Val Gln Arg Gln Ala Glu Val Arg Lys Asn Gly Thr Leu Pro Trp Leu Arg Pro Asp Ala Lys Ser Gln Val Thr Phe Gln Tyr Asp Asp Gly Lys Ile Val Gly Ile Asp Ala Val Val Leu Ser Thr Gln His Ser Glu 0050/48792 CA 02321264 2000-os-i4 Glu Ile Asp Gln Lys Ser Leu Gln Glu Ala Val Met Glu Glu Ile Ile Lys Pro Ile Leu Pro Ala Glu Trp Leu Thr Ser Ala Thr Lys Phe Phe Ile Asn Pro Thr Gly Arg Phe Val Ile Gly Gly Pro Met Gly Asp Cys Gly Leu Thr Gly Arg Lys Ile Ile Val Asp Thr Tyr Gly Gly Met Ala Arg His Gly Gly Gly Ala Phe Ser Gly Lys Asp Pro Ser Lys Val Asp Arg Ser Ala Ala Tyr Ala Ala Arg Tyr Val Ala Lys Asn Ile Val Ala Ala Gly Leu Ala Asp Arg Cys Glu Ile Gln Val Ser Tyr Ala Ile Gly Val Ala Glu Pro Thr Ser Ile Met Val Glu Thr Phe Gly Thr Glu Lys Val Pro Ser Glu Gln Leu Thr Leu Leu Val Arg Glu Phe Phe Asp Leu Arg Pro Tyr Gly Leu Ile Gln Met Leu Asp Leu Leu His Pro Ile Tyr Lys Glu Thr Ala Ala Tyr Gly His Phe Gly Arg Glu His Phe Pro Trp Glu Lys Thr Asp Lys Ala Gln Leu Leu Arg Asp Ala Ala Gly Leu Lys T~ 1155 (2) INFORMATION FOR SEQ ID No: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 384 Amino acids (B) TYPE: Amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Protein (xi) SEQUENCE DESCRIPTION: SEQ ID No: 2:
Met Ala Lys His Leu Phe Thr Ser Glu Ser Val Ser Glu Gly His Pro Asp Lys Ile Ala Asp Gln Ile Ser Asp Ala Val Leu Asp Ala Ile Leu Glu Gln Asp Pro Lys Ala Arg Val Ala Cys Glu Thr Tyr Val Lys Thr Gly Met Val Leu Val Gly Gly Glu Ile Thr Thr Ser Ala Trp Val Asp Ile Glu Glu Ile Thr Arg Asn Thr Val Arg Glu Ile Gly Tyr Val His Ser Asp Met Gly Phe Asp Ala Asn Ser Cys Ala Val Leu Ser Ala Ile Gly Lys Gln Ser Pro Asp Ile Asn Gln Gly Val Asp Arg Ala Asp Pro Leu Glu Gln Gly Ala Gly Asp Gln Gly Leu Met Phe Gly Tyr Ala Thr Asn Glu Thr Asp Val Leu Met Pro Ala Pro Ile Thr Tyr Ala His Arg Leu Val Gln Arg Gln Ala Glu Val Arg Lys Asn Gly Thr Leu Pro Trp Leu Arg Pro Asp Ala Lys Ser Gln Val Thr Phe Gln Tyr Asp Asp Gly Lys Ile Val Gly Ile Asp Ala Val Val Leu Ser Thr Gln His Ser Glu 0050/48792 ~ CA 02321264 2000-08-14 Glu Ile Asp Gln Lys Ser Leu Gln Glu Ala Val Met Glu Glu Ile Ile Lys Pro Ile Leu Pro Ala Glu Trp Leu Thr Ser Ala Thr Lys Phe Phe Ile Asn Pro Thr Gly Arg Phe Val Ile Gly Gly Pro Met Gly Asp Cys Gly Leu Thr Gly Arg Lys Ile Ile Val Asp Thr Tyr Gly Gly Met Ala Arg His Gly Gly Gly Ala Phe Ser Gly Lys Asp Pro Ser Lys Val Asp Arg Ser Ala Ala Tyr Ala Ala Arg Tyr Val Ala Lys Asn Ile Val Ala Ala Gly Leu Ala Asp Arg Cys Glu Ile Gln Val Ser Tyr Ala Ile Gly Val~Ala Glu Pro Thr Ser Ile Met Val Glu Thr Phe Gly Thr Glu Lys Val Pro Ser Glu Gln Leu Thr Leu Leu Val Arg Glu Phe Phe Asp Leu Arg Pro Tyr Gly Leu Ile Gln Met Leu Asp Leu Leu His Pro Ile Tyr Lys Glu Thr Ala Ala Tyr Gly His Phe Gly Arg Glu His Phe Pro Trp Glu Lys Thr Asp Lys Ala Gln Leu Leu Arg Asp Ala Ala Gly Leu Lys (2) INFORMATION FOR SEQ ID No: 3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1206 Base pairs (B) TYPE: Nucleic acid (C) STRANDEDNESS: Single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iii) ANTISENSE: NO
0050/48792 CA 02321264 2000-os-i4 (vi) ORIGINAL SOURCE:
(B) STRAIN: Escherichia coli (vii) IMMEDIATE SOURCE:
(B) CLONE: bioS1 (ix) FEATURES:
(A) NAME/KEY: CDS
(B) LOCATION: 1..1206 (xi) SEQUENCE DESCRIPTION: SEQ ID No: 3:
Met Asn Val Phe Asn Pro Ala Gln Phe Arg Ala Gln Phe Pro Ala Leu Gln Asp Ala Gly Val Tyr Leu Asp Ser Ala Ala Thr Ala Leu Lys Pro Glu Ala Val Val Glu Ala Thr Gln Gln Phe Tyr Ser Leu Ser Ala Gly Asn Val His Arg Ser Gln Phe Ala Glu Ala Gln Arg Leu Thr Ala Arg Tyr Glu Ala Ala Arg Glu Lys Val Ala Gln Leu Leu Asn Ala Pro Asp Asp Lys Thr Ile Val Trp Thr Arg Gly Thr Thr Glu Ser Ile Asn Met Val Ala Gln Cys Tyr Ala Arg Pro Arg Leu Gln Pro Gly Asp Glu Ile Ile Val Ser Val Ala Glu His His Ala Asn Leu Val Pro Trp Leu Met Val Ala Gln Gln Thr Gly Ala Lys Val Val Lys Leu Pro Leu Asn Ala 0050/48792 CA 02321264 2000-os-i4 Gln Arg Leu Pro Asp Val Asp Leu Leu Pro Glu Leu Ile Thr Pro Arg Ser Arg Ile Leu Ala Leu Gly Gln Met Ser Asn Val Thr Gly Gly Cys Pro Asp Leu Ala Arg Ala Ile Thr Phe Ala His Ser Ala Gly Met Val Val Met Val Asp Gly Ala Gln Gly Ala Val His Phe Pro Ala Asp Val Gln Gln Leu Asp Ile Asp Phe Tyr Ala Phe Ser Gly His Lys Leu Tyr Gly Pro Thr Gly Ile Gly Val Leu Tyr Gly Lys Ser Glu Leu Leu Glu Ala Met Ser Pro Trp Leu Gly Gly Gly Lys Met Val His Glu Val Ser Phe Asp Gly Phe Thr Thr Gln Ser Ala Pro Trp Lys Leu Glu Ala Gly Thr Pro Asn Val Ala Gly Val Ile Gly Leu Ser Ala Ala Leu Glu Trp Leu Ala Asp Tyr Asp Ile Asn Gln Ala Glu Ser Trp Ser Arg Ser Leu Ala Thr Leu Ala Glu Asp Ala Leu Ala Lys Arg Pro Gly Phe Arg Ser Phe Arg Cys Gln Asp Ser Ser Leu Leu Ala Phe Asp Phe Ala Gly Val 0050/48792 CA 02321264 2000-os-i4 His His Ser Asp Met Val Thr Leu Leu Ala Glu Tyr Gly Ile Ala Leu Arg Ala Gly Gln His Cys Ala Gln Pro Leu Leu Ala Glu Leu Gly Val Thr Gly Thr Leu Arg Ala Ser Phe Ala Pro Tyr Asn Thr Lys Ser Asp Val Asp Ala Leu Val Asn Ala Val Asp Arg Ala Leu Glu Leu Leu Val 385' 390 395 400 Asp (2) INFORMATION FOR SEQ ID No: 4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 401 Amino acids (B) TYPE: Amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Protein (xi) SEQUENCE DESCRIPTION: SEQ ID No: 4:
Met Asn Val Phe Asn Pro Ala Gln Phe Arg Ala Gln Phe Pro Ala Leu Gln Asp Ala Gly Val Tyr Leu Asp Ser Ala Ala Thr Ala Leu Lys Pro Glu Ala Val Val Glu Ala Thr Gln Gln Phe Tyr Ser Leu Ser Ala Gly Asn Val His Arg Ser Gln Phe Ala Glu Ala Gln Arg Leu Thr Ala Arg Tyr Glu Ala Ala Arg Glu Lys Val Ala Gln Leu Leu Asn Ala Pro Asp Asp Lys Thr Ile Val Trp Thr Arg Gly Thr Thr Glu Ser Ile Asn Met 0050/48792 CA 02321264 2000-os-i4 Val Ala Gln Cys Tyr Ala Arg Pro Arg Leu Gln Pro Gly Asp Glu Ile Ile Val Ser Val Ala Glu His His Ala Asn Leu Val Pro Trp Leu Met Val Ala Gln Gln Thr Gly Ala Lys Val Val Lys Leu Pro Leu Asn Ala Gln Arg Leu Pro Asp Val Asp Leu Leu Pro Glu Leu Ile Thr Pro Arg Ser Arg Ile Leu Ala Leu Gly Gln Met Ser Asn Val Thr Gly Gly Cys Pro Asp Leu Ala Arg Ala Ile Thr Phe Ala His Ser Ala Gly Met Val Val Met Val Asp Gly Ala Gln Gly Ala Val His Phe Pro Ala Asp Val Gln Gln Leu Asp Ile Asp Phe Tyr Ala Phe Ser Gly His Lys Leu Tyr Gly Pro Thr Gly Ile Gly Val Leu Tyr Gly Lys Ser Glu Leu Leu Glu Ala Met Ser Pro Trp Leu Gly Gly Gly Lys Met Val His Glu Val Ser Phe Asp Gly Phe Thr Thr Gln Ser Ala Pro Trp Lys Leu Glu Ala Gly Thr Pro Asn Val Ala Gly Val Ile Gly Leu Ser Ala Ala Leu Glu Trp Leu Ala Asp Tyr Asp Ile Asn Gln Ala Glu Ser Trp Ser Arg Ser Leu Ala Thr Leu Ala Glu Asp Ala Leu Ala Lys Arg Pro Gly Phe Arg Ser Phe Arg Cys Gln Asp Ser Ser Leu Leu Ala Phe Asp Phe Ala Gly Val His His Ser Asp Met Val Thr Leu Leu Ala Glu Tyr Gly Ile Ala Leu Arg Ala Gly Gln His Cys Ala Gln Pro Leu Leu Ala Glu Leu Gly Val 0050/48792 CA 02321264 2000-os-i4 Thr Gly Thr Leu Arg Ala Ser Phe Ala Pro Tyr Asn Thr Lys Ser Asp Val Asp Ala Leu Val Asn Ala Val Asp Arg Ala Leu Glu Leu Leu Val Asp (2) INFORMATION FOR SEQ ID No: 5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1215 Base pairs (B) TYPE: Nucleic acid (C) STRANDEDNESS: Single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iii) ANTISENSE: NO
(vi) ORGINAL SOURCE:
(B) STRAIN: Escherichia coli (vii) IMMEDIATE SOURCE:
(B) CLONE: bioS2 (ix) FEATURES:
(A) NAME/KEY: CDS
(B) LOCATION: 1..1215 (xi) SEQUENCE DESCRIPTION: SEQ ID No: 5:
Met Lys Leu Pro Ile Tyr Leu Asp Tyr Ser Ala Thr Thr Pro Val Asp Pro Arg Val Ala Glu Lys Met Met Gln Phe Met Thr Met Asp Gly Thr Phe Gly Asn Pro Ala Ser Arg Ser His Arg Phe Gly Trp Gln Ala Glu Glu Ala Val Asp Ile Ala Arg Asn Gln Ile Ala Asp Leu Val Gly Ala 0050/48792 CA 02321264 2000-os-i4 Asp Pro Arg Glu Ile Val Phe Thr Ser Gly Ala Thr Glu Ser Asp Asn Leu Ala Ile Lys Gly Ala Ala Asn Phe Tyr Gln Lys Lys Gly Lys His Ile Ile Thr Ser Lys Thr Glu His Lys Ala Val Leu Asp Thr Cys Arg Gln Leu Glu Arg Glu Gly Phe Glu Val Thr Tyr Leu Ala Pro Gln Arg Asn Gly Ile Ile Asp Leu Lys Glu Leu Glu Ala Ala Met Arg Asp Asp Thr Ile Leu Val Ser Ile Met His Val Asn Asn Glu Ile Gly Val Val Gln Asp Ile Ala Ala Ile Gly Glu Met Cys Arg Ala Arg Gly Ile Ile Tyr His Val Asp Ala Thr Gln Ser Val Gly Lys Leu Pro Ile Asp Leu Ser Gln Leu Lys Val Asp Leu Met Ser Phe Ser Gly His Lys Ile Tyr Gly Pro Lys Gly Ile Gly Ala Leu Tyr Val Arg Arg Lys Pro Arg Val Arg Ile Glu Ala Gln Met His Gly Gly Gly His Glu Arg Gly Met Arg Ser Gly Thr Leu Pro Val His Gln Ile Val Gly Met Gly Glu Ala Tyr 0050/48792 CA 02321264 2000-os-i4 Arg Ile Ala Lys Glu Glu Met Ala Thr Glu Met Glu Arg Leu Arg Gly Leu Arg Asn Arg Leu Trp Asn Gly Ile Lys Asp Ile Glu Glu Val Tyr Leu Asn Gly Asp Leu Glu His Gly Ala Pro Asn Ile Leu Asn Val Ser Phe Asn Tyr Val Glu Gly Glu Ser Leu Ile Met Ala Leu Lys Asp Leu Ala Val Ser Ser Gly Ser Ala Cys Thr Ser Ala Ser Leu Glu Pro Ser Tyr Val Leu Arg Ala Leu Gly Leu Asn Asp Glu Leu Ala His Ser Ser Ile Arg Phe Ser Leu Gly Arg Phe Thr Thr Glu Glu Glu Ile Asp Tyr Thr Ile Glu Leu Val Arg Lys Ser Ile Gly Arg Leu Arg Asp Leu Ser Pro Leu Trp Glu Met Tyr Lys Gln Gly Val Asp Leu Asn Ser Ile Glu Trp Ala His His (2) INFORMATION FOR SEQ ID No: 6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 404 Amino acids (B) TYPE: Amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Protein 0050/48792 CA 02321264 2000-os-i4 (xi) SEQUENCE DESCRIPTION: SEQ ID No: 6:
Met Lys Leu Pro Ile Tyr Leu Asp Tyr Ser Ala Thr Thr Pro Val Asp Pro Arg Val Ala Glu Lys Met Met Gln Phe Met Thr Met Asp Gly Thr Phe Gly Asn Pro Ala Ser Arg Ser His Arg Phe Gly Trp Gln Ala Glu Glu Ala Val Asp Ile Ala Arg Asn Gln Ile Ala Asp Leu Val Gly Ala Asp Pro Arg Glu Ile Val Phe Thr Ser Gly Ala Thr Glu Ser Asp Asn Leu Ala Ile Lys Gly Ala Ala Asn Phe Tyr Gln Lys Lys Gly Lys His Ile Ile Thr Ser Lys Thr Glu His Lys Ala Val Leu Asp Thr Cys Arg Gln Leu Glu Arg Glu Gly Phe Glu Val Thr Tyr Leu Ala Pro Gln Arg Asn Gly Ile Ile Asp Leu Lys Glu Leu Glu Ala Ala Met Arg Asp Asp Thr Ile Leu Val Ser Ile Met His Val Asn Asn Glu Ile Gly Val Val Gln Asp Ile Ala Ala Ile Gly Glu Met Cys Arg Ala Arg Gly Ile Ile Tyr His Val Asp Ala Thr Gln Ser Val Gly Lys Leu Pro Ile Asp Leu Ser Gln Leu Lys Val Asp Leu Met Ser Phe Ser Gly His Lys Ile Tyr Gly Pro Lys Gly Ile Gly Ala Leu Tyr Val Arg Arg Lys Pro Arg Val Arg Ile Glu Ala Gln Met His Gly Gly Gly His Glu Arg Gly Met Arg Ser Gly Thr Leu Pro Val His Gln Ile Val Gly Met Gly Glu Ala Tyr 0050/48792 CA 02321264 2000-os-i4 Arg Ile Ala Lys Glu Glu Met Ala Thr Glu Met Glu Arg Leu Arg Gly Leu Arg Asn Arg Leu Trp Asn Gly Ile Lys Asp Ile Glu Glu Val Tyr Leu Asn Gly Asp Leu Glu His Gly Ala Pro Asn Ile Leu Asn Val Ser Phe Asn Tyr Val Glu Gly Glu Ser Leu Ile Met Ala Leu Lys Asp Leu Ala Val Ser Ser Gly Ser Ala Cys Thr Ser Ala Ser Leu Glu Pro Ser Tyr Val Leu Arg Ala Leu Gly Leu Asn Asp Glu Leu Ala His Ser Ser Ile Arg Phe Ser Leu Gly Arg Phe Thr Thr Glu Glu Glu Ile Asp Tyr Thr Ile Glu Leu Val Arg Lys Ser Ile Gly Arg Leu Arg Asp Leu Ser Pro Leu Trp Glu Met Tyr Lys Gln Gly Val Asp Leu Asn Ser Ile Glu Trp Ala His His (2) INFORMATION FOR SEQ ID No: 7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1221 Base pairs (B) TYPE: Nucleic acid (C) STRANDEDNESS: Single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNS (genomic) (iii) HYPOTHETICAL: NO
(iii) ANTISENSE: NO
(vi) ORIGINAL SOURCE:
(B) STRAIN: Escherichia coli (vii) IMMEDIATE SOURCE:
(B) CLONE: bioS3 0050/48792 CA 02321264 2000-os-i4 (ix) FEATURES:
(A) NAME/KEY: CDS
(B) LOCATION: 1..1221 (xi) SEQUENCE DESCRIPTION: SEQ ID No: 7:
Met Ile Phe Ser Val Asp Lys Val Arg Ala Asp Phe Pro Val Leu Ser Arg Glu Val Asn Gly Leu Pro Leu Ala Tyr Leu Asp Ser Ala Ala Ser Ala Gln Lys Pro Ser Gln Val Ile Asp Ala Glu Ala Glu Phe Tyr Arg His Gly Tyr Ala Ala Val His Arg Gly Ile His Thr Leu Ser Ala Gln Ala Thr Glu Lys Met Glu Asn Val Arg Lys Arg Ala Ser Leu Phe Ile Asn Ala Arg Ser Ala Glu Glu Leu Val Phe Val Arg Gly Thr Thr Glu Gly Ile Asn Leu Val Ala Asn Ser Trp Gly Asn Ser Asn Val Arg Ala Gly Asp Asn Ile Ile Ile Ser Gln Met Glu His His Ala Asn Ile Val Pro Trp Gln Met Leu Cys Ala Arg Val Gly Ala Glu Leu Arg Val I1e Pro Leu Asn Pro Asp Gly Thr Leu Gln Leu Glu Thr Leu Pro Thr Leu Phe Asp Glu Lys Thr Arg Leu Leu Ala Ile Thr His Val Ser Asn Val 0050/48792 CA 02321264 2000-os-i4 Leu Gly Thr Glu Asn Pro Leu Ala Glu Met Ile Thr Leu Ala His Gln His Gly Ala Lys Val Leu Val Asp Gly Ala Gln Ala Val Met His His Pro Val Asp Val Gln Ala Leu Asp Cys Asp Phe Tyr Val Phe Ser Gly His Lys Leu Tyr Gly Pro Thr Gly Ile Gly Ile Leu Tyr Val Lys Glu Ala Leu Leu Gln Glu Met Pro Pro Trp Glu Gly Gly Gly Ser Met Ile Ala Thr Val Ser Leu Ser Glu Gly Thr Thr Trp Thr Lys Ala Pro Trp Arg Phe Glu Ala Gly Thr Pro Asn Thr Gly Gly Ile Ile Gly Leu Gly Ala Ala Leu Glu Tyr Val Ser Ala Leu Gly Leu Asn Asn Ile Ala Glu Tyr Glu Gln Asn Leu Met His Tyr Ala Leu Ser Gln Leu Glu Ser Val Pro Asp Leu Thr Leu Tyr Gly Pro Gln Asn Arg Leu Gly Val Ile Ala Phe Asn Leu Gly Lys His His Ala Tyr Asp Val Gly Ser Phe Leu Asp Asn Tyr Gly Ile Ala Val Arg Thr Gly His His Cys Ala Met Prv Leu 0050/48792 CA 02321264 2000-os-i4 Met Ala Tyr Tyr Asn Val Pro Ala Met Cys Arg Ala Ser Leu Ala Met Tyr Asn Thr His Glu Glu Val Asp Arg Leu Val Thr Gly Leu Gln Arg Ile His Arg Leu Leu Gly (2) INFORMATION FOR SEQ ID No: 8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 406 Amino acids (B) TYPE: Amino acid (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: Protein (xi) SEQUENCE DESCRIPTION: SEQ ID No: 8:
Met Ile Phe Ser Val Asp Lys Val Arg Ala Asp Phe Pro Val Leu Ser Arg Glu Val Asn Gly Leu Pro Leu Ala Tyr Leu Asp Ser Ala Ala Ser Ala Gln Lys Pro Ser Gln Val Ile Asp Ala Glu Ala Glu Phe Tyr Arg His Gly Tyr Ala Ala Val His Arg Gly Ile His Thr Leu Ser Ala Gln Ala Thr Glu Lys Met Glu Asn Val Arg Lys Arg Ala Ser Leu Phe Ile Asn Ala Arg Ser Ala Glu Glu Leu Val Phe Val Arg Gly Thr Thr Glu Gly Ile Asn Leu Val Ala Asn Ser Trp Gly Asn Ser Asn Val Arg Ala Gly Asp Asn Ile Ile Ile Ser Gln Met Glu His His Ala Asn Ile Val Pro Trp Gln Met Leu Cys Ala Arg Val Gly Ala Glu Leu Arg Val Ile 0050/48792 CA 02321264 2000-os-i4 Pro Leu Asn Pro Asp Gly Thr Leu Gln Leu Glu Thr Leu Pro Thr Leu Phe Asp Glu Lys Thr Arg Leu Leu Ala Ile Thr His Val Ser Asn Val Leu Gly Thr Glu Asn Pro Leu Ala Glu Met Ile Thr Leu Ala His Gln His Gly Ala Lys Val Leu Val Asp Gly Ala Gln Ala Val Met His His Pro Val Asp Val Gln Ala Leu Asp Cys Asp Phe Tyr Val Phe Ser Gly His Lys Leu Tyr Gly Pro Thr Gly Ile Gly Ile Leu Tyr Val Lys Glu Ala Leu Leu Gln Glu Met Pro Pro Trp Glu Gly Gly Gly Ser Met Ile Ala Thr Val Ser Leu Ser Glu Gly Thr Thr Trp Thr Lys Ala Pro Trp Arg Phe Glu Ala Gly Thr Pro Asn Thr Gly Gly Ile Ile Gly Leu Gly Ala Ala Leu Glu Tyr Val Ser Ala Leu Gly Leu Asn Asn Ile Ala Glu Tyr Glu Gln Asn Leu Met His Tyr Ala Leu Ser Gln Leu Glu Ser Val Pro Asp Leu Thr Leu Tyr Gly Pro Gln Asn Arg Leu Gly Val Ile Ala Phe Asn Leu Gly Lys His His Ala Tyr Asp Val Gly Ser Phe Leu Asp Asn Tyr Gly Ile Ala Val Arg Thr Gly His His Cys Ala Met Pro Leu Met Ala Tyr Tyr Asn Val Pro Ala Met Cys Arg Ala Ser Leu Ala Met Tyr Asn Thr His Glu Glu Val Asp Arg Leu Val Thr Gly Leu Gln Arg Ile His Arg Leu Leu Gly 0050/48792 CA 02321264 2000-os-i4 (2) INFORMATION FOR SEQ ID No: 9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 3720 Base pairs (B) TYPE: Nucleic acid (C) STRANDEDNESS: Single (D) TOPOLOGY: circular (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iii) ANTISENSE: NO
(vii) IMMEDIATE SOURCE:
(B) CLONE: pHSl metK
(ix) FEATURES:
(A) NAME/KEY: CDS
(B) LOCATION: 530..1684 (xi) SEQUENCE DESCRIPTION: SEQ ID No: 9:
AGGTAGTTAT CCTTAAAAAP. GCCACAGCAT ACATCCTGTC CGTCCAAGCA GAGGAGCAAA 240 Met Ala Lys His Leu Phe Thr Ser Glu Ser Val Ser Glu Gly His Pro Asp Lys Ile Ala Asp Gln Ile Ser Asp Ala Val Leu Asp Ala Ile Leu Glu Gln 0050/48792 CA 02321264 2000-os-i4 Asp Pro Lys Ala Arg Val Ala Cys Glu Thr Tyr Val Lys Thr Gly Met Val Leu Val Gly Gly Glu Ile Thr Thr Ser Ala Trp Val Asp Ile Glu Glu Ile Thr Arg Asn Thr Val Arg Glu Ile Gly Tyr Val His Ser Asp Met Gly Phe Asp Ala Asn Ser Cys Ala Val Leu Ser Ala Ile Gly Lys Gln Ser Pro Asp Ile Asn Gln Gly Val Asp Arg Ala Asp Pro Leu Glu Gln Gly Ala Gly Asp Gln Gly Leu Met Phe Gly Tyr Ala Thr Asn Glu Thr Asp Val Leu Met Pro Ala Pro Ile Thr Tyr Ala His Arg Leu Val Gln Arg Gln Ala Glu Val Arg Lys Asn Gly Thr Leu Pro Trp Leu Arg Pro Asp Ala Lys Ser Gln Val Thr Phe Gln Tyr Asp Asp Gly Lys Ile Val Gly Ile Asp Ala Val Val Leu Ser Thr Gln His Ser Glu Glu Ile Asp Gln Lys Ser Leu Gln Glu Ala Val Met Glu Glu Ile Ile Lys Pro Ile Leu Pro Ala Glu Trp Leu Thr Ser Ala Thr Lys Phe Phe Ile Asn 0050/48792 CA 02321264 2000-os-i4 Pro Thr Gly Arg Phe Val Ile Gly Gly Pro Met Gly Asp Cys Gly Leu Thr Gly Arg Lys Ile Ile Val Asp Thr Tyr Gly Gly Met Ala Arg His Gly Gly Gly Ala Phe Ser Gly Lys Asp Pro Ser Lys Val Asp Arg Ser Ala Ala Tyr Ala Ala Arg Tyr Val Ala Lys Asn Ile Val Ala Ala Gly Leu Ala Asp Arg Cys Glu Ile Gln Val Ser Tyr Ala Ile Gly Val Ala Glu Pro Thr Ser Ile Met Val Glu Thr Phe Gly Thr Glu Lys Val Pro Ser Glu Gln Leu Thr Leu Leu Val Arg Glu Phe Phe Asp Leu Arg Pro Tyr Gly Leu Ile Gln Met Leu Asp Leu Leu His Pro Ile Tyr Lys Glu Thr Ala Ala Tyr Gly His Phe Gly Arg Glu His Phe Pro Trp Glu Lys Thr Asp Lys Ala Gln Leu Leu Arg Asp Ala Ala Gly Leu Lys 0050/48792 CA 02321264 2000-os-i4 0050/48792 CA 02321264 2000-os-i4 (2) INFORMATION FOR SEQ ID No: 10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 384 Amino acids (B) TYPE: Amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Protein (xi) SEQUENCE DESCRIPTION: SEQ ID No: 10:
Met Ala Lys His Leu Phe Thr Ser Glu Ser Val Ser Glu Gly His Pro Asp Lys Ile Ala Asp Gln Ile Ser Asp Ala Val Leu Asp Ala Ile Leu Glu Gln Asp Pro Lys Ala Arg Val Ala Cys Glu Thr Tyr Val Lys Thr Gly Met Val Leu Val Gly Gly Glu Ile Thr Thr Ser Ala Trp Val Asp Ile Glu Glu Ile Thr Arg Asn Thr Val Arg Glu Ile Gly Tyr Val His Ser Asp Met Gly Phe Asp Ala Asn Ser Cys Ala Val Leu Ser Ala Ile Gly Lys Gln Ser Pro Asp Ile Asn Gln Gly Val Asp Arg Ala Asp Pro Leu Glu Gln Gly Ala Gly Asp Gln Gly Leu Met Phe Gly Tyr Ala Thr Asn Glu Thr Asp Val Leu Met Pro Ala Pro Ile Thr Tyr Ala His Arg Leu Val Gln Arg Gln Ala Glu Val Arg Lys Asn Gly Thr Leu Pro Trp 0050/48792 CA 02321264 2000-os-i4 Leu Arg Pro Asp Ala Lys Ser Gln Val Thr Phe Gln Tyr Asp Asp Gly Lys Ile Val Gly Ile Asp Ala Val Val Leu Ser Thr Gln His Ser Glu Glu Ile Asp Gln Lys Ser Leu Gln Glu Ala Val Met Glu Glu Ile Ile Lys Pro Ile Leu Pro Ala Glu Trp Leu Thr Ser Ala Thr Lys Phe Phe Ile Asn Pro Thr Gly Arg Phe Val Ile Gly Gly Pro Met Gly Asp Cys Gly Leu Thr Gly Arg Lys Ile Ile Val Asp Thr Tyr Gly Gly Met Ala Arg His Gly Gly Gly Ala Phe Ser Gly Lys Asp Pro Ser Lys Val Asp Arg Ser Ala Ala Tyr Ala Ala Arg Tyr Val Ala Lys Asn Ile Val Ala Ala Gly Leu Ala Asp Arg Cys Glu Ile Gln Val Ser Tyr Ala Ile Gly Val Ala Glu Pro Thr Ser Ile Met Val Glu Thr Phe Gly Thr Glu Lys Val Pro Ser Glu Gln Leu Thr Leu Leu Val Arg Glu Phe Phe Asp Leu Arg Pro Tyr Gly Leu Ile Gln Met Leu Asp Leu Leu His Pro Ile Tyr Lys Glu Thr Ala Ala Tyr Gly His Phe Gly Arg Glu His Phe Pro Trp Glu Lys Thr Asp Lys Ala Gln Leu Leu Arg Asp Ala Ala Gly Leu Lys (2) INFORMATION FOR SEQ ID No: 11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 3794 Base pairs (B) TYPE: Nucleic acid (C) STRANDEDNESS: Single (D) TOPOLOGY: circular 0050/48792 CA 02321264 2000-os-i4 (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iii) ANTISENSE: NO
(vii) IMMEDIATE SOURCE:
(B) CLONE: pHS1 bioS1 (ix) FEATURES:
(A) NAME/KEY: CDS
(B) LOCATION: 601..1806 (xi) SEQUENCE DESCRIPTION: SEQ ID No: 11:
Met Asn Val Phe Asn Pro Ala Gln Phe Arg Ala Gln Phe Pro Ala Leu Gln Asp Ala Gly Val Tyr Leu Asp Ser Ala Ala Thr Ala Leu Lys Pro Glu Ala Val Val Glu Ala Thr Gln Gln Phe Tyr Ser Leu Ser Ala Gly Asn Val His Arg Ser Gln Phe Ala Glu Ala Gln Arg Leu Thr Ala Arg Tyr Glu Ala Ala Arg Glu Lys Val Ala Gln Leu Leu Asn Ala Pro Asp 65 70 75 g0 Asp Lys Thr Ile Val Trp Thr Arg Gly Thr Thr Glu Ser Ile Asn Met Val Ala Gln Cys Tyr Ala Arg Pro Arg Leu Gln Pro Gly Asp Glu Ile Ile Val Ser Val Ala Glu His His Ala Asn Leu Val Pro Trp Leu Met Val Ala Gln Gln Thr Gly Ala Lys Val Val Lys Leu Pro Leu Asn Ala Gln Arg Leu Pro Asp Val Asp Leu Leu Pro Glu Leu Ile Thr Pro Arg Ser Arg Ile Leu Ala Leu Gly Gln Met Ser Asn Val Thr Gly Gly Cys Pro Asp Leu Ala Arg Ala Ile Thr Phe Ala His Ser Ala Gly Met Val Val Met Val Asp Gly Ala Gln Gly Ala Val His Phe Pro Ala Asp Val Gln Gln Leu Asp Ile Asp Phe Tyr Ala Phe Ser Gly His Lys Leu Tyr Gly Pro Thr Gly Ile Gly Val Leu Tyr Gly Lys Ser Glu Leu Leu Glu Ala Met Ser Pro Trp Leu Gly Gly Gly Lys Met Val His Glu Val Ser 0050/48792 CA 02321264 2000-os-i4 Phe Asp Gly Phe Thr Thr Gln Ser Ala Pro Trp Lys Leu Glu Ala Gly Thr Pro Asn Val Ala Gly Val Ile Gly Leu Ser Ala Ala Leu Glu Trp Leu Ala Asp Tyr Asp Ile Asn Gln Ala Glu Ser Trp Ser Arg Ser Leu Ala Thr Leu Ala Glu Asp Ala Leu Ala Lys Arg Pro Gly Phe Arg Ser Phe Arg Cys Gln Asp Ser Ser Leu Leu Ala Phe Asp Phe Ala Gly Val His His Ser Asp Met Val Thr Leu Leu Ala Glu Tyr Gly Ile Ala Leu Arg Ala Gly Gln His Cys Ala Gln Pro Leu Leu Ala Glu Leu Gly Val Thr Gly Thr Leu Arg Ala Ser Phe Ala Pro Tyr Asn Thr Lys Ser Asp Val Asp Ala Leu Val Asn Ala Val Asp Arg Ala Leu Glu Leu Leu Val Asp 0050./48792 CA 02321264 2000-os-i4 0050/48792 CA 02321264 2000-os-i4 (2) INFORMATION FOR SEQ ID No: 12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 401 Amino acids (B) TYPE: Amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Protein (xi) SEQUENCE DESCRIPTION: SEQ ID No: 12:
Met Asn Val Phe Asn Pro Ala Gln Phe Arg Ala Gln Phe Pro Ala Leu Gln Asp Ala Gly Val Tyr Leu Asp Ser Ala Ala Thr Ala Leu Lys Pro Glu Ala Val Val Glu Ala Thr Gln Gln Phe Tyr Ser Leu Ser Ala Gly Asn Val His Arg Ser Gln Phe Ala Glu Ala Gln Arg Leu Thr Ala Arg 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 Val Ala Gln Cys Tyr Ala Arg Pro Arg Leu Gln Pro Gly Asp Glu Ile Ile Val Ser Val Ala Glu His His Ala Asn Leu Val Pro Trp Leu Met Val Ala Gln Gln Thr Gly Ala Lys Val Val Lys Leu Pro Leu Asn Ala Gln Arg Leu Pro Asp Val Asp Leu Leu Pro Glu Leu Ile Thr Pro Arg Ser Arg Ile Leu Ala Leu Gly Gln Met Ser Asn Val Thr Gly Gly Cys Pro Asp Leu Ala Arg Ala Ile Thr Phe Ala His Ser Ala Gly Met Val Val Met Val Asp Gly Ala Gln Gly Ala Val His Phe Pro Ala Asp Val Gln Gln Leu Asp Ile Asp Phe Tyr Ala Phe Ser Gly His Lys Leu Tyr Gly Pro Thr Gly Ile Gly Val Leu Tyr Gly Lys Ser Glu Leu Leu Glu Ala Met Ser Pro Trp Leu Gly Gly Gly Lys Met Val His Glu Val Ser Phe Asp Gly Phe Thr Thr Gln Ser Ala Pro Trp Lys Leu Glu Ala Gly Thr Pro Asn Val Ala Gly Val Ile Gly Leu Ser Ala Ala Leu Glu Trp Leu Ala Asp Tyr Asp Ile Asn Gln Ala Glu Ser Trp Ser Arg Ser Leu Ala Thr Leu Ala Glu Asp Ala Leu Ala Lys Arg Pro Gly Phe Arg Ser Phe Arg Cys Gln Asp Ser Ser Leu Leu Ala Phe Asp Phe Ala Gly Val His His Ser Asp Met Val Thr Leu Leu Ala Glu Tyr Gly Ile Ala Leu Arg Ala Gly Gln His Cys Ala Gln Pro Leu Leu Ala Glu Leu Gly Val Thr Gly Thr Leu Arg Ala Ser Phe Ala Pro Tyr Asn Thr Lys Ser Asp Val Asp Ala Leu Val Asn Ala Val Asp Arg Ala Leu Glu Leu Leu Val Asp (2) INFORMATION FOR SEQ ID No: 13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4975 Base pairs (B) TYPE: Nucleic acid 0050/48792 CA 02321264 2000-os-i4 (C) STRANDEDNESS: Single (D) TOPOLOGY: circular (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iii) ANTISENSE: NO
(vii) IMMEDIATE SOURCE:
(B) CLONE: pHS1 metK bioS1 (ix) FEATURES:
(A) NAME/KEY: CDS
(B) LOCATION: 1782..2987 (ix) FEATURES:
(A) NAME/KEY: CDS
(B) LOCATION: 530..1684 (xi) SEQUENCE DESCRIPTION: SEQ ID No: 13:
Met Ala Lys His Leu Phe Thr Ser Glu Ser Val Ser Glu Gly His Pro Asp Lys Ile Ala Asp Gln Ile Ser Asp Ala Val Leu Asp Ala Ile Leu Glu Gln 0050/48792 CA 02321264 2000-os-i4 Asp Pro Lys Ala Arg Val Ala Cys Glu Thr Tyr Val Lys Thr Gly Met Val Leu Val Gly Gly Glu Ile Thr Thr Ser Ala Trp Val Asp Ile Glu Glu Ile Thr Arg Asn Thr Val Arg Glu Ile Gly Tyr Val His Ser Asp ATG GGC TTT GAC GCT AAC TCC TGT GCG GTT CTG AGC GCT ATC GGC AAA $23 Met Gly Phe Asp Ala Asn Ser Cys Ala Val Leu Ser Ala Ile Gly Lys Gln Ser Pro Asp Ile Asn Gln Gly Val Asp Arg Ala Asp Pro Leu Glu Gln Gly Ala Gly Asp Gln Gly Leu Met Phe Gly Tyr Ala Thr Asn Glu Thr Asp Val Leu Met Pro Ala Pro Ile Thr Tyr Ala His Arg Leu Val Gln Arg Gln Ala Glu Val Arg Lys Asn Gly Thr Leu Pro Trp Leu Arg Pro Asp Ala Lys Ser Gln Val Thr Phe Gln Tyr Asp Asp Gly Lys Ile Val Gly Ile Asp Ala Val Val Leu Ser Thr Gln His Ser Glu Glu Ile Asp Gln Lys Ser Leu Gln Glu Ala Val Met Glu Glu Ile Ile Lys Pro Ile Leu Pro Ala Glu Trp Leu Thr Ser Ala Thr Lys Phe Phe Ile Asn 0050/48792 CA 02321264 2000-os-i4 Pro Thr Gly Arg Phe Val Ile Gly Gly Pro Met Gly Asp Cys Gly Leu Thr Gly Arg Lys Ile Ile Val Asp Thr Tyr Gly Gly Met Ala Arg His Gly Gly Gly Ala Phe Ser Gly Lys Asp Pro Ser Lys Val Asp Arg Ser Ala Ala Tyr Ala Ala Arg Tyr Val Ala Lys Asn Ile Val Ala Ala Gly Leu Ala Asp Arg Cys Glu Ile Gln Val Ser Tyr Ala Ile Gly Val Ala Glu Pro Thr Ser Ile Met Val Glu Thr Phe Gly Thr Glu Lys Val Pro Ser Glu Gln Leu Thr Leu Leu Val Arg Glu Phe Phe Asp Leu Arg Pro Tyr Gly Leu Ile Gln Met Leu Asp Leu Leu His Pro Ile Tyr Lys Glu Thr Ala Ala Tyr Gly His Phe Gly Arg Glu His Phe Pro Trp Glu Lys Thr As.p Lys Ala Gln Leu Leu Arg Asp Ala Ala Gly Leu Lys Met Asn Val Phe Asn Pro Ala Gln Phe Arg Ala Gln Phe Pro Ala Leu Gln Asp Ala Gly Val Tyr Leu Asp 0050/48792 CA 02321264 2000-os-i4 Ser Ala Ala Thr Ala Leu Lys Pro Glu Ala Val Val Glu Ala Thr Gln Gln Phe Tyr Ser Leu Ser Ala Gly Asn Val His Arg Ser Gln Phe Ala Glu Ala Gln Arg Leu Thr Ala Arg Tyr Glu Ala Ala Arg Glu Lys Val Ala Gln Leu Leu Asn Ala Pro Asp Asp Lys Thr Ile Val Trp Thr Arg Gly Thr Thr Glu Ser Ile Asn Met Val Ala Gln Cys Tyr Ala Arg Pro Arg Leu Gln Pro Gly Asp Glu Ile Ile Val Ser Val Ala Glu His His Ala Asn Leu Val Pro Trp Leu Met Val Ala Gln Gln Thr Gly Ala Lys Val Val Lys Leu Pro Leu Asn Ala Gln Arg Leu Pro Asp Val Asp Leu Leu Pro Glu Leu Ile Thr Pro Arg Ser Arg Ile Leu Ala Leu Gly Gln Met Ser Asn Val Thr Gly Gly Cys Pro Asp Leu Ala Arg Ala Ile Thr Phe Ala His Ser Ala Gly Met Val Val Met Val Asp Gly Ala Gln Gly Ala Val His Phe Pro Ala Asp Val Gln Gln Leu Asp Ile Asp Phe Tyr 0050/48792 CA 02321264 2000-os-i4 Ala Phe Ser Gly His Lys Leu Tyr Gly Pro Thr Gly Ile Gly Val Leu Tyr Gly Lys Ser Glu Leu Leu Glu Ala Met Ser Pro Trp Leu Gly Gly Gly Lys Met Val His Glu Val Ser Phe Asp Gly Phe Thr Thr Gln Ser Ala Pro Trp Lys Leu Glu Ala Gly Thr Pro Asn Val Ala Gly Val Ile Gly Leu Ser Ala Ala Leu Glu Trp Leu Ala Asp Tyr Asp Ile Asn Gln Ala Glu Ser Trp Ser Arg Ser Leu Ala Thr Leu Ala Glu Asp Ala Leu Ala Lys Arg Pro Gly Phe Arg Ser Phe Arg Cys Gln Asp Ser Ser Leu Leu Ala Phe Asp Phe Ala Gly Val His His Ser Asp Met Val Thr Leu Leu Ala Glu Tyr Gly Ile Ala Leu Arg Ala Gly Gln His Cys Ala Gln Pro Leu Leu Ala Glu Leu Gly Val Thr Gly Thr Leu Arg Ala Ser Phe Ala Prv Tyr Asn Thr Lys Ser Asp Val Asp Ala Leu Val Asn Ala Val Asp Arg Ala Leu Glu Leu Leu Val Asp 0050/48792 CA 02321264 2000-os-i4 0050/48792 CA 02321264 2000-os-i4 (2) INFORMATION FOR SEQ ID No: 14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 384 Amino acids (B) TYPE: Amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Protein (xi) SEQUENCE DESCRIPTION: SEQ ID No:
14:
Met Ala Lys His Leu Phe Thr Ser Glu Ser Ser Glu His Pro Val Gly Asp Lys Ile Ala Asp Gln Ile Ser Asp Ala Leu Asp Ile Leu Val Ala Glu Gln Asp Pro Lys Ala Arg Val Ala Cys Thr Tyr Lys Thr Glu Val Gly Met Val Leu Val Gly Gly Glu Ile Thr Ser Ala Val Asp Thr Trp Ile Glu Glu Ile Thr Arg Asn Thr Val Arg Ile Gly Val His Glu Tyr Ser Asp Met Gly Phe Asp Ala Asn Ser Cys Val Leu Ala Ile Ala Ser Gly Lys Gln Ser Pro Asp Ile Asn Gln Gly Val Asp Arg Ala Asp Pro Leu Glu Gln Gly Ala Gly Asp Gln Gly Leu Met Phe Gly Tyr Ala Thr 0050/48792 CA 02321264 2000-os-i4 Asn Glu Thr Asp Val Leu Met Pro Ala Pro Ile Thr Tyr Ala His Arg Leu Val Gln Arg Gln Ala Glu Val Arg Lys Asn Gly Thr Leu Pro Trp Leu Arg Pro Asp Ala Lys Ser Gln Val Thr Phe Gln Tyr Asp Asp Gly Lys Ile Val Gly Ile Asp Ala Val Val Leu Ser Thr Gln His Ser Glu Glu Ile Asp Gln Lys Ser Leu Gln Glu Ala Val Met Glu Glu Ile Ile Lys Pro Ile Leu Pro Ala Glu Trp Leu Thr Ser Ala Thr Lys Phe Phe Ile Asn Pro Thr Gly Arg Phe Val Ile Gly Gly Pro Met Gly Asp Cys Gly Leu Thr Gly Arg Lys Ile Ile Val Asp Thr Tyr Gly Gly Met Ala Arg His Gly Gly Gly Ala Phe Ser Gly Lys Asp Pro Ser Lys Val Asp Arg Ser Ala Ala Tyr Ala Ala Arg Tyr Val Ala Lys Asn Ile Val Ala Ala Gly Leu Ala Asp Arg Cys Glu Ile Gln Val Ser Tyr Ala Ile Gly Val Ala Glu Pro Thr Ser Ile Met Val Glu Thr Phe Gly Thr Glu Lys Val Pro Ser Glu Gln Leu Thr Leu Leu Val Arg Glu Phe Phe Asp Leu Arg Pro Tyr Gly Leu Ile Gln Met Leu Asp Leu Leu His Pro Ile Tyr Lys Glu Thr Ala Ala Tyr Gly His Phe Gly Arg Glu His Phe Pro Trp Glu Lys Thr Asp Lys Ala Gln Leu Leu Arg Asp Ala Ala Gly Leu Lys (2) INFORMATION FOR SEQ ID No: 15:
0050/48792 CA 02321264 2000-os-i4 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 401 Amino acids (B) TYPE: Amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Protein (xi) SEQUENCE DESCRIPTION: SEQ ID No: 15:
Met Asn Val Phe Asn Pro Ala Gln Phe Arg Ala Gln Phe Pro Ala Leu Gln Asp Ala Gly Val Tyr Leu Asp Ser Ala Ala Thr Ala Leu Lys Pro Glu Ala Val Val Glu Ala Thr Gln Gln Phe Tyr Ser Leu Ser Ala Gly Asn Val His Arg Ser Gln Phe Ala Glu Ala Gln Arg Leu Thr Ala Arg Tyr Glu Ala Ala Arg Glu Lys Val Ala Gln Leu Leu Asn Ala Pro Asp Asp Lys Thr Ile Val Trp Thr Arg Gly Thr Thr Glu Ser Ile Asn Met Val Ala Gln Cys Tyr Ala Arg Pro Arg Leu Gln Pro Gly Asp Glu Ile Ile Val Ser Val Ala Glu His His Ala Asn Leu Val Pro Trp Leu Met Val Ala Gln Gln Thr Gly Ala Lys Val Val Lys Leu Pro Leu Asn Ala Gln Arg Leu Pro Asp Val Asp Leu Leu Pro Glu Leu Ile Thr Pro Arg Ser Arg Ile Leu Ala Leu Gly Gln Met Ser Asn Val Thr Gly Gly Cys Pro Asp Leu Ala Arg Ala Ile Thr Phe Ala His Ser Ala Gly Met Val Val Met Val Asp Gly Ala Gln Gly Ala Val His Phe Pro Ala Asp Val Gln Gln Leu Asp Ile Asp Phe Tyr Ala Phe Ser Gly His Lys Leu Tyr 0050/48792 CA 02321264 2000-os-i4 Gly Pro Thr Gly Ile Gly Val Leu Tyr Gly Lys Ser Glu Leu Leu Glu Ala Met Ser Pro Trp Leu Gly Gly Gly Lys Met Val His Glu Val Ser Phe Asp Gly Phe Thr Thr Gln Ser Ala Pro Trp Lys Leu Glu Ala Gly Thr Pro Asn Val Ala Gly Val Ile Gly Leu Ser Ala Ala Leu Glu Trp Leu Ala Asp Tyr Asp Ile Asn Gln Ala Glu Ser Trp Ser Arg Ser Leu Ala Thr Leu Ala Glu Asp Ala Leu Ala Lys Arg Pro Gly Phe Arg Ser Phe Arg Cys Gln Asp Ser Ser Leu Leu Ala Phe Asp Phe Ala Gly Val His His Ser Asp Met Val Thr Leu Leu Ala Glu Tyr Gly Ile Ala Leu Arg Ala Gly Gln His Cys Ala Gln Pro Leu Leu Ala Glu Leu Gly Val Thr Gly Thr Leu Arg Ala Ser Phe Ala Pro Tyr Asn Thr Lys Ser Asp Val Asp Ala Leu Val Asn Ala Val Asp Arg Ala Leu Glu Leu Leu Val Asp
Spontaneously rifampicin-resistant colonies were isolated from strain BM4086 (Ketner and Campbell J. Molec. Biology 1975 96:13) 10 by plating on rifampicin plates. A P1 lysate was generated from one of these resistant strains. The strain W3110 was transduced with this P1 lysate and clones were subsequently selected using rifampicin. The resulting strain was transformed with plasmid pHBbiol4 using the CaClz method (Maniatis et al. Molecular Cloning 15 Cols Spring Harbour Laboratory Press 1989) and grown on LB
containing 100 ~tg of ampicillin/ml. The isolated, transformed strain (LU5560) was in each case transformed with plasmid pHSl, pHS1 metK, pHS1 bioS1 or pHS1 metK bioSl using the CaCl2 method and then selected on LB agar containing 100 ~,g of ampicillin/ml and 25 ~,g of kanamycin/ml.
One colony from each of the transformants was in each case inoculated into a DYT culture containing the appropriate antibiotics and incubated for 12 h. The overnight culture (= ONC) was used to inoculate a 10 ml culture in TB medium (Sambrook, J.
Fritsch, E F. Maniatis, T. 2nd ed. Cold Spring Harbor Laboratory Press., 1989 ISBN 0-87969-373-8), which contained g of glycerol/1 and the appropriate antibiotics. In the cases where plasmids pHSl, pHS1 metK, pHSlbioSl and pHS1 metK bioS1 30 were present, imM IPTG and 0.5~ arabinose were added simultaneously in order to induce expression of the metK and bioS1 genes or, respectively, the combination of the two genes.
After 24 h, the cells were separated off from the culture supernatant by centrifugation and the biotin concentration in the Supernatant was determined by means of a competitive ELISA
employing streptavidin. The results of this determination are shown in Table I.
Table I: Determination of the biotin concentration Strain Plasmid I Plasmid II Biotin mg/1 LU5580 pHBbiol4 Control, without ~ 11 plasmid LU5580 pHBbiol4 pHSl 25 LU5580 pHBbiol4 pHS1 bioSl 45 LU5580 pHBbiol4 pHSl metK 37 LU5580 pHBbiol4 pHSl metK bioSi 52 0050/48792 CA 02321264 2000-os-i4 SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT:
(A) NAME: BASF Aktiengesellschaft (B) STREET: Karl Bosch Strasse (C) CITY: Ludwigshafen (D) FEDERAL STATE: Rheinland-Pfalz (E) COUNTRY: Germany (F) POSTAL CODE: 67056 (ii) TITLE OF APPLICATION: Process for preparing biotin (iii) NUMBER OF SEQUENCES: 15 (iv) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.25 (EPO) (2) INFORMATION FOR SEQ ID No: 1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1155 Base pairs (B) TYPE: Nucleic acid (C) STRANDEDNESS: Single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iii) ANTISENSE: NO
(vi) ORIGINAL SOURCE:
(B) STRAIN: Escherichia coli (vii) IMMEDIATE SOURCE:
(B) CLONE: metK
(ix) FEATURES:
(A) NAME/KEY: CDS
(B) LOCATION: 1..1155 (xi) SEQUENCE DESCRIPTION: SEQ ID No: 1:
0050/48792 CA 02321264 2000-os-i4 Met Ala Lys His Leu Phe Thr Ser Glu Ser Val Ser Glu Gly His Pro Asp Lys Ile Ala Asp Gln Ile Ser Asp Ala Val Leu Asp Ala Ile Leu Glu Gln Asp Pro Lys Ala Arg Val Ala Cys Glu Thr Tyr Val Lys Thr Gly Met Val Leu Val Gly Gly Glu Ile Thr Thr Ser Ala Trp Val Asp Ile Glu Glu Ile Thr Arg Asn Thr Val Arg Glu Ile Gly Tyr Val His Ser Asp Met Gly Phe Asp Ala Asn Ser Cys Ala Val Leu Ser Ala Ile Gly Lys Gln Ser Pro Asp Ile Asn Gln Gly Val Asp Arg Ala Asp Pro Leu Glu Gln Gly Ala Gly Asp Gln Gly Leu Met Phe Gly Tyr Ala Thr Asn Glu Thr Asp Val Leu Met Pro Ala Pro Ile Thr Tyr Ala His Arg Leu Val Gln Arg Gln Ala Glu Val Arg Lys Asn Gly Thr Leu Pro Trp Leu Arg Pro Asp Ala Lys Ser Gln Val Thr Phe Gln Tyr Asp Asp Gly Lys Ile Val Gly Ile Asp Ala Val Val Leu Ser Thr Gln His Ser Glu 0050/48792 CA 02321264 2000-os-i4 Glu Ile Asp Gln Lys Ser Leu Gln Glu Ala Val Met Glu Glu Ile Ile Lys Pro Ile Leu Pro Ala Glu Trp Leu Thr Ser Ala Thr Lys Phe Phe Ile Asn Pro Thr Gly Arg Phe Val Ile Gly Gly Pro Met Gly Asp Cys Gly Leu Thr Gly Arg Lys Ile Ile Val Asp Thr Tyr Gly Gly Met Ala Arg His Gly Gly Gly Ala Phe Ser Gly Lys Asp Pro Ser Lys Val Asp Arg Ser Ala Ala Tyr Ala Ala Arg Tyr Val Ala Lys Asn Ile Val Ala Ala Gly Leu Ala Asp Arg Cys Glu Ile Gln Val Ser Tyr Ala Ile Gly Val Ala Glu Pro Thr Ser Ile Met Val Glu Thr Phe Gly Thr Glu Lys Val Pro Ser Glu Gln Leu Thr Leu Leu Val Arg Glu Phe Phe Asp Leu Arg Pro Tyr Gly Leu Ile Gln Met Leu Asp Leu Leu His Pro Ile Tyr Lys Glu Thr Ala Ala Tyr Gly His Phe Gly Arg Glu His Phe Pro Trp Glu Lys Thr Asp Lys Ala Gln Leu Leu Arg Asp Ala Ala Gly Leu Lys T~ 1155 (2) INFORMATION FOR SEQ ID No: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 384 Amino acids (B) TYPE: Amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Protein (xi) SEQUENCE DESCRIPTION: SEQ ID No: 2:
Met Ala Lys His Leu Phe Thr Ser Glu Ser Val Ser Glu Gly His Pro Asp Lys Ile Ala Asp Gln Ile Ser Asp Ala Val Leu Asp Ala Ile Leu Glu Gln Asp Pro Lys Ala Arg Val Ala Cys Glu Thr Tyr Val Lys Thr Gly Met Val Leu Val Gly Gly Glu Ile Thr Thr Ser Ala Trp Val Asp Ile Glu Glu Ile Thr Arg Asn Thr Val Arg Glu Ile Gly Tyr Val His Ser Asp Met Gly Phe Asp Ala Asn Ser Cys Ala Val Leu Ser Ala Ile Gly Lys Gln Ser Pro Asp Ile Asn Gln Gly Val Asp Arg Ala Asp Pro Leu Glu Gln Gly Ala Gly Asp Gln Gly Leu Met Phe Gly Tyr Ala Thr Asn Glu Thr Asp Val Leu Met Pro Ala Pro Ile Thr Tyr Ala His Arg Leu Val Gln Arg Gln Ala Glu Val Arg Lys Asn Gly Thr Leu Pro Trp Leu Arg Pro Asp Ala Lys Ser Gln Val Thr Phe Gln Tyr Asp Asp Gly Lys Ile Val Gly Ile Asp Ala Val Val Leu Ser Thr Gln His Ser Glu 0050/48792 ~ CA 02321264 2000-08-14 Glu Ile Asp Gln Lys Ser Leu Gln Glu Ala Val Met Glu Glu Ile Ile Lys Pro Ile Leu Pro Ala Glu Trp Leu Thr Ser Ala Thr Lys Phe Phe Ile Asn Pro Thr Gly Arg Phe Val Ile Gly Gly Pro Met Gly Asp Cys Gly Leu Thr Gly Arg Lys Ile Ile Val Asp Thr Tyr Gly Gly Met Ala Arg His Gly Gly Gly Ala Phe Ser Gly Lys Asp Pro Ser Lys Val Asp Arg Ser Ala Ala Tyr Ala Ala Arg Tyr Val Ala Lys Asn Ile Val Ala Ala Gly Leu Ala Asp Arg Cys Glu Ile Gln Val Ser Tyr Ala Ile Gly Val~Ala Glu Pro Thr Ser Ile Met Val Glu Thr Phe Gly Thr Glu Lys Val Pro Ser Glu Gln Leu Thr Leu Leu Val Arg Glu Phe Phe Asp Leu Arg Pro Tyr Gly Leu Ile Gln Met Leu Asp Leu Leu His Pro Ile Tyr Lys Glu Thr Ala Ala Tyr Gly His Phe Gly Arg Glu His Phe Pro Trp Glu Lys Thr Asp Lys Ala Gln Leu Leu Arg Asp Ala Ala Gly Leu Lys (2) INFORMATION FOR SEQ ID No: 3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1206 Base pairs (B) TYPE: Nucleic acid (C) STRANDEDNESS: Single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iii) ANTISENSE: NO
0050/48792 CA 02321264 2000-os-i4 (vi) ORIGINAL SOURCE:
(B) STRAIN: Escherichia coli (vii) IMMEDIATE SOURCE:
(B) CLONE: bioS1 (ix) FEATURES:
(A) NAME/KEY: CDS
(B) LOCATION: 1..1206 (xi) SEQUENCE DESCRIPTION: SEQ ID No: 3:
Met Asn Val Phe Asn Pro Ala Gln Phe Arg Ala Gln Phe Pro Ala Leu Gln Asp Ala Gly Val Tyr Leu Asp Ser Ala Ala Thr Ala Leu Lys Pro Glu Ala Val Val Glu Ala Thr Gln Gln Phe Tyr Ser Leu Ser Ala Gly Asn Val His Arg Ser Gln Phe Ala Glu Ala Gln Arg Leu Thr Ala Arg Tyr Glu Ala Ala Arg Glu Lys Val Ala Gln Leu Leu Asn Ala Pro Asp Asp Lys Thr Ile Val Trp Thr Arg Gly Thr Thr Glu Ser Ile Asn Met Val Ala Gln Cys Tyr Ala Arg Pro Arg Leu Gln Pro Gly Asp Glu Ile Ile Val Ser Val Ala Glu His His Ala Asn Leu Val Pro Trp Leu Met Val Ala Gln Gln Thr Gly Ala Lys Val Val Lys Leu Pro Leu Asn Ala 0050/48792 CA 02321264 2000-os-i4 Gln Arg Leu Pro Asp Val Asp Leu Leu Pro Glu Leu Ile Thr Pro Arg Ser Arg Ile Leu Ala Leu Gly Gln Met Ser Asn Val Thr Gly Gly Cys Pro Asp Leu Ala Arg Ala Ile Thr Phe Ala His Ser Ala Gly Met Val Val Met Val Asp Gly Ala Gln Gly Ala Val His Phe Pro Ala Asp Val Gln Gln Leu Asp Ile Asp Phe Tyr Ala Phe Ser Gly His Lys Leu Tyr Gly Pro Thr Gly Ile Gly Val Leu Tyr Gly Lys Ser Glu Leu Leu Glu Ala Met Ser Pro Trp Leu Gly Gly Gly Lys Met Val His Glu Val Ser Phe Asp Gly Phe Thr Thr Gln Ser Ala Pro Trp Lys Leu Glu Ala Gly Thr Pro Asn Val Ala Gly Val Ile Gly Leu Ser Ala Ala Leu Glu Trp Leu Ala Asp Tyr Asp Ile Asn Gln Ala Glu Ser Trp Ser Arg Ser Leu Ala Thr Leu Ala Glu Asp Ala Leu Ala Lys Arg Pro Gly Phe Arg Ser Phe Arg Cys Gln Asp Ser Ser Leu Leu Ala Phe Asp Phe Ala Gly Val 0050/48792 CA 02321264 2000-os-i4 His His Ser Asp Met Val Thr Leu Leu Ala Glu Tyr Gly Ile Ala Leu Arg Ala Gly Gln His Cys Ala Gln Pro Leu Leu Ala Glu Leu Gly Val Thr Gly Thr Leu Arg Ala Ser Phe Ala Pro Tyr Asn Thr Lys Ser Asp Val Asp Ala Leu Val Asn Ala Val Asp Arg Ala Leu Glu Leu Leu Val 385' 390 395 400 Asp (2) INFORMATION FOR SEQ ID No: 4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 401 Amino acids (B) TYPE: Amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Protein (xi) SEQUENCE DESCRIPTION: SEQ ID No: 4:
Met Asn Val Phe Asn Pro Ala Gln Phe Arg Ala Gln Phe Pro Ala Leu Gln Asp Ala Gly Val Tyr Leu Asp Ser Ala Ala Thr Ala Leu Lys Pro Glu Ala Val Val Glu Ala Thr Gln Gln Phe Tyr Ser Leu Ser Ala Gly Asn Val His Arg Ser Gln Phe Ala Glu Ala Gln Arg Leu Thr Ala Arg Tyr Glu Ala Ala Arg Glu Lys Val Ala Gln Leu Leu Asn Ala Pro Asp Asp Lys Thr Ile Val Trp Thr Arg Gly Thr Thr Glu Ser Ile Asn Met 0050/48792 CA 02321264 2000-os-i4 Val Ala Gln Cys Tyr Ala Arg Pro Arg Leu Gln Pro Gly Asp Glu Ile Ile Val Ser Val Ala Glu His His Ala Asn Leu Val Pro Trp Leu Met Val Ala Gln Gln Thr Gly Ala Lys Val Val Lys Leu Pro Leu Asn Ala Gln Arg Leu Pro Asp Val Asp Leu Leu Pro Glu Leu Ile Thr Pro Arg Ser Arg Ile Leu Ala Leu Gly Gln Met Ser Asn Val Thr Gly Gly Cys Pro Asp Leu Ala Arg Ala Ile Thr Phe Ala His Ser Ala Gly Met Val Val Met Val Asp Gly Ala Gln Gly Ala Val His Phe Pro Ala Asp Val Gln Gln Leu Asp Ile Asp Phe Tyr Ala Phe Ser Gly His Lys Leu Tyr Gly Pro Thr Gly Ile Gly Val Leu Tyr Gly Lys Ser Glu Leu Leu Glu Ala Met Ser Pro Trp Leu Gly Gly Gly Lys Met Val His Glu Val Ser Phe Asp Gly Phe Thr Thr Gln Ser Ala Pro Trp Lys Leu Glu Ala Gly Thr Pro Asn Val Ala Gly Val Ile Gly Leu Ser Ala Ala Leu Glu Trp Leu Ala Asp Tyr Asp Ile Asn Gln Ala Glu Ser Trp Ser Arg Ser Leu Ala Thr Leu Ala Glu Asp Ala Leu Ala Lys Arg Pro Gly Phe Arg Ser Phe Arg Cys Gln Asp Ser Ser Leu Leu Ala Phe Asp Phe Ala Gly Val His His Ser Asp Met Val Thr Leu Leu Ala Glu Tyr Gly Ile Ala Leu Arg Ala Gly Gln His Cys Ala Gln Pro Leu Leu Ala Glu Leu Gly Val 0050/48792 CA 02321264 2000-os-i4 Thr Gly Thr Leu Arg Ala Ser Phe Ala Pro Tyr Asn Thr Lys Ser Asp Val Asp Ala Leu Val Asn Ala Val Asp Arg Ala Leu Glu Leu Leu Val Asp (2) INFORMATION FOR SEQ ID No: 5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1215 Base pairs (B) TYPE: Nucleic acid (C) STRANDEDNESS: Single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iii) ANTISENSE: NO
(vi) ORGINAL SOURCE:
(B) STRAIN: Escherichia coli (vii) IMMEDIATE SOURCE:
(B) CLONE: bioS2 (ix) FEATURES:
(A) NAME/KEY: CDS
(B) LOCATION: 1..1215 (xi) SEQUENCE DESCRIPTION: SEQ ID No: 5:
Met Lys Leu Pro Ile Tyr Leu Asp Tyr Ser Ala Thr Thr Pro Val Asp Pro Arg Val Ala Glu Lys Met Met Gln Phe Met Thr Met Asp Gly Thr Phe Gly Asn Pro Ala Ser Arg Ser His Arg Phe Gly Trp Gln Ala Glu Glu Ala Val Asp Ile Ala Arg Asn Gln Ile Ala Asp Leu Val Gly Ala 0050/48792 CA 02321264 2000-os-i4 Asp Pro Arg Glu Ile Val Phe Thr Ser Gly Ala Thr Glu Ser Asp Asn Leu Ala Ile Lys Gly Ala Ala Asn Phe Tyr Gln Lys Lys Gly Lys His Ile Ile Thr Ser Lys Thr Glu His Lys Ala Val Leu Asp Thr Cys Arg Gln Leu Glu Arg Glu Gly Phe Glu Val Thr Tyr Leu Ala Pro Gln Arg Asn Gly Ile Ile Asp Leu Lys Glu Leu Glu Ala Ala Met Arg Asp Asp Thr Ile Leu Val Ser Ile Met His Val Asn Asn Glu Ile Gly Val Val Gln Asp Ile Ala Ala Ile Gly Glu Met Cys Arg Ala Arg Gly Ile Ile Tyr His Val Asp Ala Thr Gln Ser Val Gly Lys Leu Pro Ile Asp Leu Ser Gln Leu Lys Val Asp Leu Met Ser Phe Ser Gly His Lys Ile Tyr Gly Pro Lys Gly Ile Gly Ala Leu Tyr Val Arg Arg Lys Pro Arg Val Arg Ile Glu Ala Gln Met His Gly Gly Gly His Glu Arg Gly Met Arg Ser Gly Thr Leu Pro Val His Gln Ile Val Gly Met Gly Glu Ala Tyr 0050/48792 CA 02321264 2000-os-i4 Arg Ile Ala Lys Glu Glu Met Ala Thr Glu Met Glu Arg Leu Arg Gly Leu Arg Asn Arg Leu Trp Asn Gly Ile Lys Asp Ile Glu Glu Val Tyr Leu Asn Gly Asp Leu Glu His Gly Ala Pro Asn Ile Leu Asn Val Ser Phe Asn Tyr Val Glu Gly Glu Ser Leu Ile Met Ala Leu Lys Asp Leu Ala Val Ser Ser Gly Ser Ala Cys Thr Ser Ala Ser Leu Glu Pro Ser Tyr Val Leu Arg Ala Leu Gly Leu Asn Asp Glu Leu Ala His Ser Ser Ile Arg Phe Ser Leu Gly Arg Phe Thr Thr Glu Glu Glu Ile Asp Tyr Thr Ile Glu Leu Val Arg Lys Ser Ile Gly Arg Leu Arg Asp Leu Ser Pro Leu Trp Glu Met Tyr Lys Gln Gly Val Asp Leu Asn Ser Ile Glu Trp Ala His His (2) INFORMATION FOR SEQ ID No: 6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 404 Amino acids (B) TYPE: Amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Protein 0050/48792 CA 02321264 2000-os-i4 (xi) SEQUENCE DESCRIPTION: SEQ ID No: 6:
Met Lys Leu Pro Ile Tyr Leu Asp Tyr Ser Ala Thr Thr Pro Val Asp Pro Arg Val Ala Glu Lys Met Met Gln Phe Met Thr Met Asp Gly Thr Phe Gly Asn Pro Ala Ser Arg Ser His Arg Phe Gly Trp Gln Ala Glu Glu Ala Val Asp Ile Ala Arg Asn Gln Ile Ala Asp Leu Val Gly Ala Asp Pro Arg Glu Ile Val Phe Thr Ser Gly Ala Thr Glu Ser Asp Asn Leu Ala Ile Lys Gly Ala Ala Asn Phe Tyr Gln Lys Lys Gly Lys His Ile Ile Thr Ser Lys Thr Glu His Lys Ala Val Leu Asp Thr Cys Arg Gln Leu Glu Arg Glu Gly Phe Glu Val Thr Tyr Leu Ala Pro Gln Arg Asn Gly Ile Ile Asp Leu Lys Glu Leu Glu Ala Ala Met Arg Asp Asp Thr Ile Leu Val Ser Ile Met His Val Asn Asn Glu Ile Gly Val Val Gln Asp Ile Ala Ala Ile Gly Glu Met Cys Arg Ala Arg Gly Ile Ile Tyr His Val Asp Ala Thr Gln Ser Val Gly Lys Leu Pro Ile Asp Leu Ser Gln Leu Lys Val Asp Leu Met Ser Phe Ser Gly His Lys Ile Tyr Gly Pro Lys Gly Ile Gly Ala Leu Tyr Val Arg Arg Lys Pro Arg Val Arg Ile Glu Ala Gln Met His Gly Gly Gly His Glu Arg Gly Met Arg Ser Gly Thr Leu Pro Val His Gln Ile Val Gly Met Gly Glu Ala Tyr 0050/48792 CA 02321264 2000-os-i4 Arg Ile Ala Lys Glu Glu Met Ala Thr Glu Met Glu Arg Leu Arg Gly Leu Arg Asn Arg Leu Trp Asn Gly Ile Lys Asp Ile Glu Glu Val Tyr Leu Asn Gly Asp Leu Glu His Gly Ala Pro Asn Ile Leu Asn Val Ser Phe Asn Tyr Val Glu Gly Glu Ser Leu Ile Met Ala Leu Lys Asp Leu Ala Val Ser Ser Gly Ser Ala Cys Thr Ser Ala Ser Leu Glu Pro Ser Tyr Val Leu Arg Ala Leu Gly Leu Asn Asp Glu Leu Ala His Ser Ser Ile Arg Phe Ser Leu Gly Arg Phe Thr Thr Glu Glu Glu Ile Asp Tyr Thr Ile Glu Leu Val Arg Lys Ser Ile Gly Arg Leu Arg Asp Leu Ser Pro Leu Trp Glu Met Tyr Lys Gln Gly Val Asp Leu Asn Ser Ile Glu Trp Ala His His (2) INFORMATION FOR SEQ ID No: 7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1221 Base pairs (B) TYPE: Nucleic acid (C) STRANDEDNESS: Single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNS (genomic) (iii) HYPOTHETICAL: NO
(iii) ANTISENSE: NO
(vi) ORIGINAL SOURCE:
(B) STRAIN: Escherichia coli (vii) IMMEDIATE SOURCE:
(B) CLONE: bioS3 0050/48792 CA 02321264 2000-os-i4 (ix) FEATURES:
(A) NAME/KEY: CDS
(B) LOCATION: 1..1221 (xi) SEQUENCE DESCRIPTION: SEQ ID No: 7:
Met Ile Phe Ser Val Asp Lys Val Arg Ala Asp Phe Pro Val Leu Ser Arg Glu Val Asn Gly Leu Pro Leu Ala Tyr Leu Asp Ser Ala Ala Ser Ala Gln Lys Pro Ser Gln Val Ile Asp Ala Glu Ala Glu Phe Tyr Arg His Gly Tyr Ala Ala Val His Arg Gly Ile His Thr Leu Ser Ala Gln Ala Thr Glu Lys Met Glu Asn Val Arg Lys Arg Ala Ser Leu Phe Ile Asn Ala Arg Ser Ala Glu Glu Leu Val Phe Val Arg Gly Thr Thr Glu Gly Ile Asn Leu Val Ala Asn Ser Trp Gly Asn Ser Asn Val Arg Ala Gly Asp Asn Ile Ile Ile Ser Gln Met Glu His His Ala Asn Ile Val Pro Trp Gln Met Leu Cys Ala Arg Val Gly Ala Glu Leu Arg Val I1e Pro Leu Asn Pro Asp Gly Thr Leu Gln Leu Glu Thr Leu Pro Thr Leu Phe Asp Glu Lys Thr Arg Leu Leu Ala Ile Thr His Val Ser Asn Val 0050/48792 CA 02321264 2000-os-i4 Leu Gly Thr Glu Asn Pro Leu Ala Glu Met Ile Thr Leu Ala His Gln His Gly Ala Lys Val Leu Val Asp Gly Ala Gln Ala Val Met His His Pro Val Asp Val Gln Ala Leu Asp Cys Asp Phe Tyr Val Phe Ser Gly His Lys Leu Tyr Gly Pro Thr Gly Ile Gly Ile Leu Tyr Val Lys Glu Ala Leu Leu Gln Glu Met Pro Pro Trp Glu Gly Gly Gly Ser Met Ile Ala Thr Val Ser Leu Ser Glu Gly Thr Thr Trp Thr Lys Ala Pro Trp Arg Phe Glu Ala Gly Thr Pro Asn Thr Gly Gly Ile Ile Gly Leu Gly Ala Ala Leu Glu Tyr Val Ser Ala Leu Gly Leu Asn Asn Ile Ala Glu Tyr Glu Gln Asn Leu Met His Tyr Ala Leu Ser Gln Leu Glu Ser Val Pro Asp Leu Thr Leu Tyr Gly Pro Gln Asn Arg Leu Gly Val Ile Ala Phe Asn Leu Gly Lys His His Ala Tyr Asp Val Gly Ser Phe Leu Asp Asn Tyr Gly Ile Ala Val Arg Thr Gly His His Cys Ala Met Prv Leu 0050/48792 CA 02321264 2000-os-i4 Met Ala Tyr Tyr Asn Val Pro Ala Met Cys Arg Ala Ser Leu Ala Met Tyr Asn Thr His Glu Glu Val Asp Arg Leu Val Thr Gly Leu Gln Arg Ile His Arg Leu Leu Gly (2) INFORMATION FOR SEQ ID No: 8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 406 Amino acids (B) TYPE: Amino acid (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: Protein (xi) SEQUENCE DESCRIPTION: SEQ ID No: 8:
Met Ile Phe Ser Val Asp Lys Val Arg Ala Asp Phe Pro Val Leu Ser Arg Glu Val Asn Gly Leu Pro Leu Ala Tyr Leu Asp Ser Ala Ala Ser Ala Gln Lys Pro Ser Gln Val Ile Asp Ala Glu Ala Glu Phe Tyr Arg His Gly Tyr Ala Ala Val His Arg Gly Ile His Thr Leu Ser Ala Gln Ala Thr Glu Lys Met Glu Asn Val Arg Lys Arg Ala Ser Leu Phe Ile Asn Ala Arg Ser Ala Glu Glu Leu Val Phe Val Arg Gly Thr Thr Glu Gly Ile Asn Leu Val Ala Asn Ser Trp Gly Asn Ser Asn Val Arg Ala Gly Asp Asn Ile Ile Ile Ser Gln Met Glu His His Ala Asn Ile Val Pro Trp Gln Met Leu Cys Ala Arg Val Gly Ala Glu Leu Arg Val Ile 0050/48792 CA 02321264 2000-os-i4 Pro Leu Asn Pro Asp Gly Thr Leu Gln Leu Glu Thr Leu Pro Thr Leu Phe Asp Glu Lys Thr Arg Leu Leu Ala Ile Thr His Val Ser Asn Val Leu Gly Thr Glu Asn Pro Leu Ala Glu Met Ile Thr Leu Ala His Gln His Gly Ala Lys Val Leu Val Asp Gly Ala Gln Ala Val Met His His Pro Val Asp Val Gln Ala Leu Asp Cys Asp Phe Tyr Val Phe Ser Gly His Lys Leu Tyr Gly Pro Thr Gly Ile Gly Ile Leu Tyr Val Lys Glu Ala Leu Leu Gln Glu Met Pro Pro Trp Glu Gly Gly Gly Ser Met Ile Ala Thr Val Ser Leu Ser Glu Gly Thr Thr Trp Thr Lys Ala Pro Trp Arg Phe Glu Ala Gly Thr Pro Asn Thr Gly Gly Ile Ile Gly Leu Gly Ala Ala Leu Glu Tyr Val Ser Ala Leu Gly Leu Asn Asn Ile Ala Glu Tyr Glu Gln Asn Leu Met His Tyr Ala Leu Ser Gln Leu Glu Ser Val Pro Asp Leu Thr Leu Tyr Gly Pro Gln Asn Arg Leu Gly Val Ile Ala Phe Asn Leu Gly Lys His His Ala Tyr Asp Val Gly Ser Phe Leu Asp Asn Tyr Gly Ile Ala Val Arg Thr Gly His His Cys Ala Met Pro Leu Met Ala Tyr Tyr Asn Val Pro Ala Met Cys Arg Ala Ser Leu Ala Met Tyr Asn Thr His Glu Glu Val Asp Arg Leu Val Thr Gly Leu Gln Arg Ile His Arg Leu Leu Gly 0050/48792 CA 02321264 2000-os-i4 (2) INFORMATION FOR SEQ ID No: 9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 3720 Base pairs (B) TYPE: Nucleic acid (C) STRANDEDNESS: Single (D) TOPOLOGY: circular (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iii) ANTISENSE: NO
(vii) IMMEDIATE SOURCE:
(B) CLONE: pHSl metK
(ix) FEATURES:
(A) NAME/KEY: CDS
(B) LOCATION: 530..1684 (xi) SEQUENCE DESCRIPTION: SEQ ID No: 9:
AGGTAGTTAT CCTTAAAAAP. GCCACAGCAT ACATCCTGTC CGTCCAAGCA GAGGAGCAAA 240 Met Ala Lys His Leu Phe Thr Ser Glu Ser Val Ser Glu Gly His Pro Asp Lys Ile Ala Asp Gln Ile Ser Asp Ala Val Leu Asp Ala Ile Leu Glu Gln 0050/48792 CA 02321264 2000-os-i4 Asp Pro Lys Ala Arg Val Ala Cys Glu Thr Tyr Val Lys Thr Gly Met Val Leu Val Gly Gly Glu Ile Thr Thr Ser Ala Trp Val Asp Ile Glu Glu Ile Thr Arg Asn Thr Val Arg Glu Ile Gly Tyr Val His Ser Asp Met Gly Phe Asp Ala Asn Ser Cys Ala Val Leu Ser Ala Ile Gly Lys Gln Ser Pro Asp Ile Asn Gln Gly Val Asp Arg Ala Asp Pro Leu Glu Gln Gly Ala Gly Asp Gln Gly Leu Met Phe Gly Tyr Ala Thr Asn Glu Thr Asp Val Leu Met Pro Ala Pro Ile Thr Tyr Ala His Arg Leu Val Gln Arg Gln Ala Glu Val Arg Lys Asn Gly Thr Leu Pro Trp Leu Arg Pro Asp Ala Lys Ser Gln Val Thr Phe Gln Tyr Asp Asp Gly Lys Ile Val Gly Ile Asp Ala Val Val Leu Ser Thr Gln His Ser Glu Glu Ile Asp Gln Lys Ser Leu Gln Glu Ala Val Met Glu Glu Ile Ile Lys Pro Ile Leu Pro Ala Glu Trp Leu Thr Ser Ala Thr Lys Phe Phe Ile Asn 0050/48792 CA 02321264 2000-os-i4 Pro Thr Gly Arg Phe Val Ile Gly Gly Pro Met Gly Asp Cys Gly Leu Thr Gly Arg Lys Ile Ile Val Asp Thr Tyr Gly Gly Met Ala Arg His Gly Gly Gly Ala Phe Ser Gly Lys Asp Pro Ser Lys Val Asp Arg Ser Ala Ala Tyr Ala Ala Arg Tyr Val Ala Lys Asn Ile Val Ala Ala Gly Leu Ala Asp Arg Cys Glu Ile Gln Val Ser Tyr Ala Ile Gly Val Ala Glu Pro Thr Ser Ile Met Val Glu Thr Phe Gly Thr Glu Lys Val Pro Ser Glu Gln Leu Thr Leu Leu Val Arg Glu Phe Phe Asp Leu Arg Pro Tyr Gly Leu Ile Gln Met Leu Asp Leu Leu His Pro Ile Tyr Lys Glu Thr Ala Ala Tyr Gly His Phe Gly Arg Glu His Phe Pro Trp Glu Lys Thr Asp Lys Ala Gln Leu Leu Arg Asp Ala Ala Gly Leu Lys 0050/48792 CA 02321264 2000-os-i4 0050/48792 CA 02321264 2000-os-i4 (2) INFORMATION FOR SEQ ID No: 10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 384 Amino acids (B) TYPE: Amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Protein (xi) SEQUENCE DESCRIPTION: SEQ ID No: 10:
Met Ala Lys His Leu Phe Thr Ser Glu Ser Val Ser Glu Gly His Pro Asp Lys Ile Ala Asp Gln Ile Ser Asp Ala Val Leu Asp Ala Ile Leu Glu Gln Asp Pro Lys Ala Arg Val Ala Cys Glu Thr Tyr Val Lys Thr Gly Met Val Leu Val Gly Gly Glu Ile Thr Thr Ser Ala Trp Val Asp Ile Glu Glu Ile Thr Arg Asn Thr Val Arg Glu Ile Gly Tyr Val His Ser Asp Met Gly Phe Asp Ala Asn Ser Cys Ala Val Leu Ser Ala Ile Gly Lys Gln Ser Pro Asp Ile Asn Gln Gly Val Asp Arg Ala Asp Pro Leu Glu Gln Gly Ala Gly Asp Gln Gly Leu Met Phe Gly Tyr Ala Thr Asn Glu Thr Asp Val Leu Met Pro Ala Pro Ile Thr Tyr Ala His Arg Leu Val Gln Arg Gln Ala Glu Val Arg Lys Asn Gly Thr Leu Pro Trp 0050/48792 CA 02321264 2000-os-i4 Leu Arg Pro Asp Ala Lys Ser Gln Val Thr Phe Gln Tyr Asp Asp Gly Lys Ile Val Gly Ile Asp Ala Val Val Leu Ser Thr Gln His Ser Glu Glu Ile Asp Gln Lys Ser Leu Gln Glu Ala Val Met Glu Glu Ile Ile Lys Pro Ile Leu Pro Ala Glu Trp Leu Thr Ser Ala Thr Lys Phe Phe Ile Asn Pro Thr Gly Arg Phe Val Ile Gly Gly Pro Met Gly Asp Cys Gly Leu Thr Gly Arg Lys Ile Ile Val Asp Thr Tyr Gly Gly Met Ala Arg His Gly Gly Gly Ala Phe Ser Gly Lys Asp Pro Ser Lys Val Asp Arg Ser Ala Ala Tyr Ala Ala Arg Tyr Val Ala Lys Asn Ile Val Ala Ala Gly Leu Ala Asp Arg Cys Glu Ile Gln Val Ser Tyr Ala Ile Gly Val Ala Glu Pro Thr Ser Ile Met Val Glu Thr Phe Gly Thr Glu Lys Val Pro Ser Glu Gln Leu Thr Leu Leu Val Arg Glu Phe Phe Asp Leu Arg Pro Tyr Gly Leu Ile Gln Met Leu Asp Leu Leu His Pro Ile Tyr Lys Glu Thr Ala Ala Tyr Gly His Phe Gly Arg Glu His Phe Pro Trp Glu Lys Thr Asp Lys Ala Gln Leu Leu Arg Asp Ala Ala Gly Leu Lys (2) INFORMATION FOR SEQ ID No: 11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 3794 Base pairs (B) TYPE: Nucleic acid (C) STRANDEDNESS: Single (D) TOPOLOGY: circular 0050/48792 CA 02321264 2000-os-i4 (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iii) ANTISENSE: NO
(vii) IMMEDIATE SOURCE:
(B) CLONE: pHS1 bioS1 (ix) FEATURES:
(A) NAME/KEY: CDS
(B) LOCATION: 601..1806 (xi) SEQUENCE DESCRIPTION: SEQ ID No: 11:
Met Asn Val Phe Asn Pro Ala Gln Phe Arg Ala Gln Phe Pro Ala Leu Gln Asp Ala Gly Val Tyr Leu Asp Ser Ala Ala Thr Ala Leu Lys Pro Glu Ala Val Val Glu Ala Thr Gln Gln Phe Tyr Ser Leu Ser Ala Gly Asn Val His Arg Ser Gln Phe Ala Glu Ala Gln Arg Leu Thr Ala Arg Tyr Glu Ala Ala Arg Glu Lys Val Ala Gln Leu Leu Asn Ala Pro Asp 65 70 75 g0 Asp Lys Thr Ile Val Trp Thr Arg Gly Thr Thr Glu Ser Ile Asn Met Val Ala Gln Cys Tyr Ala Arg Pro Arg Leu Gln Pro Gly Asp Glu Ile Ile Val Ser Val Ala Glu His His Ala Asn Leu Val Pro Trp Leu Met Val Ala Gln Gln Thr Gly Ala Lys Val Val Lys Leu Pro Leu Asn Ala Gln Arg Leu Pro Asp Val Asp Leu Leu Pro Glu Leu Ile Thr Pro Arg Ser Arg Ile Leu Ala Leu Gly Gln Met Ser Asn Val Thr Gly Gly Cys Pro Asp Leu Ala Arg Ala Ile Thr Phe Ala His Ser Ala Gly Met Val Val Met Val Asp Gly Ala Gln Gly Ala Val His Phe Pro Ala Asp Val Gln Gln Leu Asp Ile Asp Phe Tyr Ala Phe Ser Gly His Lys Leu Tyr Gly Pro Thr Gly Ile Gly Val Leu Tyr Gly Lys Ser Glu Leu Leu Glu Ala Met Ser Pro Trp Leu Gly Gly Gly Lys Met Val His Glu Val Ser 0050/48792 CA 02321264 2000-os-i4 Phe Asp Gly Phe Thr Thr Gln Ser Ala Pro Trp Lys Leu Glu Ala Gly Thr Pro Asn Val Ala Gly Val Ile Gly Leu Ser Ala Ala Leu Glu Trp Leu Ala Asp Tyr Asp Ile Asn Gln Ala Glu Ser Trp Ser Arg Ser Leu Ala Thr Leu Ala Glu Asp Ala Leu Ala Lys Arg Pro Gly Phe Arg Ser Phe Arg Cys Gln Asp Ser Ser Leu Leu Ala Phe Asp Phe Ala Gly Val His His Ser Asp Met Val Thr Leu Leu Ala Glu Tyr Gly Ile Ala Leu Arg Ala Gly Gln His Cys Ala Gln Pro Leu Leu Ala Glu Leu Gly Val Thr Gly Thr Leu Arg Ala Ser Phe Ala Pro Tyr Asn Thr Lys Ser Asp Val Asp Ala Leu Val Asn Ala Val Asp Arg Ala Leu Glu Leu Leu Val Asp 0050./48792 CA 02321264 2000-os-i4 0050/48792 CA 02321264 2000-os-i4 (2) INFORMATION FOR SEQ ID No: 12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 401 Amino acids (B) TYPE: Amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Protein (xi) SEQUENCE DESCRIPTION: SEQ ID No: 12:
Met Asn Val Phe Asn Pro Ala Gln Phe Arg Ala Gln Phe Pro Ala Leu Gln Asp Ala Gly Val Tyr Leu Asp Ser Ala Ala Thr Ala Leu Lys Pro Glu Ala Val Val Glu Ala Thr Gln Gln Phe Tyr Ser Leu Ser Ala Gly Asn Val His Arg Ser Gln Phe Ala Glu Ala Gln Arg Leu Thr Ala Arg 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 Val Ala Gln Cys Tyr Ala Arg Pro Arg Leu Gln Pro Gly Asp Glu Ile Ile Val Ser Val Ala Glu His His Ala Asn Leu Val Pro Trp Leu Met Val Ala Gln Gln Thr Gly Ala Lys Val Val Lys Leu Pro Leu Asn Ala Gln Arg Leu Pro Asp Val Asp Leu Leu Pro Glu Leu Ile Thr Pro Arg Ser Arg Ile Leu Ala Leu Gly Gln Met Ser Asn Val Thr Gly Gly Cys Pro Asp Leu Ala Arg Ala Ile Thr Phe Ala His Ser Ala Gly Met Val Val Met Val Asp Gly Ala Gln Gly Ala Val His Phe Pro Ala Asp Val Gln Gln Leu Asp Ile Asp Phe Tyr Ala Phe Ser Gly His Lys Leu Tyr Gly Pro Thr Gly Ile Gly Val Leu Tyr Gly Lys Ser Glu Leu Leu Glu Ala Met Ser Pro Trp Leu Gly Gly Gly Lys Met Val His Glu Val Ser Phe Asp Gly Phe Thr Thr Gln Ser Ala Pro Trp Lys Leu Glu Ala Gly Thr Pro Asn Val Ala Gly Val Ile Gly Leu Ser Ala Ala Leu Glu Trp Leu Ala Asp Tyr Asp Ile Asn Gln Ala Glu Ser Trp Ser Arg Ser Leu Ala Thr Leu Ala Glu Asp Ala Leu Ala Lys Arg Pro Gly Phe Arg Ser Phe Arg Cys Gln Asp Ser Ser Leu Leu Ala Phe Asp Phe Ala Gly Val His His Ser Asp Met Val Thr Leu Leu Ala Glu Tyr Gly Ile Ala Leu Arg Ala Gly Gln His Cys Ala Gln Pro Leu Leu Ala Glu Leu Gly Val Thr Gly Thr Leu Arg Ala Ser Phe Ala Pro Tyr Asn Thr Lys Ser Asp Val Asp Ala Leu Val Asn Ala Val Asp Arg Ala Leu Glu Leu Leu Val Asp (2) INFORMATION FOR SEQ ID No: 13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4975 Base pairs (B) TYPE: Nucleic acid 0050/48792 CA 02321264 2000-os-i4 (C) STRANDEDNESS: Single (D) TOPOLOGY: circular (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iii) ANTISENSE: NO
(vii) IMMEDIATE SOURCE:
(B) CLONE: pHS1 metK bioS1 (ix) FEATURES:
(A) NAME/KEY: CDS
(B) LOCATION: 1782..2987 (ix) FEATURES:
(A) NAME/KEY: CDS
(B) LOCATION: 530..1684 (xi) SEQUENCE DESCRIPTION: SEQ ID No: 13:
Met Ala Lys His Leu Phe Thr Ser Glu Ser Val Ser Glu Gly His Pro Asp Lys Ile Ala Asp Gln Ile Ser Asp Ala Val Leu Asp Ala Ile Leu Glu Gln 0050/48792 CA 02321264 2000-os-i4 Asp Pro Lys Ala Arg Val Ala Cys Glu Thr Tyr Val Lys Thr Gly Met Val Leu Val Gly Gly Glu Ile Thr Thr Ser Ala Trp Val Asp Ile Glu Glu Ile Thr Arg Asn Thr Val Arg Glu Ile Gly Tyr Val His Ser Asp ATG GGC TTT GAC GCT AAC TCC TGT GCG GTT CTG AGC GCT ATC GGC AAA $23 Met Gly Phe Asp Ala Asn Ser Cys Ala Val Leu Ser Ala Ile Gly Lys Gln Ser Pro Asp Ile Asn Gln Gly Val Asp Arg Ala Asp Pro Leu Glu Gln Gly Ala Gly Asp Gln Gly Leu Met Phe Gly Tyr Ala Thr Asn Glu Thr Asp Val Leu Met Pro Ala Pro Ile Thr Tyr Ala His Arg Leu Val Gln Arg Gln Ala Glu Val Arg Lys Asn Gly Thr Leu Pro Trp Leu Arg Pro Asp Ala Lys Ser Gln Val Thr Phe Gln Tyr Asp Asp Gly Lys Ile Val Gly Ile Asp Ala Val Val Leu Ser Thr Gln His Ser Glu Glu Ile Asp Gln Lys Ser Leu Gln Glu Ala Val Met Glu Glu Ile Ile Lys Pro Ile Leu Pro Ala Glu Trp Leu Thr Ser Ala Thr Lys Phe Phe Ile Asn 0050/48792 CA 02321264 2000-os-i4 Pro Thr Gly Arg Phe Val Ile Gly Gly Pro Met Gly Asp Cys Gly Leu Thr Gly Arg Lys Ile Ile Val Asp Thr Tyr Gly Gly Met Ala Arg His Gly Gly Gly Ala Phe Ser Gly Lys Asp Pro Ser Lys Val Asp Arg Ser Ala Ala Tyr Ala Ala Arg Tyr Val Ala Lys Asn Ile Val Ala Ala Gly Leu Ala Asp Arg Cys Glu Ile Gln Val Ser Tyr Ala Ile Gly Val Ala Glu Pro Thr Ser Ile Met Val Glu Thr Phe Gly Thr Glu Lys Val Pro Ser Glu Gln Leu Thr Leu Leu Val Arg Glu Phe Phe Asp Leu Arg Pro Tyr Gly Leu Ile Gln Met Leu Asp Leu Leu His Pro Ile Tyr Lys Glu Thr Ala Ala Tyr Gly His Phe Gly Arg Glu His Phe Pro Trp Glu Lys Thr As.p Lys Ala Gln Leu Leu Arg Asp Ala Ala Gly Leu Lys Met Asn Val Phe Asn Pro Ala Gln Phe Arg Ala Gln Phe Pro Ala Leu Gln Asp Ala Gly Val Tyr Leu Asp 0050/48792 CA 02321264 2000-os-i4 Ser Ala Ala Thr Ala Leu Lys Pro Glu Ala Val Val Glu Ala Thr Gln Gln Phe Tyr Ser Leu Ser Ala Gly Asn Val His Arg Ser Gln Phe Ala Glu Ala Gln Arg Leu Thr Ala Arg Tyr Glu Ala Ala Arg Glu Lys Val Ala Gln Leu Leu Asn Ala Pro Asp Asp Lys Thr Ile Val Trp Thr Arg Gly Thr Thr Glu Ser Ile Asn Met Val Ala Gln Cys Tyr Ala Arg Pro Arg Leu Gln Pro Gly Asp Glu Ile Ile Val Ser Val Ala Glu His His Ala Asn Leu Val Pro Trp Leu Met Val Ala Gln Gln Thr Gly Ala Lys Val Val Lys Leu Pro Leu Asn Ala Gln Arg Leu Pro Asp Val Asp Leu Leu Pro Glu Leu Ile Thr Pro Arg Ser Arg Ile Leu Ala Leu Gly Gln Met Ser Asn Val Thr Gly Gly Cys Pro Asp Leu Ala Arg Ala Ile Thr Phe Ala His Ser Ala Gly Met Val Val Met Val Asp Gly Ala Gln Gly Ala Val His Phe Pro Ala Asp Val Gln Gln Leu Asp Ile Asp Phe Tyr 0050/48792 CA 02321264 2000-os-i4 Ala Phe Ser Gly His Lys Leu Tyr Gly Pro Thr Gly Ile Gly Val Leu Tyr Gly Lys Ser Glu Leu Leu Glu Ala Met Ser Pro Trp Leu Gly Gly Gly Lys Met Val His Glu Val Ser Phe Asp Gly Phe Thr Thr Gln Ser Ala Pro Trp Lys Leu Glu Ala Gly Thr Pro Asn Val Ala Gly Val Ile Gly Leu Ser Ala Ala Leu Glu Trp Leu Ala Asp Tyr Asp Ile Asn Gln Ala Glu Ser Trp Ser Arg Ser Leu Ala Thr Leu Ala Glu Asp Ala Leu Ala Lys Arg Pro Gly Phe Arg Ser Phe Arg Cys Gln Asp Ser Ser Leu Leu Ala Phe Asp Phe Ala Gly Val His His Ser Asp Met Val Thr Leu Leu Ala Glu Tyr Gly Ile Ala Leu Arg Ala Gly Gln His Cys Ala Gln Pro Leu Leu Ala Glu Leu Gly Val Thr Gly Thr Leu Arg Ala Ser Phe Ala Prv Tyr Asn Thr Lys Ser Asp Val Asp Ala Leu Val Asn Ala Val Asp Arg Ala Leu Glu Leu Leu Val Asp 0050/48792 CA 02321264 2000-os-i4 0050/48792 CA 02321264 2000-os-i4 (2) INFORMATION FOR SEQ ID No: 14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 384 Amino acids (B) TYPE: Amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Protein (xi) SEQUENCE DESCRIPTION: SEQ ID No:
14:
Met Ala Lys His Leu Phe Thr Ser Glu Ser Ser Glu His Pro Val Gly Asp Lys Ile Ala Asp Gln Ile Ser Asp Ala Leu Asp Ile Leu Val Ala Glu Gln Asp Pro Lys Ala Arg Val Ala Cys Thr Tyr Lys Thr Glu Val Gly Met Val Leu Val Gly Gly Glu Ile Thr Ser Ala Val Asp Thr Trp Ile Glu Glu Ile Thr Arg Asn Thr Val Arg Ile Gly Val His Glu Tyr Ser Asp Met Gly Phe Asp Ala Asn Ser Cys Val Leu Ala Ile Ala Ser Gly Lys Gln Ser Pro Asp Ile Asn Gln Gly Val Asp Arg Ala Asp Pro Leu Glu Gln Gly Ala Gly Asp Gln Gly Leu Met Phe Gly Tyr Ala Thr 0050/48792 CA 02321264 2000-os-i4 Asn Glu Thr Asp Val Leu Met Pro Ala Pro Ile Thr Tyr Ala His Arg Leu Val Gln Arg Gln Ala Glu Val Arg Lys Asn Gly Thr Leu Pro Trp Leu Arg Pro Asp Ala Lys Ser Gln Val Thr Phe Gln Tyr Asp Asp Gly Lys Ile Val Gly Ile Asp Ala Val Val Leu Ser Thr Gln His Ser Glu Glu Ile Asp Gln Lys Ser Leu Gln Glu Ala Val Met Glu Glu Ile Ile Lys Pro Ile Leu Pro Ala Glu Trp Leu Thr Ser Ala Thr Lys Phe Phe Ile Asn Pro Thr Gly Arg Phe Val Ile Gly Gly Pro Met Gly Asp Cys Gly Leu Thr Gly Arg Lys Ile Ile Val Asp Thr Tyr Gly Gly Met Ala Arg His Gly Gly Gly Ala Phe Ser Gly Lys Asp Pro Ser Lys Val Asp Arg Ser Ala Ala Tyr Ala Ala Arg Tyr Val Ala Lys Asn Ile Val Ala Ala Gly Leu Ala Asp Arg Cys Glu Ile Gln Val Ser Tyr Ala Ile Gly Val Ala Glu Pro Thr Ser Ile Met Val Glu Thr Phe Gly Thr Glu Lys Val Pro Ser Glu Gln Leu Thr Leu Leu Val Arg Glu Phe Phe Asp Leu Arg Pro Tyr Gly Leu Ile Gln Met Leu Asp Leu Leu His Pro Ile Tyr Lys Glu Thr Ala Ala Tyr Gly His Phe Gly Arg Glu His Phe Pro Trp Glu Lys Thr Asp Lys Ala Gln Leu Leu Arg Asp Ala Ala Gly Leu Lys (2) INFORMATION FOR SEQ ID No: 15:
0050/48792 CA 02321264 2000-os-i4 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 401 Amino acids (B) TYPE: Amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Protein (xi) SEQUENCE DESCRIPTION: SEQ ID No: 15:
Met Asn Val Phe Asn Pro Ala Gln Phe Arg Ala Gln Phe Pro Ala Leu Gln Asp Ala Gly Val Tyr Leu Asp Ser Ala Ala Thr Ala Leu Lys Pro Glu Ala Val Val Glu Ala Thr Gln Gln Phe Tyr Ser Leu Ser Ala Gly Asn Val His Arg Ser Gln Phe Ala Glu Ala Gln Arg Leu Thr Ala Arg Tyr Glu Ala Ala Arg Glu Lys Val Ala Gln Leu Leu Asn Ala Pro Asp Asp Lys Thr Ile Val Trp Thr Arg Gly Thr Thr Glu Ser Ile Asn Met Val Ala Gln Cys Tyr Ala Arg Pro Arg Leu Gln Pro Gly Asp Glu Ile Ile Val Ser Val Ala Glu His His Ala Asn Leu Val Pro Trp Leu Met Val Ala Gln Gln Thr Gly Ala Lys Val Val Lys Leu Pro Leu Asn Ala Gln Arg Leu Pro Asp Val Asp Leu Leu Pro Glu Leu Ile Thr Pro Arg Ser Arg Ile Leu Ala Leu Gly Gln Met Ser Asn Val Thr Gly Gly Cys Pro Asp Leu Ala Arg Ala Ile Thr Phe Ala His Ser Ala Gly Met Val Val Met Val Asp Gly Ala Gln Gly Ala Val His Phe Pro Ala Asp Val Gln Gln Leu Asp Ile Asp Phe Tyr Ala Phe Ser Gly His Lys Leu Tyr 0050/48792 CA 02321264 2000-os-i4 Gly Pro Thr Gly Ile Gly Val Leu Tyr Gly Lys Ser Glu Leu Leu Glu Ala Met Ser Pro Trp Leu Gly Gly Gly Lys Met Val His Glu Val Ser Phe Asp Gly Phe Thr Thr Gln Ser Ala Pro Trp Lys Leu Glu Ala Gly Thr Pro Asn Val Ala Gly Val Ile Gly Leu Ser Ala Ala Leu Glu Trp Leu Ala Asp Tyr Asp Ile Asn Gln Ala Glu Ser Trp Ser Arg Ser Leu Ala Thr Leu Ala Glu Asp Ala Leu Ala Lys Arg Pro Gly Phe Arg Ser Phe Arg Cys Gln Asp Ser Ser Leu Leu Ala Phe Asp Phe Ala Gly Val His His Ser Asp Met Val Thr Leu Leu Ala Glu Tyr Gly Ile Ala Leu Arg Ala Gly Gln His Cys Ala Gln Pro Leu Leu Ala Glu Leu Gly Val Thr Gly Thr Leu Arg Ala Ser Phe Ala Pro Tyr Asn Thr Lys Ser Asp Val Asp Ala Leu Val Asn Ala Val Asp Arg Ala Leu Glu Leu Leu Val Asp
Claims (14)
1. A process for producing biotin wherein an S-adenosylmethionine synthase gene, having the sequence SEQ
ID No. 1, and at least one further biotin biosynthesis gene bioS1, bioS2 or bioS3, having the sequences SEQ ID No. 3, SEQ
ID No. 5 or SEQ ID No. 7, and also their functional variants, analogues or derivatives, are expressed in a prokaryotic or eukaryotic host organism which is able to synthesize biotin, this organism is cultured and the synthesized biotin is used directly after separating off the biomass or after purifying the biotin.
ID No. 1, and at least one further biotin biosynthesis gene bioS1, bioS2 or bioS3, having the sequences SEQ ID No. 3, SEQ
ID No. 5 or SEQ ID No. 7, and also their functional variants, analogues or derivatives, are expressed in a prokaryotic or eukaryotic host organism which is able to synthesize biotin, this organism is cultured and the synthesized biotin is used directly after separating off the biomass or after purifying the biotin.
2. A process as claimed in claim 1, wherein the variants of the genes having the sequences SEQ ID No.1, SEQ ID No. 3, SEQ ID
No. 5 and SEQ ID No. 7 are genes which, on the amino acid level deduced from the sequences as claimed in claim 1, exhibit a homology of from 30 to 100% and enable an increased synthesis of biotin to be achieved.
No. 5 and SEQ ID No. 7 are genes which, on the amino acid level deduced from the sequences as claimed in claim 1, exhibit a homology of from 30 to 100% and enable an increased synthesis of biotin to be achieved.
3. A process as claimed in claim 1 or 2, wherein an organism selected from the group of the genera Escherichia, Citrobacter, Serratia, Klebsiella, Salmonella, Pseudomonas, Comamonas, Acinetobacter, Azotobacter, Chromobacterium, Bacillus, Clostridium, Arthrobacter, Corynebacterium, Brevibacterium, Lactococcus, Lactobacillus, Streptomyces, Rhizobium, Agrobacterium, Staphylococcus, Rhodotorula, Sporobolomyces, Yarrowia, Schizosaccharomyces or Saccharomyces is used as the host organism.
4. A process as claimed in any of claims 1 to 3, wherein a regulation-defective biotin mutant is used as the host organism.
5. A process as claimed in any of claims 1 to 4, wherein at least one copy of the genes having the sequences SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 5 and SEQ ID No. 7 as claimed in claim 1 is expressed in a prokaryotic or eukaryotic host organism either alone or together with one or more copies of at least one further biotin gene selected from the group bioA, bioB, bioF, bioC, bioD, bioH, bioP, biota, bioX, bioY or bioR.
6. A process as claimed in any of claims 1 to 5, wherein at least one copy of the genes having the sequences SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 5 and SEQ ID No. 7 as claimed in claim 1 is expressed in a prokaryotic or eukaryotic host organism either alone or, on a shared vector or on separate vectors, together with one or more copies at least one further biotin gene selected from the group bioA, bioB, bioF, bioC, bioD, bioH, bioP, bioW, bioX, bioY or bioR.
7. A gene construct which comprises an S-adenosylmethionine synthase gene, having the sequence SEQ ID No. 1, and at least one further biotin biosynthesis gene bioS1, bioS2 or bioS3, having the sequences SEQ ID No. 3, SEQ ID No. 5 and SEQ ID
No. 7, and also their functional variants, analogues or derivatives, and which is functionally linked to one or more regulatory signals for the purpose of increasing gene expression and/or protein expression and/or whose natural regulation has been switched off.
No. 7, and also their functional variants, analogues or derivatives, and which is functionally linked to one or more regulatory signals for the purpose of increasing gene expression and/or protein expression and/or whose natural regulation has been switched off.
8. A gene construct as claimed in claim 7, which has been inserted into a vector which is suitable for expressing the gene in a prokaryotic or eukaryotic host organism.
9. A gene construct as claimed in claim 7 or 8, wherein the genes having the sequences SEQ ID No. 1, SEQ ID No. 3, SEQ ID
No. 5 and SEQ ID No. 7, and also their functional variants, analogues or derivatives, are present in several copies in the gene construct.
No. 5 and SEQ ID No. 7, and also their functional variants, analogues or derivatives, are present in several copies in the gene construct.
10. A gene construct as claimed in any of claims 7 to 9, wherein the S-adenosylmethionine synthase gene, SEQ ID No. 1, and at least one further biotin biosynthesis gene bioS1, bioS2 or bioS3, having the sequences SEQ ID No. 3, SEQ ID No. 5 and SEQ ID No. 7, and also their functional variants, analogues or derivatives, as claimed in claim 7, are present in the gene construct or vector together with one or more copies of at least one further gene selected from the group bioA, bioB, bioF, bioC, bioD, bioH, bioP, bioW, bioX, bioY or bioR.
11. An organism which comprises a gene construct as claimed in any of claims 7 to 10.
12. The use of the sequences as claimed in claim 1 for producing biotin.
13. The use of the bioS3 gene, having the sequence SEQ ID No. 7, or of its functional variants, analogues or derivatives, either alone or in combination with at least one further gene selected from the group S-adenosylmethionine synthase gene, bioS1, bioS2, bioA, bioB, bioF, bioC, bioD, bioH, bioP, bioW, bioX, bioY or bioR, for producing biotin.
14. The use of a gene construct as claimed in any of claims 7 to for producing biotin.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE19806872A DE19806872A1 (en) | 1998-02-19 | 1998-02-19 | Production of biotin by expressing S-adenosyl-methionine synthase and second biotin synthesis gene in host cells |
| DE19806872.7 | 1998-02-19 | ||
| PCT/EP1999/001052 WO1999042591A1 (en) | 1998-02-19 | 1999-02-17 | Method for producing biotin |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2321264A1 true CA2321264A1 (en) | 1999-08-26 |
Family
ID=7858233
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002321264A Abandoned CA2321264A1 (en) | 1998-02-19 | 1999-02-17 | Method for producing biotin |
Country Status (8)
| Country | Link |
|---|---|
| EP (1) | EP1054977A1 (en) |
| JP (1) | JP2002504338A (en) |
| KR (1) | KR20010041062A (en) |
| CN (1) | CN1210403C (en) |
| CA (1) | CA2321264A1 (en) |
| DE (1) | DE19806872A1 (en) |
| IL (1) | IL137310A0 (en) |
| WO (1) | WO1999042591A1 (en) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR101023788B1 (en) * | 2008-04-18 | 2011-03-21 | 주식회사 콧데 | Recombinant plasmid, biotin producing microorganism transformed by the recombinant plasmid and method for manufacturing biotin using the transformed biotin producing microorganism |
| AU2014233711B2 (en) * | 2013-03-15 | 2020-05-28 | Monsanto Technology Llc | Compositions and Methods for the Production and Delivery of RNA |
| CN107099497B (en) * | 2017-06-09 | 2020-08-11 | 浙江大学 | Plasmid and cell for promoting biotin synthesis and promoting method thereof |
| CN112473700A (en) * | 2020-12-07 | 2021-03-12 | 南昌航空大学 | Preparation method and application of bismuth oxybromide/biochar composite visible-light-driven photocatalyst |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH09224690A (en) * | 1996-02-21 | 1997-09-02 | Shiseido Co Ltd | Production of biotin |
| DK0806479T3 (en) * | 1996-05-06 | 2003-10-06 | Hoffmann La Roche | Production of biotin by fermentation |
| DE19731274A1 (en) * | 1997-07-22 | 1999-01-28 | Basf Ag | Process for the production of biotin |
-
1998
- 1998-02-19 DE DE19806872A patent/DE19806872A1/en not_active Withdrawn
-
1999
- 1999-02-17 JP JP2000532531A patent/JP2002504338A/en not_active Withdrawn
- 1999-02-17 CN CNB998031534A patent/CN1210403C/en not_active Expired - Fee Related
- 1999-02-17 IL IL13731099A patent/IL137310A0/en unknown
- 1999-02-17 WO PCT/EP1999/001052 patent/WO1999042591A1/en not_active Application Discontinuation
- 1999-02-17 KR KR1020007009100A patent/KR20010041062A/en not_active Application Discontinuation
- 1999-02-17 EP EP99908923A patent/EP1054977A1/en not_active Withdrawn
- 1999-02-17 CA CA002321264A patent/CA2321264A1/en not_active Abandoned
Also Published As
| Publication number | Publication date |
|---|---|
| CN1210403C (en) | 2005-07-13 |
| CN1291232A (en) | 2001-04-11 |
| EP1054977A1 (en) | 2000-11-29 |
| JP2002504338A (en) | 2002-02-12 |
| DE19806872A1 (en) | 1999-08-26 |
| KR20010041062A (en) | 2001-05-15 |
| WO1999042591A1 (en) | 1999-08-26 |
| IL137310A0 (en) | 2001-07-24 |
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| Date | Code | Title | Description |
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| FZDE | Discontinued |