Biotin biosynthetic genes

BACKGROUND OF THE INVENTION
The present invention relates to the production process of biotin by fermentation using a genetically engineered organism.
Biotin is one of the essential vitamins for nutrition of animals, plants, and microorganisms, and very important as medicine or food additives.
Biotin biosynthesis of Escherichia coli has been studied well, and it has been clarified that biotin is synthesized from pimelyl CoA via 7 keto-8-amino pelargonic acid (KAPA), 7,8-diamino pelargonic acid (DAPA) and desthiobiotin (DTB) [Escherichia coli and Salmonella typhimurium, Cellular and Molecular Biology, 544, (1987)]. The analysis of genetic information involved in the biosynthesis of biotin has been advanced on Escherichia coli [J. Biol. Chem., 263, 19577, (1988)] and Bacillus sphaericus (U.S. Pat. No. 5,096,823). At least four enzymes are known to be involved in this biosynthetic pathway. These four enzymes are encoded by the bioA, bioB, bioD and bioF genes. The bioF gene codes for KAPA synthetase which catalyzes the conversion of pimelyl CoA to KAPA. The bioA gene codes for DAPA aminotransferase which converts KAPA to DAPA. The bioD gene codes for DTB synthetase which converts DAPA to DTB. The bioB gene codes for biotin synthase which converts DTB to biotin. It has been also reported that the bioC and bioH genes are involved in the synthesis of pimelyl CoA in Escherichia coli.
There are many studies on fermentative production of biotin. Escherichia coli (Japanese Patent Kokai No. 149091/1986 and Japanese Patent Kokai No. 155081/1987), Bacillus sphaericus (Japanese Patent Kokai No. 180174/1991), Serratia marcescens (Japanese Patent Kokai No. 27980/1990) and Brevibacterium flavum (Japanese Patent Kokai No. 240489/1991) have been used. But these processes have not yet been suitable for use in an industrial production process because of a low productivity. Moreover, large amounts of DTB, a biotin precursor, accumulates in the fermentation of these bacteria. Therefore, it has been assumed that the last step of the biotin biosynthetic pathway, from DTB to biotin, is a rate limiting step.
On the other hand, it was found that a bacterial strain belonging to the genus Kurthia produces DTB and small amounts of biotin. Also mutants which produce much larger amounts of biotin were derived from wild type strains of the genus Kurthia by selection for resistance to biotin antimetabolites acidomycin (ACM), 5-(2-thienyl)-valeric acid (TVA) and alpha-methyl desthiobiotin (MeDTB). However, in view of the still low biotin titers it is desirable to apply genetic engineering to improve the biotin productivity of such mutants.
SUMMARY OF THE INVENTION
The present invention relates therefore to the chromosomal DNA fragments carrying the genes involved in the biotin biosynthesis of Kurthia sp. The isolated chromosomal DNA fragments carry 8 genes, the bioA, bioB, bioC, bioD, bioF, bioFII, bioH and bioHII genes, and transcriptional regulatory sequences. The bioFII gene codes for an isozyme of the bioF gene product. The bioHII gene codes for an isozyme of the bioH gene product.
The present invention fuirther relates to Kurthia sp. strains in which at least one gene involved in biotin biosynthesis is amplified, and also to the production process of biotin by this genetically engineered Kurthia sp. strain.
Although the DNA fragment mentioned above may be of various origins, it is preferable to use the strains belonging to the genus Kurthia. Specific examples of such strains include, for example, Kurthia sp. 538-6 (DSM No. 9454) and its mutant strains by selection for resistance to biotin antimetabolites such as Kurthia sp. 538-KA26 (DSM No. 10609).

BRIEF DESCRIPTION OF THE FIGURES
Before the present invention is explained in more detail by referring to the following examples a short description of the enclosed Figures is given:
FIG. 1: Restriction maps of pKB100, pKB200 and pKB300.
FIG. 2: Structure of pKB100.
FIG. 3: Structure of pKB200.
FIG. 4: Restriction maps and complementation results of pKH100, pKH101 and pKH102.
FIG. 5: Structure of pKH100.
FIG. 6: Restriction maps of pKC100, pKC101 and pKC102.
FIG. 7: Structure of pKC100.
FIG. 8: Structures of derived plasmids from pKB100, pKB200 and pKB300.
FIG. 9: Gene organizations of the gene clusters involved in biotin biosynthesis of Kurthia sp. 538-KA26.
FIG. 10: Nucleotide sequence between the ORF1 and ORF2 genes of Kurthia sp. 538-KA26 SEQ ID NO: 17.
FIG. 11: Nucleotide sequence of the promoter region of the bioH gene cluster SEQ ID NO: 20.
FIG. 12: Nucleotide sequence of the promoter region of the bioFII gene cluster SEQ ID NO: 22.
FIG. 13: Construction of the shuttle vector pYK1.
FIG. 14: Construction of the bioB expression plasmid pYK114.

DETAILED DESCRIPTION OF THE INVENTION
Generally speaking the present invention is directed to DNA molecules comprising polynucleotides encoding polypeptides represented by SEQ ID Nos. 2, 4, 6, 8, 10, 12, 14 or 16, and functional derivatives of these polypeptides which contain addition, insertion, deletion and/or substitution of one or more amino acid residue(s), and to DNA molecules comprising polynucleotides which hybridize under stringent hybridizing conditions to polynucleotides which encode such polypeptides and functional derivatives. The invention is also directed to vectors comprising one or more such DNA sequences, for example, a vector wherein said DNA sequences are functionally linked to promoter sequence(s). The invention is further directed to biotin-expressing cells, said cells having been transformed by one or more DNA sequences or vector(s) as defined above, and a process for the production of biotin which comprises cultivating a biotin-expressing cell as defined above in a culture medium to express biotin into the culture medium, and isolating the resulting biotin from the culture medium by methods known in the art. Any conventional culture medium and culturing conditions may be used in accordance with the invention. Preferably, such a process is carried out wherein the cultivation is effected from 1 to 10 days, preferably from 2 to 7 days, at a pH from 5 to 9, preferably from 6 to 8, and a temperature range from 10 to 45.degree. C., preferably from 25 to 30.degree. C.
Finally, the present invention is also directed to a process for the preparation of pharmaceutical, food or feed compositions characterized therein that biotin obtained by such processes is mixed with one or more generally used additives with which a man skilled in the art is familiar.
The DNA molecules of the invention may be produced by any conventional means, such as by the techniques of genetic engineering and automated gene synthesis known in the art.
A detailed method for isolation of DNA fragments carrying the genes coding for the enzymes involved in the biotin biosynthesis from these bacterial strains is described below.
Therefore, DNA can be extracted from Kurthia sp. 538-KA26 by the known phenol method. Such DNA is then partially digested by Sau3AI and ligated with pBR322 digested by BamHI to construct a genomic library of Kurthia sp. 538-KA26.
Biotin auxotrophic mutants which lack the biosynthetic ability to produce biotin are transformed with the genomic library obtained above, and transformants showing biotin prototrophy are selected. The selected transformants have the genornic DNA fragments complementing deficient genes in the biotin auxotrophic mutants. As biotin auxotrophic mutants, Escherichia coli R875 (bioB.sup.-), R877 (bioD.sup.-), BM7086 (bioH.sup.-) and R878 (bioC.sup.-) (J. Bacteriol., 112, 830-839, (1972) and J. Bacteriol., 143, 789-800, (1980) can be used. The transformation of such Escherichia coli strains can be carried out according to a conventional method such as the competent cell method [Molecular Cloning, Cold Spring Harbor Laboratory Press, 252, (1982)].
In the present invention, a hybrid plasmid which complements the bioB deficient mutant of Escherichia coli was obtained in the manner described above. The obtained hybrid plasmid is named pKB100. The pKB100 corresponds to plasmid pBR322 carrying a 5.58 Kb of a genomic DNA fragment from Kurthia sp. 538-KA26, and its restriction cleavage map is shown in FIGS. 1 and 2.
The hybrid plasmid named pKB200 which complements the bioD deficient mutant of Escherichia coli was also obtained as described above. The pKB200 corresponds to plasmid pBR322 carrying a 7.87 Kb of genomic DNA fragment from Kurthia sp. 538-KA26, and its restriction cleavage map is shown in FIGS. 1 and 3. The genomic DNA fragment in pKB200 completely overlapps with the inserted fragment of the pKB100 and carries the bioF, bioB, bioD, ORF1 and ORF2 genes and a part of the bioA gene of Kurthia sp. 538-KA26 as shown in FIG. 9-A.
The complete bioA gene of Kurthia sp. 538-KA26 can be isolated by conventional methods, such as colony hybridization using a part of the genomic DNA fragment in pKB200 as a probe. The whole DNA of Kurthia sp. 538-KA26 is digested with a restriction enzyme such as HindIII and ligated with a plasmid vector cleaved by the same restriction enzyme. Then, Escherichia coli is transformed with the hybrid plasmids carrying genomnic DNA fragments of Kurthia sp. 538-KA26 to construct a genomic library. As a vector and Escherichia coli strain, the pUC19 [Takara Shuzo Co.(Higashiiru, Higashinotohin, Shijodohri, Shimogyo-ku, Kyoto-shi, Japan)] and Escherichia coli JM109 (Takara Shuzo Co.) can be used, respectively.
The hybrid plasmid named pKB300 carrying a 8.44 Kb genomic DNA fragment from Kurthia sp. 538-KA26 was obtained by colony hybridization and its restriction cleavage map is shown in FIG. 1. The genomic DNA fragment in the pKB300 carries two gene clusters involved in the biotin biosynthesis of Kurthia sp. 538-KA26 as shown in FIG. 9-A. One cluster consists of the ORF1, bioD and bioA genes. Another cluster consists of the ORF2, bioF and bioB genes. The nucleotide sequences of the bioD and bioA genes are shown in SEQ ID No. 1 and SEQ ID NO: 3, respectively. The predicted amino acid sequences of the bioD and bioA gene products are shown in SEQ ID NO: 2 and SEQ ID NO: 4, respectively. The bioD gene codes for a polypeptide of 236 amino acid residues with a molecular weight of 26,642. The bioA gene codes for a polypeptide of 460 amino acid residues with a molecular weight of 51,731. The ORF1 gene codes for a polypeptide of 194 amino acid residues with a molecular weight of 21,516, but the biological function of this gene product is unknown.
The nucleotide sequences of the bioF and bioB genes are shown in SEQ ID NO: 5 and SEQ ID NO: 7, respectively. The predicted amino acid sequences of the bioF and bioB gene products are SEQ ID NO: 6 and SEQ ID NO: 8, respectively. The bioF gene codes for a polypeptide of 387 amino acid residues with a molecular weight of 42,619. The bioB gene codes for a polypeptide of 338 amino acid residues with a molecular weight of 37,438. The ORF2 gene codes for a polypeptide of 63 amino acid residues with a molecular weight of 7,447, but the biological function of this gene product is unknown. Inverted repeat sequences which are transcriptional terminator signals are found downstream of the bioA and bioB genes. As shown in FIG. 10 SEQ ID NO: 17, two transcriptional promoter sequences which initiate transcriptions in both directions are found between the ORF1 and ORF2 genes. Furthermore, there are two inverted repeat sequences named Box1 and Box2 involved in the negative control of the transcriptions between each promoter sequence and each translational start codon.
In addition, two hybrid plasmids which complement the biotin auxotrophic mutants of Escherichia coli were obtained in the manner described above. The hybrid plasmid named pKH100 complements the bioH deficient mutant, and the hybrid plasrnid named pKC100 the bioC mutant. pKH100 (FIGS. 4 and 5) has a 1.91 Kb genomic DNA fragment from Kurthia sp. 538-KA26 carrying a gene cluster consisting of the bioH and ORF3 genes as shown in FIG. 9-B. The nucleotide sequence of the bioH gene and the predicted amino acid sequence of this gene product are shown in SEQ ID NO: 9 and SEQ ID NO: 10, respectively. The bioH gene codes for a polypeptide of 267 amino acid residues with a molecular weight of 29,423. The ORF3 gene codes for a polypeptide of 86 amino acid residues with a molecular weight of 9,955, but the biological function of this gene product is unknown. A promoter sequence is found upstream of the bioH gene as shown in FIG. 11 SEQ ID NO: 20, and there is an inverted repeat sequence which is the transcriptional terminator downstream of the ORF3 genes. Since the promoter region has no inverted sequence such as Box1 and Box2, it is expected that the expressions of these genes are not regulated.
On the other hand, pKC100 carries a 6.76 Kb genomic DNA fragment from Kurthia sp. 538-KA26 as shown in FIGS. 6 and 7. The genomic DNA fragment in pKC100 carries a gene cluster consisting of the bioFII, bioHII and bioC genes as shown in FIG. 9-C. The bioHII and bioFII genes are genes for isozymes of the bioH and bioF genes, respectively, because the bioHII and bioFII genes complement the bioH deficient and the bioF deficient mutants of Escherichia coli, respectively. The nucleotide sequences of the bioFII, bioHII and bioC genes are shown in SEQ ID NO: 11, SEQ ID NO: 13 and SEQ ID NO: 15, respectively. The predicted amino acid sequences of the bioFII, bioHII and bioC gene products are shown in SEQ ID NO: 12, SEQ ID NO: 14 and SEQ ID NO: 16, respectively. The bioFII gene codes for a polypeptide of 398 amino acid residues with a molecular weight of 44,776. The bioHII gene codes for a polypeptide of 248 amino acid residues with a molecular weight of 28,629. The bioC gene codes for a polypeptide of 276 amino acid residues with a molecular weight of 31,599. A promoter sequence is found upstream of the bioFII gene, and there is an inverted repeat sequence named Box3 in the promoter region as shown in FIG. 12 SEQ ID NO: 22. The transcription of these genes terminates at an inverted repeat sequence existing downstream of the bioC gene. Since the nucleotide sequence of Box3 is significantly similar to those of Box1 and Box2. expressions of these genes is estimated to be regulated similarly to the bioA and bioB gene clusters.
Needless to say, the nucleotide sequences and amino acid sequences of the genes isolated above are artificially changed in some cases, e.g., the initiation codon GTG or TTG may be converted into an ATG codon.
Therefore the present invention is also directed to functional derivatives of the polypeptides of the present case. Such functional derivatives are defined on the basis of the amino acid sequence of the present invention by addition, insertion, deletion and/or substitution of one or more amino acid residues of such sequences wherein such derivatives still have the same type of enzymatic activity as the corresponding polypeptides of the present invention. Such activities can be measured by any assays known in the art or specifically described herein. Such functional derivatives can be made either by chemical peptide synthesis known in the art or by recombinant means on the basis of the DNA sequences as disclosed herein by methods known in the state of the art, such as, e.g., that disclosed by Sambrook et al. (Molecular Cloning, Cold Spring Harbour Laboratory Press, New York, USA, second edition 1989). Amino acid exchanges in proteins and peptides which do not generally alter the activity of such molecules are known in the state of the art and are described, for example, by H. Neurath and R. L. Hill in "The Proteins" (Academix Press, New York, 1979, see especially FIG. 6, page 14). The most commonly occurring exchanges are: Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Thy/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, Asp/Gly as well as the reverse.
Furthermore the present invention is not only directed to the DNA sequences as disclosed e.g., in the sequence listing as well as their complementary strands, but also to those which include these sequences, DNA sequences which hybridize under Standard Conditions with such sequences or fragments thereof and DNA sequences, which because of the degeneration of the genetic code, do not hybridize under Standard Conditions with such sequences but which code for polypeptides having exactly the same amino acid sequence.
"Standard Conditions" for hybridization mean in this context the conditions which are generally used by a man skilled in the art to detect specific hybridization signals and which are described, e.g., by Sambrook et al., (s.a.) or preferably so-called stringent hybridization and non-stringent washing conditions, or more preferably so-called stringent hybridization and stringent washing conditions a man skilled in the art is familiar with and which are described, e.g., in Sambrook et al. (s.a.).
DNA sequences which are derived from the DNA sequences of the present invention either because they hybridize with such DNA sequences (see above) or can be constructed by the polymerase chain reaction by using primers designed on the basis of such DNA sequences can be prepared either as indicated namely by the PCR reaction, or by site directed mutagenesis [see e.g., Smith, Ann. Rev. Genet. 19, 423 (1985)] or synthetically as decribed, e.g., in EP 747 483 or by the usual methods of Molecular Cloning as described, e.g., in Sambrook et al. (s.a.).
As a host strain for the expression and/or amplification of the DNA sequences of the present invention, any microorganism may be used, e.g., those identified in EP 635 572, but it is preferable to use the strains belonging to the genus Kurthia, especially Kurthia sp. 538-6 (DSM No. 9454) and Kurthia sp. 538-51F9 (DSM No. 10610).
In order to obtain a transformant with a high biotin productivity, the DNA sequences of the present invention are used under the control of a promoter which is effective in such host cells. The DNA sequences of the present invention can be introduced into the host cell by transformation with a plasmid carrying such DNA sequences or by integration into the chromosome of the host cell.
When Kurthia sp. 538-51F9 is used as the host cell, Kurthia sp. 538-51F9 may be transformed with a hybrid plasmid carrying at least one gene involved in biotin biosynthesis isolated above from a Kurthia sp. strain. As a vector plasmid for the hybrid plasmid, pUB110 [J. Bacteriol., 154, 1184-1194, (1983)], pHP13 (Mol. Gen. Genet., 209, 335-342, 1987), or other plasmids comprising the origin of replication functioning in Kurthia sp. strain can be used. As DNA sequences for amplification and/or expression in Kurthia sp. any DNA sequence of the present invention can be used, but the DNA sequence corresponding to the bioB gene coding for biotin synthase is prefered. One example of such a hybrid plasmid is pYK114 shown in FIG. 14. In this plasmid the bioB gene is under the control of the promoter for the bioH gene and carries the replicating origin of pUB110.
Kurthia sp. 538-51F9 may be transformed with pYK114 obtained as described above by the protoplast transformation method [Molecular Biological Methods for Bacillus, 150, (1990)]. However, since Kurthia sp. 538-51F9 has a low efficiency of regeneration from protoplasts, transformation efficiency of this strain is very low. Therefore, it is preferred to use a strain having high efficiency of regeneration from protoplasts should be used, e.g., Kurthia sp. 538-51F9-RG21 which ca be prepared as described in Example 14 of the present case.
The present invention also provides a process for the production of biotin by the cultivation of the thus obtained transformants, and separation and purification of the produced biotin.
Cultivation of the biotin-expressing cells of the present invention can be done by methods known in the art. The culturing conditions are not critical so long as they are sufficient for the expression of biotin by the biotin-expresing cells to occur. A culture medium containing an assimilable carbon source, a digestible nitrogen source, an inorganic salt, and other nutrients necessary for the growth of the biotin-expressing cell can be used. As the carbon source, for example, glucose, fructose, lactose, galactose, sucrose, maltose, starch, dextrin or glycerol may be employed. As the nitrogen source, for example, peptone, soybean powder, corn steep liquor, meat extract, ammonium sulfate, ammonium nitrate, urea or a mixture thereof may be employed. Further, as an inorganic salt, sulfates, hydrochlorides or phosphates of calcium, magnesium, zinc, manganese, cobalt and iron can be employed. And, if necessary, conventional nutrient factors or an antifoaming agent, such as animal oil, vegetable oil or mineral oil can also be added. If the obtained biotin-expressing cell has an antibiotic resistant marker, the respective antibiotic should be supplemented into the medium. The pH of the culture medium may be between 5 to 9, preferably 6 to 8. The cultivation temperature can be 10 to 45.degree. C., preferably 25 to 30.degree. C. The cultivation time can be 1 to 10 days, preferably 2 to 7 days.
The biotin produced under the conditions as described above can easily be isolated from the culture medium by methods known in the art. Thus, for example, after solid materials have been removed from the culture medium by filtration, the biotin in the filtrate may be absorbed on active carbon, then eluted and purified further with an ion exchange resin. Alternatively, the filtrate may be applied directly to an ion exchange resin and, after the elution, the biotin is recrystallized from a mixture of alcohol and water.
EXAMPLES
Example 1
Cloning bioB and bioF Genes of Kurthia sp. 538-KA26
1. Preparation of the Genomic Library.
The acidomycin-resistant strain of Kurthia sp., 538-KA26 (DSM No. 10609), was cultivated in 100 ml of nutrient broth (Kyokuto Seiyaku Co; Honcho 3-1-1, Nihonbashi, Chuoh-Ku, Tokyo, Japan) at 30.degree. C. overnight, and bacterial cells were recovered by centrifugation. The whole DNA was extracted from the bacterial cells by the phenol extraction method [Experiments with gene fusions, Cold Spring Harbor Laboratory, 137-138, (1984)], and 1.9 mg of the whole DNA was obtained.
The whole DNA (10 .mu.g) was partially digested with 1.2 units of Sau3AI at 37.degree. C. for 1 hour to yield fragments with around 10 Kb in length. 5-15 Kb DNA fragments were obtained by agarose gel electrophoresis.
The vector pBR322 (Takara Shuzo Co.) was completely digested with BamHI, and then treated with alkaline phosphatase to avoid self ligation. The DNA fragments were ligated with the cleaved pBR322 using a DNA ligation Kit (Takara Shuzo Co.) according to the instruction of the manufacturer. The ligation mixture was transferred to Escherichia coli JM109 (Takara Shuzo Co.) by the competent cell method [Molecular Cloning, Cold Spring Harbor Laboratory, 252-253, (1982)], and the strains were selected for ampicillin resistance (100 .mu.g/ml) on agar plate LB medium (1% Bacto-tryptone, 0.5% Bacto-yeast extract, 0.5% NaCl, pH 7.5). About 5,000 individual clones having the genomic DNA fragments were obtained as a genomic library.
The ampicillin-resistant strains of the genomic library of Kurthia sp. 538-KA26 were cultivated at 37.degree. C. overnight in 50 ml of LB medium containing 100 .mu.g/ml ampicillin, and bacterial cells were collected by centrifugation. Plasmid DNA was extracted from the bacterial cells by the alkaline-denaturation method [Molecular Cloning, Cold Spring Harbor Laboratory, 90-91, (1982)].
2. Selection of the Clone Carrying the bioB Gene from the Genomic Library.
The plasmid DNA was transferred by the competent cell method into Escherichia coli bioB deficient mutant R875 (J. Bacteriol. 112, 830-839, 1972) without a biotin synthetase activity. The transformed Escherichia coli R875 cells were washed twice with 0.85% NaCl and streaked on 1.5% agar plates of M9CT medium (0.6% Na.sub.2 HPO.sub.4, 0.3% HK.sub.2 PO.sub.4, 0.05% NaCl, 0.1% NH.sub.4 Cl, 2 mM MgSO.sub.4, 0.1 mM CaCl.sub.2, 0.2% glucose, 0.6% vitamin-free casamino acid, 1 .mu.g/ml thiamin) containing 100 .mu.g/ml of ampicillin, and the plates were incubated at 37.degree. C. for 40 hours. One transformant with the phenotype of the biotin prototrophy was obtained. The transformant was cultivated in LB medium containing 100 .mu.g/ml ampicillin, and the hybrid plasmid was extracted from the cells. The hybrid plasmid carries an insert of 5.58 Kb and was designated pKB100. The restriction map is shown in FIGS. 1 and 2.
3. Complementation of Biotin Deficient Mutants of Escherichia coli with pKB100.
pKB100 was transferred to biotin deficient mutants of Escherichia coli, R875 (bioB.sup.-), W602 (bioA.sup.-), R878 (bioC.sup.-), R877 (bioD.sup.-), R874 (bioF.sup.-) or BM7086 (bioH.sup.-) [J. Bacteriol., 112, 830-839, (1972) and J. Bacteriol., 143, 789-800, (1980)], by the competent cell method. The transformed mutants were washed with 0.85% NaCl three times and plated on M9CT agar plates containing 100 .mu.g/ml of ampicillin and 0.1 ng/ml biotin, and the plates were incubated at 37.degree. C. overnight. Colonies on the plates were replicated on M9CT agar plates with 100 .mu.g/ml ampicillin in the presence or absence of 0.1 ng/ml biotin, the plates were incubated at 37.degree. C. for 24 hours to perform the complementation analysis. As shown in Table 1, the pKB100 could complement not only the bioB but also the bioF mutant. In contrast, bioA, bioC, bioD and bioH mutants were not complemented by pKB100. From this results, it was confirmed that the pKB100 carried the bioB and bioF genes of Kurthia sp. 538-KA26.
TABLE 1______________________________________Escherichia coli biotin deficient mutantPlasmid bioA.sup.- bioB.sup.- bioC.sup.- bioD.sup.- bioF.sup.- bioH.sup.-______________________________________pKB100 - + - - + - pKB200 - + - + - - pKB300 + pKH100 - - - - - + pKC100 - - + - + +______________________________________
Example 2
Isolation of Hybrid Plasmid Carrying of the bioD Gene of Kurthia sp. 538-KA26
1. Isolation of the Hybrid Plasmid Carrying the bioD Gene.
The genomic library of Kurthia sp. 538-KA26 of Example 1-1 was transferred into the Escherichia coli bioD deficient mutant R877, and transformants having an ampicillin resistance and biotin prototrophy phenotype were selected in the same manner as described in Example 1-2. The transformant were cultivated at 37.degree. C. overnight in LB medium with 100 .mu.g/ml ampicillin, and the bacterial cells were collected by centrifugation. The hybrid plasmid was extracted from the cells by the alkaline-denaturation method. The hybrid plasmid had a 7.87 Kb insert DNA fragment and was designated pKB200. Cleavage patterns of pKB200 were analyzed using various restriction endonucleases (HindIII, NcoI, EcoRI, BglII, SalI, and PstI) and compared with that of pKB100. Restriction endonuclease analysis revealed that the two hybrid plasmids had exactly the same cleavage sites and that the 1.5 Kb DNA fragment was extended to the left side of pKB100 and the 0.8 Kb fragment was stretched out to the right side in the pKB200 (FIGS. 1 and 3).
2. Complementation of Biotin Deficient Mutant of Escherichia coli with pKB200.
The pKB200 was transferred to the biotin deficient mutants of Escherichia coli, R875 (bioB.sup.-), W602 (bioA.sup.-), R878 (bioC.sup.-), R877 (bioD.sup.-), R874 (bioF.sup.-) or BM7086 (bioH.sup.-). Complementation analysis was performed by the method described in Example 1-3. The pKB200 complemented the bioD and bioB mutants, but not the bioA, bioC, bioF and bioH mutants as shown in Table 1. Although the pKB200 overlapped on the whole length of pKB100, pKB200 did not complement the bioF mutant.
Example 3
Isolation of the Hybrid Plasmid Carrying the bioH Gene of Kurthia sp. 538-KA26
1. Isolation of the Hybrid Plasmid Carrying the bioH Gene.
The genomic library of Kurthia sp. 538-KA26 of Example 1-1 was transferred to the Escherichia coli bioH deficient mutant BM7086. Transformants having the bioH clone were selected for biotin prototrophy in the same manner as in Example 1-2. The hybrid plasmid were extracted from the transformed cells by the alkaline-denaturation method and analyzed by restriction enzymes. The hybrid plasmid had 1.91 Kb inserted DNA fragment and was designated pKH100. Since the genomic library used above has 5-15 Kb of the genomic DNA fragments, the pKH100 was thought to be subjected to a modification, such as deletion, in Escherichia coli strain. The restriction map of the pKH100 is shown in FIGS. 4 and 5. Cleavage patterns of the pKH100 were completely different from those of pKB100 and pKB200. Therefore, pKH100 carried a DNA fragment of the Kurthia chromosome which differed from those in pKB100 and pKB200.
2. Complementation of the Biotin Deficient Mutant of Escherichia coli with pKH100.
Complementation analysis was performed by the method described in Example 1-3. The pKH100 was transferred to the biotin deficient mutants of Escherichia coli, R875 (bioB.sup.-), W602 (bioA.sup.-), R878 (bioC.sup.-), R877 (bioD.sup.-), R874 (bioF.sup.-) or BM7086 (bioH.sup.-). pKH100 complemented only the bioH mutant, but not the bioB, bioA, bioC, bioD and bioF mutants as shown in Table 1. Thus, pKH100 carries the bioH gene.
Example 4
Isolation of the Hybrid Plasmid Carrying the bioC Gene of Kurthia sp. 538-KA26
1. Isolation of the Hybrid Plasmid Carrying the bioC Gene.
The genomic library of Kurthia sp. 538-KA26 of Example 1-1 was transferred to the Escherichia coli bioC deficient mutant R878. Transformants with the bioC clone were selected for biotin prototrophy in the same manner as describedin Example 1-2. The hybrid plasmid was extracted from the transformant cells by the alkaline-denaturation method and analyzed with restriction enzymes. The hybrid plasmid had a 6.76 Kb inserted DNA fragment and was designated pKC100. The restriction map of pKC100 is shown in FIGS. 6 and 7. Cleavage patterns of pKC100 were completely different from those of pKB100, pKB200 and pKH100. Therefore, pKC100 carries a different region of the Kurthia chromosome from those of pKB100, pKB200 and pKH100.
2. Complementation of the Botin Deficient Mutant of Escherichia coli with pKC100.
The complementation analysis was performed by the method described in Example 1-3. pKC100 was transferred to the biotin deficient mutants of Escherichia coli, R875 (bioB.sup.-), W602 (bioA.sup.-), R878 (bioC.sup.-), R877 (bioD.sup.-), R874 (bioF.sup.-) or BM7086 (bioH.sup.-). pKC100 complemented the bioC, bioF and bioH mutants as shown in Table 1. Since the inserted DNA fragment in pKH100 was different from those in pKB100 and pKH100, pKC100 carried not only the bioC gene but also genes for isozymes of the bioF gene product (KAPA synthetase) and the bioH gene product.
Example 5
Isolation of the Hybrid Plasmid Carrying the bioA Gene of Kurthia sp. 538-KA26
1. Isolation of the Left Region of the Chromosomal DNA in pKB200.
We isolated the left region of the chromosomal DNA in pKB200 from Kurthia sp. 538-KA26 chromosomal DNA by the hybridization method. The whole DNA of Kurthia sp. 538-KA26 was completely digested with HindIII and subjected to agarose gel electrophoresis. The DNA fragments on the gel were denatured and then transferred to a nylon membrane (Hybond-N, Amersham) according to the recommendations of the manufacturer.
pKB200 was completely digested with NcoI, and a 2.1 Kb NcoI fragment was isolated by agarose gel electrophoresis (FIG. 1). The NcoI fragment was labeled with .sup.32 P by the Multiprime DNA labeling system (Amersham) and used as a hybridization probe. The hybridization was performed on the membrane prepared above using the "Rapid hybridization buffer" (Amersham) according to the instructions of the manufacturer. The probe strongly hybridized to a HindIII fragment of about 8.5 Kb.
In order to isolate the 8.5 Kb fragment, the whole DNA of Kurthia sp. 538-KA26 was completely digested with HindIII, and 7.5-9.5 Kb DNA fragments were obtained by agarose gel electrophoresis. The vector plasmid pUC19 (Takara Shuzo Co.) was completely digested with HindIII and treated with alkaline phosphatase to avoid self ligation. The 7.5-9.5 Kb DNA fragments were ligated with the cleaved the pUC19 using a DNA ligation Kit (Takara Shuzo Co.), and the reaction mixture was transferred to Escherichia coli JM109 by the competent cell method. About 1,000 individual clones carrying such genomic DNA fragments were obtained as a genomic library.
The selection was carried out by the colony hybridization method according to the protocol described by Maniatis et al. [Molecular Cloning, Cold Spring Harbor Laboratory, 312-328, (1982)]. The grown colonies on the agar plates were transferred to nylon membranes (Hybond-N, Amersham) and lysed by alkali. The denatured DNA was immobilized on the membranes. .sup.32 P labeled NcoI fragments prepared as described above were used as a hybridization probe, and the hybridization was performed using the "Rapid hybridization buffer" (Amersham) according to the instructions of the manufacturer. Three colonies which hybridized with the probe DNA were obtained, and hybrid plasmids in these colonies were extracted by the alkaline-denaturation method.
The structure analysis was performed with restriction enzymes (BamHI, HindIII, NcoI, EcoRI, BglII, SalI and PstI). All of the three hybrid plasmids had a 8.44 Kb inserted DNA fragment, and the three hybrid plasmids had exactly the same cleavage patterns. These results indicated that they were identical. This hybrid plasmid was designated pKB300. The restriction map of pKB300 is shown in FIG. 1. About half length of the genomic DNA fragment in pKB300 overlappes with that of pKB200.
2. Complementation of the bioA Deficient Mutant of Escherichia coli with pKB300.
The complementation analysis of Escherichia coli W602 (bioA.sup.-) with pKB300 was performed by the method described in Example 1-3. Since pKB300 complemented the bioA mutation (Table 1), pKB300 carries the bioA gene of Kurthia sp.
Example 6
Subcloning of the bioA, B, D and F Genes of Kurthia sp. 538-KA26
1. Construction of the Hybrid Plasmid pKB103 and pKB104.
pKB100 was completely digested with HindIII, and a 3.3 Kb HindIII fragment was isolated. The 3.3 Kb fragment was ligated with the vector pUC18 (Takara Shuzo Co.) cleaved with HindIII using a DNA ligation Kit to construct the hybrid plasmids pKB103 and pKB104. In pKB103 and pKB104, the 3.3 Kb fragments were inserted in both orientations relative to the promoter-operator of the lac gene in pUC18. Their restriction map is shown in FIG. 8.
Complementation of the bioB or bioF deficient mutants of Escherichia coli (R875 or R874) were performed with pKB103 and pKB104 in the same manner as described in Example 1-3. pKB103 and pKB104 complemented the bioB and bioF mutants (Table 2).
2. Construction of Derivatives of pKB200.
Since pKB200 complemented the bioD mutation and covered the whole length of pKB100 carrying the bioB and bioF genes, a series of deletion mutations of pKB200 were constructed to localize more precisely bioB, bioD and bioF. A 4.0 Kb SalI-HindIII fragment of pKB200 was inserted into the SalI and HindIII sites of pUC18 and pUC19 to give pKB221 and pKB222 in which the SalI-HindIII fragment is placed in both orientations.
pKB200 was completely digested with NruI, and a 7.5 Kb NruI fragment was isolated by agarose gel electrophoresis. The NruI fragment was recirculated by the DNA ligation Kit, and pKB223 was obtained.
pKB200 was completely digested with HindIII. A 4.8 Kb HindIII fragment was isolated by agarose gel electrophoresis and cloned into the HindIII site of pUC18 in both orientations to generate pKB224 and pKB225.
pKB200 was partially digested with NcoI, and a 3.1 Kb NcoI fragment was isolated by agarose gel electrophoresis. The ends of the NcoI fragment were made blunt by using the Klenow fragment of the DNA polymerase I (Takara Shuzo Co.) and ligated with HindIII linker (Takara Shuzo Co.) The 3.1 Kb HindIII fragment was obtained by treatment with HindIII and cloned into the HindIII site of the pUC19 in both orientation to give pKB228 and pKB229.
In the same manner, both ends of a 2.1 Kb NcoI fragment of pKB200 were converted to HindIII sites by treatment with the Klenow fragment and addition of HindIII linkers. Then the obtained HindIII fragment was inserted into the HindIII site of pUC19 in both orientations to give pKB230 and pKB231.
pKB234 and pKB235 were generated by insertion of a 1.6 Kb HindIII-NruI fragment of pKB230 into the HindIII and SmaI sites of pUC19 and pUC18, respectively.
The restriction maps of the pKB200 derivatives are shown in FIG. 8.
3. Complementation Analysis of Biotin Deficient Mutants of Escherichia coli with pKB200 Derivatives.
Complementation analysis was performed with the pKB200 derivatives in the same manner as described in Example 1-3. The complementation results are summarized in Table 2. The bioB deficient mutant was complemented by pKB221, pKB222, pKB224 and pKB225, but not by pKB223, pKB228, pKB229, pKB230, pKB231, pKB234 and pKB235. The bioF deficient mutant was complemented by pKB223, pKB224, pKB225, pKB228 and pKB229, but not by pKB221, pKB222, pKB230, pKB231, pKB234 and pKB235. On the other hand, the bioD deficient mutant was complemented by pKB223, pKB224 and pKB225, but not by pKB221 and pKB222.
Together with the complementation analysis with pKB103 and pKB104, these results support that the bioF gene is present at the left side of the first NruI site on pKB103 while the bioB gene is located on the right side of the same NruI site with a short overlap to the left and that the bioD gene is present on at most 1.5 Kb left side region of the pKB200. Thus, the complementation results with various derivatives of pKB100 and pKB200 showed that the bioD, bioF and bioB genes lie in turn on the 4.4 Kb region at the left side of the HindIII site of pKB200.
4. Construction of the Hybrid Plasmid pKB361.
To determine the location of the bioA gene, the derivative of pKB300 was constructed. pKB361 was generated by insertion of a 2.8 Kb BamHI-SalI fragment of pKB300 into the BamHI and SalI sites of pUC19 (FIG. 8).
pKB361 was transferred to the bioA deficient mutant of Escherichia coli (W602), and complementation analysis was performed in the same manner as described in Example 1-3. The bioA mutant was complemented by pKB361 (Table 2), suggesting the presence of the bioA gene within the 2.8 Kb region between the BamHI and SalI sites of pKB300.
TABLE 2______________________________________ Escherichia coli biotin deficient mutantPlasmid bioA.sup.- bioD.sup.- bioF.sup.- bioB.sup.-______________________________________pKB103, 104 + + pKB221, 222 - - + pKB223 + + - pKB224, 225 + + + pKB228, 229 + - pKB230, 231 - - pKB234, 235 - - pKB361 +______________________________________
Example 7
Subcloning of the bioH gene of Kurthia sp. 538-KA26
1. Construction of the Hybrid Plasmids pKH101 and pKH102.
pKH100 was completely digested with BamHI and recirculated with a DNA ligation Kit to generate pKH101 in which a 0.75 Kb BamHI fragment was deleted from pKH100 (FIG. 4). pKH102 was constructed from pKH100 by treatment with HindIII followed by recirculation with a DNA ligation Kit. The pKH102 lacked a 1.07 Kb HindIII fragment in pKH100 (FIG. 4).
Complementation analysis of the Escherichia coli bioH mutant (R878) was performed with pKH101 and pKH102 in the same manner as in Example 1-3. pKH101 complemented the bioH mutant, but not pKH102 (FIG. 4). This result indicated that the bioH gene is located in the left region (1.16 Kb) of the BamHI site on pKH100.
Example 8
Subcloning of the bioC Gene of Kurthia sp. 538-KA26
pKC100 was completely digested with BamHI, and a 1.81 Kb BamHI fragment was isolated by agarose gel electrophoresis. The BamHI fragment was ligated with pBR322 treated with BamHI and the Klenow fragment by a DNA ligation Kit. Finally, pKC101 and pKC102 in which the BamHI fragment was inserted in both orientations were obtained (FIG. 6).
pKC101 and pKC102 were transferred to the Escherichia coli bioC mutant R878, and complementation analysis was carried out in the same manner as in Example 1-3. The bioC mutant was complemented with pKC101 and pKC102, and the bioC gene was confirmed to lie in the 1.81 Kb BamHI fragment.
Example 9
Nucleotide Sequence of the Inserted DNA Fragments on pKB100, pKB200 and pKB300
For nucleotide sequencing analysis of the inserted DNA fragments of pKB100, pKB200 and pKB300, several subclones overlapping mutually were constructed using pUC18, pUC19, M13mp18 and M13mp19 (Takara Shuzo Co.) and a series of deletion derivatives of the subclones were obtained by the Kilo-Sequencing Deletion Kit (Takara Shuzo Co.). Then, nucleotide sequencing analysis of the deletion derivatives was carried out by the dideoxy-chain termination technique (Sequenase version 2.0 DNA sequencing kit using 7-deaza-dGTP, United States Biochemical Co.). The results were analyzed by the computer program (GENETYX) from Software Development Co.
Computer analysis of this sequence revealed that the cloned DNA fragment has the capacity to code for six open reading frames (ORF). This gene operon has two gene clusters proceeding to both directions (FIG. 9-A).
The first ORF in the left gene cluster starts with the TTG codon preceded by a ribosomal binding site (RBS) with homology to the 3' end of the Bacillus subtilis 16S rRNA and codes for a protein of 194 amino acid residues having a molecular weight of 21,516. It was not possible to determine the function of the gene product by the complementation analysis, accordingly, this ORF was named ORF1.
The nucleotide sequence of the second ORF in the left gene cluster is shown in SEQ ID NO: 1. This gene codes for a protein of 236 amino acid residues with a molecular weight of 26,642. The predicted amino acid sequence of this gene product is shown in SEQ ID NO: 2. A putative RBS is found upstream of the ATG initiation codon. The complementation analysis (Example 6-3) showed that this ORF is the bioD gene.
The third ORF in the left gene cluster has a putative RBS upstream of the ATG initiation codon, and the nucleotide sequence of this gene is shown in SEQ ID NO: 3. This gene codes for a protein of 460 amino acid residues with a molecular weight of 51,731. The predicted amino acid sequence of this gene product is shown in SEQ ID NO: 4. This ORF was confirmed to correspond to the bioA gene (Example 6-3). An inverted repeat sequence was found to be located approximately 3 bp downstream from the termination codon. This structure may act as a transcriptional terminator.
The first ORF in the right gene cluster, named ORF2 starts at the ATG codon preceded by a putative RBS. This gene product is a protein consisting of 63 amino acid residues, and the calculated molecular weight is 7,447. We could not identify the function of this gene product by the complementation analysis and the amino acid sequence homology search. Accordingly, this ORF was named ORF2.
The nucleotide sequence of the second ORF in the right gene cluster is shown in SEQ ID NO: 5. This gene has three potential ATG initiation codons corresponding to the first, twenty-fifth and thirty-second amino acid residues. The complementation analysis (Example 6-3) showed that this ORF corresponds to the bioF gene. The predicted amino acid sequence of this gene product is shown in SEQ ID NO: 6. The molecular weight of the predicted protein with 387 amino acid residues was calculated to be 42,619, starting from the first initiation codon.
The third ORF in the right gene cluster as shown in SEQ ID NO: 7 has three potential initiation codons, two ATG codons (the first and eighteenth amino acid residues) and a GTG codon (the twelfth amino acid residue). The predicted amino acid sequence of this-gene product is shown in SEQ ID NO: 8. The molecular weight of the predicted protein with 338 amino acid residues translated from the first initiation codon was calculated to be 37,438. The complementation analysis (Example 6-3) showed that this ORF corresponds to the bioB gene. The presence of an inverted repeat sequence 16 bp downstream from the termination codon is characteristic of a transcriptional terminator.
There were two possible promoter sequences forming face to face promoters between ORF1 and ORF2 as shown in FIG. 10 SEQ ID NO: 17. The transcriptions proceed to the left into the ORF1, bioD and bioA gene cluster, and to the right into the ORF2, bioF and bioB gene cluster. In addition, two transcriptional terminators were located downstream of the termination codons of the bioA and bioB genes. Therefore, the transcriptions in both directions generate two different mRNAs.
Two components of the inverted repeat sequences, Box1 and Box2, were found between the initiation site of the ORF1 and ORF2 genes (FIG. 10 SEQ ID NO: 17). The overall homology for the Box1 and Box2 is 82.5%. Comparison of the Box1 or Box2 with the operator of the Escherichia coli biotin operon [Nature, 276, 689-694, (1978)] showed that there is a high level of conservation (54.6% homology for both). The similarities between two inverted repeat sequences of the biotin operator of Escherichia coli suggest that the Box1 and Box2 must be involved in the negative control of the biotin synthesis by biotin.
Example 10
Nucleotide Sequence of the Inserted DNA Fragments of pKH100
The nucleotide sequence analysis of the inserted DNA fragment of pKH100 was performed in the same manner as described in Example 9. A gene cluster containing two ORFs was found on the inserted DNA fragment (FIG. 9-B). In addition, it was confirmed that a part of the vector plasmid pBR322 and the inserted DNA fragment were deleted.
The first ORF as shown in SEQ ID NO: 9 codes for a protein of 267 amino acid residues, and the calculated molecular weight is 29,423. The predicted amino acid sequence of this gene product is shown in SEQ ID NO: 10. A putative RBS is located at 6 bp upstream from the ATG initiation codon. The complementation analysis, as shown in Example 7, indicated that this ORF corresponds to the bioH gene.
The second ORF with a potential RBS was found downstream of the bioH gene. The ORF codes for a protein of 86 amino acid residues with a molecular weight of 9,955. The protein encoded by the ORF did not share homology with the biotin gene products of Escherichia coli and Bacillus sphaericus. The ORF was named ORF3.
A possible promoter sequence was found upstream from the initiation codon of the bioH gene as shown in FIG. 11 SEQ ID NO: 20. Since no inverted repeat sequence such as Box1 and Box2 was found in the 5'-noncoding region of the bioH gene, the transcription of this gene cluster must be not regulated. In addition, there is an inverted repeat sequence overlapping with the termination codon of ORF3. Since this structure is able to act as a transcriptional terminator, the putative bioH promoter would therefore allow transcription of the bioH and ORF3 genes.
Example 11
Nucleotide Sequence of the Inserted DNA Fragments of pKC100
The nucleotide sequence analysis of the inserted DNA fragment of pKC100 was performed in the same manner as described in Example 9. A gene cluster consisting of three ORFs was found on the inserted DNA fragment (FIG. 9-C).
The third ORF has a putative RBS upstream of the initiation codon and the nucleotide sequence of this gene is shown in SEQ ID NO: 15. This gene codes for a protein of 276 amino acid residues, and the calculated molecular weight is 31,599. The predicted amino acid sequence of this gene product is shown in SEQ ID NO: 16. The complementation analysis as shown in Example 8 indicating that this ORF corresponds to the bioC gene.
The first ORF as shown in SEQ ID NO: 11 codes for a protein of 398 amino acid residues with a molecular weight of 44,776. A putative RBS is located upstream of the initiation codon. The predicted amino acid sequence of this gene product as shown in SEQ ID NO: 12 has 43.0% homology with that of the bioF gene product of Kurthia sp. 538-KA26 in Example 9. Moreover, the pKC100 complemented the Escherichia coli bioF mutant as shown in Example 4. Therefore, this ORF was concluded to be a gene for an isozyme of the bioF gene product, KAPA synthetase. Therefore, this ORF was named bioFII gene.
The second ORF as shown in SEQ ID NO: 13 has a putative RBS upstream of the initiation codon. This gene codes for a protein of 248 amino acid residues with a molecular weight of 28,629. The predicted amino acid sequence of this gene product as shown in SEQ ID NO: 14 has 24.2% homology with that of the bioH gene product of Kurthia sp. 538-KA26 in Example 10. As shown in Example 4, the pKC100 also complemented the Escherichia coli bioH mutant. These results showed that this ORF is a gene for isozyme of the bioH gene product therefore this ORF was named bioHII gene.
A possible promoter sequence was found upstream from the initiation codon of the bioFII gene as shown in FIG. 12 SEQ ID NO: 22. An inverted sequence is located between the promoter sequence and the RBS of the bioFII gene. This inverted repeat sequence designated Box3 was compared with the Box1 and Box2 located between the ORF1 and ORF2 genes (Example 9). The Box1, Box2 and Box3 were extremely similar to each other (homology of Box1 and Box3 was 80.0% and that of Box2 and Box3 was 77.5%). Therefore, the cluster of the bioC gene must be regulated by a negative control similarly to the bioA cluster and the bioB cluster. In addition, there is an inverted repeat sequence 254 bp downstream of the termination codon of the bioC gene. This structure is thought to act as a transcriptional terminator.
Example 12
Construction of the Shuttle Vector for Escherichia coli and Kurthia sp. Strain
A shuttle vector for Escherichia coli and Kurthia sp. was constructed by the strategy as shown in FIG. 13. The Staphylococcus aureus plasmid pUB110 (Bacillus Genetic Stock Center; The Ohio State University, Department of Biochemistry, 484 West Twelfth Avenue, Columbus, Ohio 43210, USA) was completely digested with EcoRI and PvuII. A 3.5 Kb EcoRI-PvuII fragment containing the replication origin for Kurthia sp. and the kanamycin resistant gene was isolated by agarose gel electrophoresis. The pUC19 was completely digested with EcoRI and DraI, and the 1.2 Kb EcoRI-DraI fragment having the replication origin of Escherichia coli was isolated by agarose gel electrophoresis. Then, these fragments were ligated with a DNA ligation Kit to generate the shuttle vector pYK1. pYK1 can replicate in Escherichia coli and Kurthia sp., and Escherichia coli or Kurthia sp. transformed by pYK1 show resistance to kanamycin.
Example 13
Construction of the Expression Plasmid of the bioB Gene of Kurthia sp.
pYK114 in which the Kurthia bioB gene was inserted downstream of the promoter of the Kurthia bioH gene was constructed by the strategy as shown in FIG. 14. pKH101 of Example 7 was completely digested with BanII, and ends of BanII fragments were blunted by the Klenow fragment of the DNA polymerase. Then the BanII fragments were treated with EcoRI, and a 0.6 Kb EcoRI-blunt fragment containing the bioH promoter was isolated by agarose gel electrophoresis. pKB104 of Example 6 was completely digested with KpnI, and KpnI ends were changed to blunt ends by treatment with the Klenow fragment. After digestion with HindIII, a 1.3 Kb blunt-HindIII fragment carrying the bioB gene was isolated by agarose electrophoresis. The EcoRI-blunt and blunt-HindIII fragments were ligated with pYK1 digested with EcoRI and HindIII to construct pYK114. The bioB gene is constitutively expressed under the bioH promoter from pYK114.
Example 14
Isolation of the Derivative Strain of Kurthia sp. 538-51F9 with a High Transformation Efficiency.
Kurthia sp. 538-51F9 (DSM No.10610) was cultivated at 28.degree. C. in 50 ml of Tripticase Soy Broth (Becton Dickinson) until an optical density at 600 nm (OD.sub.600) of 1.0. Grown cells were collected by centrifugation and suspended in SMM (0.5 M sucrose, 0.02M sodium maleate, 0.02 M MgCl.sub.2 6H.sub.2 O; pH 6.5) at OD.sub.600 16. Then lysozyme (Sigma) was added to the cell suspension at 200 mg/ml, and the suspension was incubated at 30.degree. C. for 90 minutes to form protoplasts. After the protoplasts have been washed with SMM twice, they were suspended in 0.5 ml of SMM. 1.5 ml of PEG solution (30% w/v polyethyleneglycol 4000 in SMM) was added to the protoplast suspension, and the suspension was incubated for 2 minutes on ice. Then 6 ml of SMM was added, and the protoplasts were collected by centrifugation. The collected protoplasts were suspended in SMM and incubated at 30.degree. C. for 90 minutes. DM3 medium (0.5 M sodium succinate pH 7.3, 0.5% w/v casamino acid, 0.5% w/v yeast extract, 0.3% w/v KH.sub.2 PO.sub.4, 0.7% w/v K.sub.2 HPO.sub.4, 0.5% w/v glucose, 0.02 M MgCl.sub.2 6H.sub.2 O, 0.01% w/v bovine serum albumin) containing 0.6% agarose (Sigma; Type VII) was added to the protoplast suspension, and the suspension was overlaid on DM3 medium agar plates. The plates were incubated at 30.degree. C. for 3 days. In total, 65 colonies regenerated on the DM3 plates were obtained.
The transformation efficiency of the regenerated strains was investigated with pYK1 of Example 12. As a result, 40 strains were selected and cultivated at 28.degree. C. in 50 ml of Tripticase Soy Broth until OD.sub.600 was 1.0. Grown cells were collected by centrifugation and suspended in SMM at OD.sub.600 16. Then the cells were treated with lysozyme by the method described above, and the protoplasts were obtained. The protoplasts were suspended in 0.5 ml SMM, and pYK1 (1 .mu.g) was added to the protoplast suspensions. After addition of 1.5 ml of a PEG solution, the suspensions were incubated for 2 minutes on ice. 6 ml of SMM was added, and the protoplasts were collected by centrifugation. Then the protoplasts were suspended in SMM and incubated at 30.degree. C. for 90 minutes. The DM3 medium containing 0.6% agarose was added to the protoplast suspensions, and the suspensions were overlaid on DM3 medium agar plates. The plates were incubated at 30.degree. C. for 3 days. The DM3-agarose including the regenerated colonies on the plates were collected and spread on the nutrient broth agar plates with 5 .mu.g/ml kanamycin to select the transfornants. The plates were incubated overnight at 30.degree. C. Finally, the derivative strain, Kurthia sp. 538-51F9-RG21, characterized by a high transformation efficiency (2,000 transformants per .mu.g of DNA) was obtained.
Example 15
Amplification of the bioB Gene in Kurthia sp. 538-51F9-RG21
1. Transformation of Kurthia sp. 538-51F9-RG21.
The expression plasmid of the bioB gene of the Kurthia strain, pYK114, was constructed as described in Example 13. Kurthia sp. 538-51F9-RG21 was transformed with pYK114 and the vector plasmid pYK1 as described in Example 14. Kurthia sp. 538-51F9-RG21 carrying pYK1 or pYK114 was named Kurthia sp. 538-51F9-RG21 (pYK1) or Kurthia sp. 538-51F9-RG21 (pYK114), respectively.
2. Biotin Production by Fermentation.
Kurthia sp. 538-51F9-RG21 (pYK1) and Kurthia sp. 538-51F9-RG21 (pYK114) were inoculated into 50 ml of the production medium (6% glycerol, 5.5% proteose peptone, 0.1% KH.sub.2 PO.sub.4, 0.05% MgSO.sub.4 7H.sub.2 O, 0.05% FeSO.sub.4 7H.sub.2 O, 0.001% MnSO.sub.4 5H.sub.2 O; pH 7.0) containing 5 .mu.g/ml kanamycin. As a control, Kurthia sp. 538-51F9-RG21 was inoculated into 50 ml of the production medium. The cultivation was carried out at 28.degree. C. for 120 hours.
After the cultivation, 2 ml of the culture broth was centrifuged to remove bacterial cells, and the supernatant was obtained. Biotin production in the supernatant was assayed by the microbiological assay using Lactobacillus plantarum (ATCC 8014). The amounts of produced biotin are given in Table 3.
TABLE 3______________________________________Strain of Kurthia sp. Biotin production (mg/L)______________________________________51F9-RG21 15.4 51F9-RG21 (pYK1) 14.3 51F9-RG21 (pYK114) 39.0______________________________________
__________________________________________________________________________# SEQUENCE LISTING - - - - <160> NUMBER OF SEQ ID NOS: 23 - - <210> SEQ ID NO 1 <211> LENGTH: 711 <212> TYPE: DNA <213> ORGANISM: Kurthia sp. <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (1)..(708) - - <400> SEQUENCE: 1 - - atg ggt caa gcc tac ttt ata acc gga act gg - #c acg gat atc ggaaaa 48 Met Gly Gln Ala Tyr Phe Ile Thr Gly Thr Gl - #y Thr Asp Ile Gly Lys 1 5 - # 10 - # 15 - - acc gtc gcc acg agt tta ctc tat atg tct ct - #t caa aca atg gga aaa 96 Thr Val Ala Thr Ser Leu Leu Tyr Met Ser Le - #u Gln Thr Met Gly Lys 20 - # 25 - # 30 - - agc gtc aca ata ttt aag ccg ttt caa aca gg - #a ttg att cac gaa acg 144 Ser Val Thr Ile Phe Lys Pro Phe Gln Thr Gl - #y Leu Ile His Glu Thr 35 - # 40 - # 45 - - aat aca tac cct gac atc tct tgg ttt gag ca - #g gaa ctt ggt gta aag 192 Asn Thr Tyr Pro Asp Ile Ser Trp Phe Glu Gl - #n Glu Leu Gly Val Lys 50 - # 55 - # 60 - - gca cct ggg ttt tac atg ctt gaa ccc gaa ac - #a tct cca cac tta gct 240 Ala Pro Gly Phe Tyr Met Leu Glu Pro Glu Th - #r Ser Pro His Leu Ala 65 - # 70 - # 75 - # 80 - - ata aaa tta aca ggg caa caa atc gac gag ca - #a aag gtc gtg gaa cga 288 Ile Lys Leu Thr Gly Gln Gln Ile Asp Glu Gl - #n Lys Val Val Glu Arg 85 - # 90 - # 95 - - gtt cac gaa ctc gaa caa atg tat gac atc gt - #g tta gtc gag ggc gct 336 Val His Glu Leu Glu Gln Met Tyr Asp Ile Va - #l Leu Val Glu Gly Ala 100 - # 105 - # 110 - - ggg gga ttg gcc gta cca ctc att gaa cga gc - #g aac agt ttc tat atg 384 Gly Gly Leu Ala Val Pro Leu Ile Glu Arg Al - #a Asn Ser Phe Tyr Met 115 - # 120 - # 125 - - aca acc gat tta att aga gat tgc aac atg cc - #a gtc att ttc gtt tct 432 Thr Thr Asp Leu Ile Arg Asp Cys Asn Met Pr - #o Val Ile Phe Val Ser 130 - # 135 - # 140 - - aca agc ggt tta gga tcg att cat aat gtc at - #a act acg cat tcg tat 480 Thr Ser Gly Leu Gly Ser Ile His Asn Val Il - #e Thr Thr His Ser Tyr 145 1 - #50 1 - #55 1 -#60 - - gcc aaa ttg cat gat att agc gtt aaa act at - #t tta tat aac cattat 528 Ala Lys Leu His Asp Ile Ser Val Lys Thr Il - #e Leu Tyr Asn His Tyr 165 - # 170 - # 175 - - cgg ccc gac gat gaa att cat cgt gac aat at - #c cta acc gtt gaa aag 576 Arg Pro Asp Asp Glu Ile His Arg Asp Asn Il - #e Leu Thr Val Glu Lys 180 - # 185 - # 190 - - ctc aca gga ctc gct gac ctc gcc tgc ata cc - #a aca ttt gtc gac gta 624 Leu Thr Gly Leu Ala Asp Leu Ala Cys Ile Pr - #o Thr Phe Val Asp Val 195 - # 200 - # 205 - - aga aaa gat ctg aga gtc tac ata ctt gat tt - #a ctt agt aat cat gaa 672 Arg Lys Asp Leu Arg Val Tyr Ile Leu Asp Le - #u Leu Ser Asn His Glu 210 - # 215 - # 220 - - ttt act caa caa cta aaa gag gtg ttc aag aa - #t gaa tag - # 711 Phe Thr Gln Gln Leu Lys Glu Val Phe Lys As - #n Glu 225 2 - #30 2 - #35 - - - - <210> SEQ ID NO 2 <211> LENGTH: 236 <212> TYPE: PRT <213> ORGANISM: Kurthia sp. - - <400> SEQUENCE: 2 - - Met Gly Gln Ala Tyr Phe Ile Thr Gly Thr Gl - #y Thr Asp Ile Gly Lys 1 5 - # 10 - # 15 - - Thr Val Ala Thr Ser Leu Leu Tyr Met Ser Le - #u Gln Thr Met Gly Lys 20 - # 25 - # 30 - - Ser Val Thr Ile Phe Lys Pro Phe Gln Thr Gl - #y Leu Ile His Glu Thr 35 - # 40 - # 45 - - Asn Thr Tyr Pro Asp Ile Ser Trp Phe Glu Gl - #n Glu Leu Gly Val Lys 50 - # 55 - # 60 - - Ala Pro Gly Phe Tyr Met Leu Glu Pro Glu Th - #r Ser Pro His Leu Ala 65 - # 70 - # 75 - # 80 - - Ile Lys Leu Thr Gly Gln Gln Ile Asp Glu Gl - #n Lys Val Val Glu Arg 85 - # 90 - # 95 - - Val His Glu Leu Glu Gln Met Tyr Asp Ile Va - #l Leu Val Glu Gly Ala 100 - # 105 - # 110 - - Gly Gly Leu Ala Val Pro Leu Ile Glu Arg Al - #a Asn Ser Phe Tyr Met 115 - # 120 - # 125 - - Thr Thr Asp Leu Ile Arg Asp Cys Asn Met Pr - #o Val Ile Phe Val Ser 130 - # 135 - # 140 - - Thr Ser Gly Leu Gly Ser Ile His Asn Val Il - #e Thr Thr His Ser Tyr 145 1 - #50 1 - #55 1 -#60 - - Ala Lys Leu His Asp Ile Ser Val Lys Thr Il - #e Leu Tyr Asn HisTyr 165 - # 170 - # 175 - - Arg Pro Asp Asp Glu Ile His Arg Asp Asn Il - #e Leu Thr Val Glu Lys 180 - # 185 - # 190 - - Leu Thr Gly Leu Ala Asp Leu Ala Cys Ile Pr - #o Thr Phe Val Asp Val 195 - # 200 - # 205 - - Arg Lys Asp Leu Arg Val Tyr Ile Leu Asp Le - #u Leu Ser Asn His Glu 210 - # 215 - # 220 - - Phe Thr Gln Gln Leu Lys Glu Val Phe Lys As - #n Glu 225 2 - #30 2 - #35 - - - - <210> SEQ ID NO 3 <211> LENGTH: 1383 <212> TYPE: DNA <213> ORGANISM: Kurthia sp. <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (1)..(1380) - - <400> SEQUENCE: 3 - - atg aat agt cat gac tta gaa aag tgg gat aa - #g gaa tat gta tgg cat 48 Met Asn Ser His Asp Leu Glu Lys Trp Asp Ly - #s Glu Tyr Val Trp His 1 5 - # 10 - # 15 - - ccg ttt aca caa atg aaa acg tat cga gaa ag - #t aaa ccg cta atc att 96 Pro Phe Thr Gln Met Lys Thr Tyr Arg Glu Se - #r Lys Pro Leu Ile Ile 20 - # 25 - # 30 - - gaa cgc ggg gaa ggg agc tac ctt ttt gac at - #a gaa ggc aat cgg tac 144 Glu Arg Gly Glu Gly Ser Tyr Leu Phe Asp Il - #e Glu Gly Asn Arg Tyr 35 - # 40 - # 45 - - ttg gac ggt tat gct tca tta tgg gtc aac gt - #a cat ggc cat aat gaa 192 Leu Asp Gly Tyr Ala Ser Leu Trp Val Asn Va - #l His Gly His Asn Glu 50 - # 55 - # 60 - - cca gag cta aac aac gct ctc att gaa caa gt - #t gaa aaa gtc gca cac 240 Pro Glu Leu Asn Asn Ala Leu Ile Glu Gln Va - #l Glu Lys Val Ala His 65 - # 70 - # 75 - # 80 - - tca aca cta cta gga tct gca aat gta cca tc - #c ata tta ctg gct aag 288 Ser Thr Leu Leu Gly Ser Ala Asn Val Pro Se - #r Ile Leu Leu Ala Lys 85 - # 90 - # 95 - - aaa tta gca gag att act cct ggt cat tta tc - #g aaa gtc ttt tac tcg 336 Lys Leu Ala Glu Ile Thr Pro Gly His Leu Se - #r Lys Val Phe Tyr Ser 100 - # 105 - # 110 - - gac act gga tca gct gct gta gaa atc tcc ct - #t aaa gtc gct tat caa 384 Asp Thr Gly Ser Ala Ala Val Glu Ile Ser Le - #u Lys Val Ala Tyr Gln 115 - # 120 - # 125 - - tat tgg aaa aat atc gat cct gta aag tat ca - #a cat aaa aat aaa ttt 432 Tyr Trp Lys Asn Ile Asp Pro Val Lys Tyr Gl - #n His Lys Asn Lys Phe 130 - # 135 - # 140 - - gtc tcc ctg aac gag gcg tac cac ggt gat ac - #a gtt gga gca gtg agt 480 Val Ser Leu Asn Glu Ala Tyr His Gly Asp Th - #r Val Gly Ala Val Ser 145 1 - #50 1 - #55 1 -#60 - - gtc ggc gga atg gat tta ttc cat aga atc tt - #t aaa cca cta ctattt 528 Val Gly Gly Met Asp Leu Phe His Arg Ile Ph - #e Lys Pro Leu Leu Phe 165 - # 170 - # 175 - - gaa cgg att cca act cct tct cct tat aca ta - #t cgc atg gct aaa cac 576 Glu Arg Ile Pro Thr Pro Ser Pro Tyr Thr Ty - #r Arg Met Ala Lys His 180 - # 185 - # 190 - - ggg gat caa gaa gca gtg aaa aac tat tgt at - #t gat gag ctg gaa aag 624 Gly Asp Gln Glu Ala Val Lys Asn Tyr Cys Il - #e Asp Glu Leu Glu Lys 195 - # 200 - # 205 - - ttg ctt caa gac caa gca gag gaa att gca gg - #a ttg att atc gaa ccg 672 Leu Leu Gln Asp Gln Ala Glu Glu Ile Ala Gl - #y Leu Ile Ile Glu Pro 210 - # 215 - # 220 - - ctt gtt caa gga gca gca ggc atc att acc ca - #c cct cct ggc ttt tta 720 Leu Val Gln Gly Ala Ala Gly Ile Ile Thr Hi - #s Pro Pro Gly Phe Leu 225 2 - #30 2 - #35 2 -#40 - - aaa gcg gtc gaa caa ttg tgc aag aag tac aa - #t ata tta ttg atttgt 768 Lys Ala Val Glu Gln Leu Cys Lys Lys Tyr As - #n Ile Leu Leu Ile Cys 245 - # 250 - # 255 - - gac gaa gta gcg gta gga ttt ggt cgc acc gg - #t aca tta ttt gcc tgt 816 Asp Glu Val Ala Val Gly Phe Gly Arg Thr Gl - #y Thr Leu Phe Ala Cys 260 - # 265 - # 270 - - gaa caa gaa gat gtc gtc cct gat att atg tg - #t atc ggt aaa gga att 864 Glu Gln Glu Asp Val Val Pro Asp Ile Met Cy - #s Ile Gly Lys Gly Ile 275 - # 280 - # 285 - - act ggc ggc tat atg cct ctg gcg gcc act at - #c atg aac gaa caa atc 912 Thr Gly Gly Tyr Met Pro Leu Ala Ala Thr Il - #e Met Asn Glu Gln Ile 290 - # 295 - # 300 - - ttt aat tct ttt tta gga gag ccc gat gaa ca - #t aaa acc ttc tat cac 960 Phe Asn Ser Phe Leu Gly Glu Pro Asp Glu Hi - #s Lys Thr Phe Tyr His 305 3 - #10 3 - #15 3 -#20 - - ggc cac acc tac aca ggg aat caa cta gcc tg - #t gcc ctg gcg ctgaag 1008 Gly His Thr Tyr Thr Gly Asn Gln Leu Ala Cy - #s Ala Leu Ala Leu Lys 325 - # 330 - # 335 - - aat atc gaa cta ata gaa aga cga gat ctc gt - #c aaa gac atc cag aag 1056 Asn Ile Glu Leu Ile Glu Arg Arg Asp Leu Va - #l Lys Asp Ile Gln Lys 340 - # 345 - # 350 - - aaa tcc aag cag cta tct gaa aaa ctg caa tc - #g cta tat gaa ctc ccg 1104 Lys Ser Lys Gln Leu Ser Glu Lys Leu Gln Se - #r Leu Tyr Glu Leu Pro 355 - # 360 - # 365 - - att gtc ggt gat atc cgc cag cgc ggc ctc at - #g att gga ata gaa atc 1152 Ile Val Gly Asp Ile Arg Gln Arg Gly Leu Me - #t Ile Gly Ile Glu Ile 370 - # 375 - # 380 - - gtt aaa gat cgc caa aca aaa gaa ccg ttc ac - #a atc caa gaa aat atc 1200 Val Lys Asp Arg Gln Thr Lys Glu Pro Phe Th - #r Ile Gln Glu Asn Ile 385 3 - #90 3 - #95 4 -#00 - - gtt tca agc atc atc caa aac gct cgg gaa aa - #t ggc ctg atc attcgg 1248 Val Ser Ser Ile Ile Gln Asn Ala Arg Glu As - #n Gly Leu Ile Ile Arg 405 - # 410 - # 415 - - gaa ctt ggc cct gtc atc aca atg atg ccc at - #t ctt tcc atg tca gaa 1296 Glu Leu Gly Pro Val Ile Thr Met Met Pro Il - #e Leu Ser Met Ser Glu 420 - # 425 - # 430 - - aag gaa ctg aat act atg gtc gaa act gtc ta - #c cgt tcg ata cag gac 1344 Lys Glu Leu Asn Thr Met Val Glu Thr Val Ty - #r Arg Ser Ile Gln Asp 435 - # 440 - # 445 - - gtt tct gtg cac aac gga tta atc cca gca gc - #a aac tga - # 1383 Val Ser Val His Asn Gly Leu Ile Pro Ala Al - #a Asn 450 - # 455 - # 460 - - - - <210> SEQ ID NO 4 <211> LENGTH: 460 <212> TYPE: PRT <213> ORGANISM: Kurthia sp. - - <400> SEQUENCE: 4 - - Met Asn Ser His Asp Leu Glu Lys Trp Asp Ly - #s Glu Tyr Val Trp His 1 5 - # 10 - # 15 - - Pro Phe Thr Gln Met Lys Thr Tyr Arg Glu Se - #r Lys Pro Leu Ile Ile 20 - # 25 - # 30 - - Glu Arg Gly Glu Gly Ser Tyr Leu Phe Asp Il - #e Glu Gly Asn Arg Tyr 35 - # 40 - # 45 - - Leu Asp Gly Tyr Ala Ser Leu Trp Val Asn Va - #l His Gly His Asn Glu 50 - # 55 - # 60 - - Pro Glu Leu Asn Asn Ala Leu Ile Glu Gln Va - #l Glu Lys Val Ala His 65 - # 70 - # 75 - # 80 - - Ser Thr Leu Leu Gly Ser Ala Asn Val Pro Se - #r Ile Leu Leu Ala Lys 85 - # 90 - # 95 - - Lys Leu Ala Glu Ile Thr Pro Gly His Leu Se - #r Lys Val Phe Tyr Ser 100 - # 105 - # 110 - - Asp Thr Gly Ser Ala Ala Val Glu Ile Ser Le - #u Lys Val Ala Tyr Gln 115 - # 120 - # 125 - - Tyr Trp Lys Asn Ile Asp Pro Val Lys Tyr Gl - #n His Lys Asn Lys Phe 130 - # 135 - # 140 - - Val Ser Leu Asn Glu Ala Tyr His Gly Asp Th - #r Val Gly Ala Val Ser 145 1 - #50 1 - #55 1 -#60 - - Val Gly Gly Met Asp Leu Phe His Arg Ile Ph - #e Lys Pro Leu LeuPhe 165 - # 170 - # 175 - - Glu Arg Ile Pro Thr Pro Ser Pro Tyr Thr Ty - #r Arg Met Ala Lys His 180 - # 185 - # 190 - - Gly Asp Gln Glu Ala Val Lys Asn Tyr Cys Il - #e Asp Glu Leu Glu Lys 195 - # 200 - # 205 - - Leu Leu Gln Asp Gln Ala Glu Glu Ile Ala Gl - #y Leu Ile Ile Glu Pro 210 - # 215 - # 220 - - Leu Val Gln Gly Ala Ala Gly Ile Ile Thr Hi - #s Pro Pro Gly Phe Leu 225 2 - #30 2 - #35 2 -#40 - - Lys Ala Val Glu Gln Leu Cys Lys Lys Tyr As - #n Ile Leu Leu IleCys 245 - # 250 - # 255 - - Asp Glu Val Ala Val Gly Phe Gly Arg Thr Gl - #y Thr Leu Phe Ala Cys 260 - # 265 - # 270 - - Glu Gln Glu Asp Val Val Pro Asp Ile Met Cy - #s Ile Gly Lys Gly Ile 275 - # 280 - # 285 - - Thr Gly Gly Tyr Met Pro Leu Ala Ala Thr Il - #e Met Asn Glu Gln Ile 290 - # 295 - # 300 - - Phe Asn Ser Phe Leu Gly Glu Pro Asp Glu Hi - #s Lys Thr Phe Tyr His 305 3 - #10 3 - #15 3 -#20 - - Gly His Thr Tyr Thr Gly Asn Gln Leu Ala Cy - #s Ala Leu Ala LeuLys 325 - # 330 - # 335 - - Asn Ile Glu Leu Ile Glu Arg Arg Asp Leu Va - #l Lys Asp Ile Gln Lys 340 - # 345 - # 350 - - Lys Ser Lys Gln Leu Ser Glu Lys Leu Gln Se - #r Leu Tyr Glu Leu Pro 355 - # 360 - # 365 - - Ile Val Gly Asp Ile Arg Gln Arg Gly Leu Me - #t Ile Gly Ile Glu Ile 370 - # 375 - # 380 - - Val Lys Asp Arg Gln Thr Lys Glu Pro Phe Th - #r Ile Gln Glu Asn Ile 385 3 - #90 3 - #95 4 -#00 - - Val Ser Ser Ile Ile Gln Asn Ala Arg Glu As - #n Gly Leu Ile IleArg 405 - # 410 - # 415 - - Glu Leu Gly Pro Val Ile Thr Met Met Pro Il - #e Leu Ser Met Ser Glu 420 - # 425 - # 430 - - Lys Glu Leu Asn Thr Met Val Glu Thr Val Ty - #r Arg Ser Ile Gln Asp 435 - # 440 - # 445 - - Val Ser Val His Asn Gly Leu Ile Pro Ala Al - #a Asn 450 - # 455 - # 460 - - - - <210> SEQ ID NO 5 <211> LENGTH: 1164 <212> TYPE: DNA <213> ORGANISM: Kurthia sp. <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (1)..(1161) - - <400> SEQUENCE: 5 - - atg att tgg gag aag gaa cta gaa aag att aa - #a gaa gga ggg ctt tac 48 Met Ile Trp Glu Lys Glu Leu Glu Lys Ile Ly - #s Glu Gly Gly Leu Tyr 1 5 - # 10 - # 15 - - aga caa ctc caa acc gtt gaa aca atg agc ga - #t caa ggg tat gcc atg 96 Arg Gln Leu Gln Thr Val Glu Thr Met Ser As - #p Gln Gly Tyr Ala Met 20 - # 25 - # 30 - - gtg aac gga aaa aaa atg atg atg ttt gcc tc - #c aat aat tac tta ggg 144 Val Asn Gly Lys Lys Met Met Met Phe Ala Se - #r Asn Asn Tyr Leu Gly 35 - # 40 - # 45 - - att gcc aat gat caa cga tta att gag gct tc - #t gtc caa gcg act caa 192 Ile Ala Asn Asp Gln Arg Leu Ile Glu Ala Se - #r Val Gln Ala Thr Gln 50 - # 55 - # 60 - - aga ttt ggt acg ggt tct act ggt tca cga tt - #a acc act ggc aat aca 240 Arg Phe Gly Thr Gly Ser Thr Gly Ser Arg Le - #u Thr Thr Gly Asn Thr 65 - # 70 - # 75 - # 80 - - att gtc cat gaa aaa cta gag aaa aga ctt gc - #a gag ttt aag caa acg 288 Ile Val His Glu Lys Leu Glu Lys Arg Leu Al - #a Glu Phe Lys Gln Thr 85 - # 90 - # 95 - - gat gca gcg ata gta tta aac aca ggg tat at - #g gct aac ata gca gcg 336 Asp Ala Ala Ile Val Leu Asn Thr Gly Tyr Me - #t Ala Asn Ile Ala Ala 100 - # 105 - # 110 - - tta acg acc ctt gtt ggt agt gac gat ctc at - #t tta tcc gat gag atg 384 Leu Thr Thr Leu Val Gly Ser Asp Asp Leu Il - #e Leu Ser Asp Glu Met 115 - # 120 - # 125 - - aat cat gcc agt att att gat ggc tgc cgt tt - #a tca cgt gcg gaa act 432 Asn His Ala Ser Ile Ile Asp Gly Cys Arg Le - #u Ser Arg Ala Glu Thr 130 - # 135 - # 140 - - atc att tat cgt cat gct gat tta ctt gac tt - #g gaa atg aaa ctc cag 480 Ile Ile Tyr Arg His Ala Asp Leu Leu Asp Le - #u Glu Met Lys Leu Gln 145 1 - #50 1 - #55 1 -#60 - - att aat acc cgc tac agg aaa aga ata att gt - #a acg gat ggc gtcttt 528 Ile Asn Thr Arg Tyr Arg Lys Arg Ile Ile Va - #l Thr Asp Gly Val Phe 165 - # 170 - # 175 - - tcg atg gat ggt gat att gcg cca ttg cca gg - #t att gtc gaa ctt gcc 576 Ser Met Asp Gly Asp Ile Ala Pro Leu Pro Gl - #y Ile Val Glu Leu Ala 180 - # 185 - # 190 - - aag cgt tat gat gca ctt gtt atg gtg gat ga - #c gca cat gcg acg ggt 624 Lys Arg Tyr Asp Ala Leu Val Met Val Asp As - #p Ala His Ala Thr Gly 195 - # 200 - # 205 - - gtt tta ggt aaa gac gga agg gga act tct ga - #a cat ttt gga ctg aag 672 Val Leu Gly Lys Asp Gly Arg Gly Thr Ser Gl - #u His Phe Gly Leu Lys 210 - # 215 - # 220 - - ggg aag ata gat atc gag atg ggg aca ctc tc - #c aaa gct gtt ggt gca 720 Gly Lys Ile Asp Ile Glu Met Gly Thr Leu Se - #r Lys Ala Val Gly Ala 225 2 - #30 2 - #35 2 -#40 - - gaa gga ggg tat atc gct gga agc agg tct tt - #a gtt gac tat gtctta 768 Glu Gly Gly Tyr Ile Ala Gly Ser Arg Ser Le - #u Val Asp Tyr Val Leu 245 - # 250 - # 255 - - aat cga gcc aga ccg ttt gtc ttc tct acc gc - #c tta tca gca gga gta 816 Asn Arg Ala Arg Pro Phe Val Phe Ser Thr Al - #a Leu Ser Ala Gly Val 260 - # 265 - # 270 - - gta gca agt gca ctt aca gca gtc gat atc at - #t caa tca gaa cct gaa 864 Val Ala Ser Ala Leu Thr Ala Val Asp Ile Il - #e Gln Ser Glu Pro Glu 275 - # 280 - # 285 - - cgc aga gta cgc att cga gcc atg agc cag cg - #t ctt tat aat gaa tta 912 Arg Arg Val Arg Ile Arg Ala Met Ser Gln Ar - #g Leu Tyr Asn Glu Leu 290 - # 295 - # 300 - - acc tcc ctt ggc tac aca gtt tcg ggg gga ga - #a act ccg att ctt gcc 960 Thr Ser Leu Gly Tyr Thr Val Ser Gly Gly Gl - #u Thr Pro Ile Leu Ala 305 3 - #10 3 - #15 3 -#20 - - att att tgc gga gaa ccg gaa cag gcc atg tt - #c ctt tcg aaa gaatta 1008 Ile Ile Cys Gly Glu Pro Glu Gln Ala Met Ph - #e Leu Ser Lys Glu Leu 325 - # 330 - # 335 - - cat aag cac gga att tat gca cca gct atc cg - #t tcg cca acg gta cct 1056 His Lys His Gly Ile Tyr Ala Pro Ala Ile Ar - #g Ser Pro Thr Val Pro 340 - # 345 - # 350 - - ctt gga act tcg cgc att cga ctt acg tta at - #g gcg aca cat caa gaa 1104 Leu Gly Thr Ser Arg Ile Arg Leu Thr Leu Me - #t Ala Thr His Gln Glu 355 - # 360 - # 365 - - gaa caa atg aat cat gtt atc gac gtg ttc ag - #a aca atc ctt acc aat 1152 Glu Gln Met Asn His Val Ile Asp Val Phe Ar - #g Thr Ile Leu Thr Asn 370 - # 375 - # 380 - - aga tac aaa tag - # - # - # 1164 Arg Tyr Lys 385 - - - - <210> SEQ ID NO 6 <211> LENGTH: 387 <212> TYPE: PRT <213> ORGANISM: Kurthia sp. - - <400> SEQUENCE: 6 - - Met Ile Trp Glu Lys Glu Leu Glu Lys Ile Ly - #s Glu Gly Gly Leu Tyr 1 5 - # 10 - # 15 - - Arg Gln Leu Gln Thr Val Glu Thr Met Ser As - #p Gln Gly Tyr Ala Met 20 - # 25 - # 30 - - Val Asn Gly Lys Lys Met Met Met Phe Ala Se - #r Asn Asn Tyr Leu Gly 35 - # 40 - # 45 - - Ile Ala Asn Asp Gln Arg Leu Ile Glu Ala Se - #r Val Gln Ala Thr Gln 50 - # 55 - # 60 - - Arg Phe Gly Thr Gly Ser Thr Gly Ser Arg Le - #u Thr Thr Gly Asn Thr 65 - # 70 - # 75 - # 80 - - Ile Val His Glu Lys Leu Glu Lys Arg Leu Al - #a Glu Phe Lys Gln Thr 85 - # 90 - # 95 - - Asp Ala Ala Ile Val Leu Asn Thr Gly Tyr Me - #t Ala Asn Ile Ala Ala 100 - # 105 - # 110 - - Leu Thr Thr Leu Val Gly Ser Asp Asp Leu Il - #e Leu Ser Asp Glu Met 115 - # 120 - # 125 - - Asn His Ala Ser Ile Ile Asp Gly Cys Arg Le - #u Ser Arg Ala Glu Thr 130 - # 135 - # 140 - - Ile Ile Tyr Arg His Ala Asp Leu Leu Asp Le - #u Glu Met Lys Leu Gln 145 1 - #50 1 - #55 1 -#60 - - Ile Asn Thr Arg Tyr Arg Lys Arg Ile Ile Va - #l Thr Asp Gly ValPhe 165 - # 170 - # 175 - - Ser Met Asp Gly Asp Ile Ala Pro Leu Pro Gl - #y Ile Val Glu Leu Ala 180 - # 185 - # 190 - - Lys Arg Tyr Asp Ala Leu Val Met Val Asp As - #p Ala His Ala Thr Gly 195 - # 200 - # 205 - - Val Leu Gly Lys Asp Gly Arg Gly Thr Ser Gl - #u His Phe Gly Leu Lys 210 - # 215 - # 220 - - Gly Lys Ile Asp Ile Glu Met Gly Thr Leu Se - #r Lys Ala Val Gly Ala 225 2 - #30 2 - #35 2 -#40 - - Glu Gly Gly Tyr Ile Ala Gly Ser Arg Ser Le - #u Val Asp Tyr ValLeu 245 - # 250 - # 255 - - Asn Arg Ala Arg Pro Phe Val Phe Ser Thr Al - #a Leu Ser Ala Gly Val 260 - # 265 - # 270 - - Val Ala Ser Ala Leu Thr Ala Val Asp Ile Il - #e Gln Ser Glu Pro Glu 275 - # 280 - # 285 - - Arg Arg Val Arg Ile Arg Ala Met Ser Gln Ar - #g Leu Tyr Asn Glu Leu 290 - # 295 - # 300 - - Thr Ser Leu Gly Tyr Thr Val Ser Gly Gly Gl - #u Thr Pro Ile Leu Ala 305 3 - #10 3 - #15 3 -#20 - - Ile Ile Cys Gly Glu Pro Glu Gln Ala Met Ph - #e Leu Ser Lys GluLeu 325 - # 330 - # 335 - - His Lys His Gly Ile Tyr Ala Pro Ala Ile Ar - #g Ser Pro Thr Val Pro 340 - # 345 - # 350 - - Leu Gly Thr Ser Arg Ile Arg Leu Thr Leu Me - #t Ala Thr His Gln Glu 355 - # 360 - # 365 - - Glu Gln Met Asn His Val Ile Asp Val Phe Ar - #g Thr Ile Leu Thr Asn 370 - # 375 - # 380 - - Arg Tyr Lys 385 - - - - <210> SEQ ID NO 7 <211> LENGTH: 1017 <212> TYPE: DNA <213> ORGANISM: Kurthia sp. <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (1)..(1014) - - <400> SEQUENCE: 7 - - atg aga aaa gag gga tta ggt ttg gaa aca tt - #g gtg aaa aag gat tgg 48 Met Arg Lys Glu Gly Leu Gly Leu Glu Thr Le - #u Val Lys Lys Asp Trp 1 5 - # 10 - # 15 - - aag atg cta gcg gaa aac gta atc aaa gga ta - #t aaa gta aca gcg gaa 96 Lys Met Leu Ala Glu Asn Val Ile Lys Gly Ty - #r Lys Val Thr Ala Glu 20 - # 25 - # 30 - - gaa gca ctt gct att gta caa gca cct gac aa - #c gag gtt tta gag att 144 Glu Ala Leu Ala Ile Val Gln Ala Pro Asp As - #n Glu Val Leu Glu Ile 35 - # 40 - # 45 - - ttg aat gca gct ttc ctt att cgt cag cac ta - #t tat gga aaa aag gtt 192 Leu Asn Ala Ala Phe Leu Ile Arg Gln His Ty - #r Tyr Gly Lys Lys Val 50 - # 55 - # 60 - - aaa ttg aat atg atc att aat acg aag tca gg - #t cta tgt cct gaa gat 240 Lys Leu Asn Met Ile Ile Asn Thr Lys Ser Gl - #y Leu Cys Pro Glu Asp 65 - # 70 - # 75 - # 80 - - tgt ggc tat tgt tcg cag tca atc gtg tcg ga - #a gct cct atc gat aaa 288 Cys Gly Tyr Cys Ser Gln Ser Ile Val Ser Gl - #u Ala Pro Ile Asp Lys 85 - # 90 - # 95 - - tat gct tgg ctg acc aaa gag aag att gtt ga - #a ggt gct caa gaa tca 336 Tyr Ala Trp Leu Thr Lys Glu Lys Ile Val Gl - #u Gly Ala Gln Glu Ser 100 - # 105 - # 110 - - att cgt cgc aaa gct ggc acg tat tgt atc gt - #t gct tct ggc cgt cgt 384 Ile Arg Arg Lys Ala Gly Thr Tyr Cys Ile Va - #l Ala Ser Gly Arg Arg 115 - # 120 - # 125 - - ccg acc aat agg gaa att gat cat gtc att ga - #a gct gtg aaa gaa att 432 Pro Thr Asn Arg Glu Ile Asp His Val Ile Gl - #u Ala Val Lys Glu Ile 130 - # 135 - # 140 - - cgc gag aca acg gat ctt aaa ata tgc tgc tg - #t cta ggt ttc tta aat 480 Arg Glu Thr Thr Asp Leu Lys Ile Cys Cys Cy - #s Leu Gly Phe Leu Asn 145 1 - #50 1 - #55 1 -#60 - - gaa acg cat gcc agt aag cta gct gaa gct gg - #g gtt cat cgc tacaag 528 Glu Thr His Ala Ser Lys Leu Ala Glu Ala Gl - #y Val His Arg Tyr Lys 165 - # 170 - # 175 - - cac aac tta aat aca tct caa gat aat tat aa - #g aat att aca tcc aca 576 His Asn Leu Asn Thr Ser Gln Asp Asn Tyr Ly - #s Asn Ile Thr Ser Thr 180 - # 185 - # 190 - - cat act tat gag gac cgt gta gat aca gtc ga - #a gct gta aaa gag gcc 624 His Thr Tyr Glu Asp Arg Val Asp Thr Val Gl - #u Ala Val Lys Glu Ala 195 - # 200 - # 205 - - gga atg tct cca tgc tcg ggt gcc att ttt gg - #t atg aat gag tct aat 672 Gly Met Ser Pro Cys Ser Gly Ala Ile Phe Gl - #y Met Asn Glu Ser Asn 210 - # 215 - # 220 - - gaa gaa gca gta gag att gcc cta tcc cta cg - #c agt ctt gac gcg gat 720 Glu Glu Ala Val Glu Ile Ala Leu Ser Leu Ar - #g Ser Leu Asp Ala Asp 225 2 - #30 2 - #35 2 -#40 - - tct att cct tgt aat ttc ctc aat gcg att ga - #c ggt aca cca cttgag 768 Ser Ile Pro Cys Asn Phe Leu Asn Ala Ile As - #p Gly Thr Pro Leu Glu 245 - # 250 - # 255 - - gga act tcc gag ttg act cca act aaa tgt tt - #g aaa tta att tcg atg 816 Gly Thr Ser Glu Leu Thr Pro Thr Lys Cys Le - #u Lys Leu Ile Ser Met 260 - # 265 - # 270 - - atg aga ttt gtt aat cca agt aag gaa atc cg - #t ctt gct gga ggt cgc 864 Met Arg Phe Val Asn Pro Ser Lys Glu Ile Ar - #g Leu Ala Gly Gly Arg 275 - # 280 - # 285 - - gag gtg aac ctc cgt tcc atg caa ccc atg gc - #a ctt tat gca gcc aat 912 Glu Val Asn Leu Arg Ser Met Gln Pro Met Al - #a Leu Tyr Ala Ala Asn 290 - # 295 - # 300 - - tct atc ttc gtc ggc gat tat cta aca aca gc - #t gga caa gaa cct acg 960 Ser Ile Phe Val Gly Asp Tyr Leu Thr Thr Al - #a Gly Gln Glu Pro Thr 305 3 - #10 3 - #15 3 -#20 - - gcg gat tgg ggc att atc gaa gac ctt ggt tt - #t gaa att gaa gaatgc 1008 Ala Asp Trp Gly Ile Ile Glu Asp Leu Gly Ph - #e Glu Ile Glu Glu Cys 325 - # 330 - # 335 - - gct ctt taa - # - #- # 1017 Ala Leu - - - - <210> SEQ ID NO 8 <211> LENGTH: 338 <212> TYPE: PRT <213> ORGANISM: Kurthia sp. - - <400> SEQUENCE: 8 - - Met Arg Lys Glu Gly Leu Gly Leu Glu Thr Le - #u Val Lys Lys AspTrp 1 5 - # 10 - # 15 - - Lys Met Leu Ala Glu Asn Val Ile Lys Gly Ty - #r Lys Val Thr Ala Glu 20 - # 25 - # 30 - - Glu Ala Leu Ala Ile Val Gln Ala Pro Asp As - #n Glu Val Leu Glu Ile 35 - # 40 - # 45 - - Leu Asn Ala Ala Phe Leu Ile Arg Gln His Ty - #r Tyr Gly Lys Lys Val 50 - # 55 - # 60 - - Lys Leu Asn Met Ile Ile Asn Thr Lys Ser Gl - #y Leu Cys Pro Glu Asp 65 - # 70 - # 75 - # 80 - - Cys Gly Tyr Cys Ser Gln Ser Ile Val Ser Gl - #u Ala Pro Ile Asp Lys 85 - # 90 - # 95 - - Tyr Ala Trp Leu Thr Lys Glu Lys Ile Val Gl - #u Gly Ala Gln Glu Ser 100 - # 105 - # 110 - - Ile Arg Arg Lys Ala Gly Thr Tyr Cys Ile Va - #l Ala Ser Gly Arg Arg 115 - # 120 - # 125 - - Pro Thr Asn Arg Glu Ile Asp His Val Ile Gl - #u Ala Val Lys Glu Ile 130 - # 135 - # 140 - - Arg Glu Thr Thr Asp Leu Lys Ile Cys Cys Cy - #s Leu Gly Phe Leu Asn 145 1 - #50 1 - #55 1 -#60 - - Glu Thr His Ala Ser Lys Leu Ala Glu Ala Gl - #y Val His Arg TyrLys 165 - # 170 - # 175 - - His Asn Leu Asn Thr Ser Gln Asp Asn Tyr Ly - #s Asn Ile Thr Ser Thr 180 - # 185 - # 190 - - His Thr Tyr Glu Asp Arg Val Asp Thr Val Gl - #u Ala Val Lys Glu Ala 195 - # 200 - # 205 - - Gly Met Ser Pro Cys Ser Gly Ala Ile Phe Gl - #y Met Asn Glu Ser Asn 210 - # 215 - # 220 - - Glu Glu Ala Val Glu Ile Ala Leu Ser Leu Ar - #g Ser Leu Asp Ala Asp 225 2 - #30 2 - #35 2 -#40 - - Ser Ile Pro Cys Asn Phe Leu Asn Ala Ile As - #p Gly Thr Pro LeuGlu 245 - # 250 - # 255 - - Gly Thr Ser Glu Leu Thr Pro Thr Lys Cys Le - #u Lys Leu Ile Ser Met 260 - # 265 - # 270 - - Met Arg Phe Val Asn Pro Ser Lys Glu Ile Ar - #g Leu Ala Gly Gly Arg 275 - # 280 - # 285 - - Glu Val Asn Leu Arg Ser Met Gln Pro Met Al - #a Leu Tyr Ala Ala Asn 290 - # 295 - # 300 - - Ser Ile Phe Val Gly Asp Tyr Leu Thr Thr Al - #a Gly Gln Glu Pro Thr 305 3 - #10 3 - #15 3 -#20 - - Ala Asp Trp Gly Ile Ile Glu Asp Leu Gly Ph - #e Glu Ile Glu GluCys 325 - # 330 - # 335 - - Ala Leu - - - - <210> SEQ ID NO 9 <211> LENGTH: 804 <212> TYPE: DNA <213> ORGANISM: Kurthia sp. <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (1)..(801) - - <400> SEQUENCE: 9 - - atg cca ttc gta aat cat gac aat gaa agc ct - #t tac tat gag gtt cac 48 Met Pro Phe Val Asn His Asp Asn Glu Ser Le - #u Tyr Tyr Glu Val His 1 5 - # 10 - # 15 - - gga caa ggt gat cct tta ttg ttg att atg gg - #g ctc ggc tat aac tct 96 Gly Gln Gly Asp Pro Leu Leu Leu Ile Met Gl - #y Leu Gly Tyr Asn Ser 20 - # 25 - # 30 - - tta tcc tgg cat aga acg gtg ccc act tta gc - #t aag cgc ttt aaa gta 144 Leu Ser Trp His Arg Thr Val Pro Thr Leu Al - #a Lys Arg Phe Lys Val 35 - # 40 - # 45 - - atc gtt ttt gat aat cgt ggt gtt ggt aag ag - #c agt aag cct gaa cag 192 Ile Val Phe Asp Asn Arg Gly Val Gly Lys Se - #r Ser Lys Pro Glu Gln 50 - # 55 - # 60 - - cca tat tct att gaa atg atg gct gag gat gc - #a aga gcg gtc ctt gat 240 Pro Tyr Ser Ile Glu Met Met Ala Glu Asp Al - #a Arg Ala Val Leu Asp 65 - # 70 - # 75 - # 80 - - gct gtt tcg gtt gac tca gca cat gta tat gg - #g att tca atg ggt gga 288 Ala Val Ser Val Asp Ser Ala His Val Tyr Gl - #y Ile Ser Met Gly Gly 85 - # 90 - # 95 - - atg att gcc caa agg ctg gca atc aca tat cc - #a gaa cgt gtt cgt tct 336 Met Ile Ala Gln Arg Leu Ala Ile Thr Tyr Pr - #o Glu Arg Val Arg Ser 100 - # 105 - # 110 - - ctt gtt cta ggt tgt acc act gcg ggt ggt ac - #t act cat att caa cct 384 Leu Val Leu Gly Cys Thr Thr Ala Gly Gly Th - #r Thr His Ile Gln Pro 115 - # 120 - # 125 - - tct cca gaa ata tct act tta atg gta tct cg - #a gcc tcc ctt aca ggt 432 Ser Pro Glu Ile Ser Thr Leu Met Val Ser Ar - #g Ala Ser Leu Thr Gly 130 - # 135 - # 140 - - tct cca agg gat aat gcc tgg tta gcg gca cc - #a ata gtt tat agt caa 480 Ser Pro Arg Asp Asn Ala Trp Leu Ala Ala Pr - #o Ile Val Tyr Ser Gln 145 1 - #50 1 - #55 1 -#60 - - gct ttt att gag aag cac cct gaa tta att ca - #g gaa gat atc caaaag 528 Ala Phe Ile Glu Lys His Pro Glu Leu Ile Gl - #n Glu Asp Ile Gln Lys 165 - # 170 - # 175 - - cga ata gaa atc att act ccg cca agc gcc ta - #t ctg tct caa cta caa 576 Arg Ile Glu Ile Ile Thr Pro Pro Ser Ala Ty - #r Leu Ser Gln Leu Gln 180 - # 185 - # 190 - - gct tgt cta act cat gat aca tcc aat gaa ct - #t gat aaa ata aac ata 624 Ala Cys Leu Thr His Asp Thr Ser Asn Glu Le - #u Asp Lys Ile Asn Ile 195 - # 200 - # 205 - - cca aca ttg att ata cac ggt gat gca gat aa - #t ttg gtt cca tat gaa 672 Pro Thr Leu Ile Ile His Gly Asp Ala Asp As - #n Leu Val Pro Tyr Glu 210 - # 215 - # 220 - - aac ggt aaa atg tta gct gaa cgt att cag gg - #t tct cag ttt cac acc 720 Asn Gly Lys Met Leu Ala Glu Arg Ile Gln Gl - #y Ser Gln Phe His Thr 225 2 - #30 2 - #35 2 -#40 - - gta tcc tgt gct ggc cac att tat tta aca ga - #a gca gct aag gaagca 768 Val Ser Cys Ala Gly His Ile Tyr Leu Thr Gl - #u Ala Ala Lys Glu Ala 245 - # 250 - # 255 - - aat gac aaa gtt ata cag ttt cta gct cat ct - #a taa- # 804 Asn Asp Lys Val Ile Gln Phe Leu Ala His Le - #u 260 - # 265 - - - - <210> SEQ ID NO 10 <211> LENGTH: 267 <212> TYPE: PRT <213> ORGANISM: Kurthia sp. - - <400> SEQUENCE: 10 - - Met Pro Phe Val Asn His Asp Asn Glu Ser Le - #u Tyr Tyr Glu ValHis 1 5 - # 10 - # 15 - - Gly Gln Gly Asp Pro Leu Leu Leu Ile Met Gl - #y Leu Gly Tyr Asn Ser 20 - # 25 - # 30 - - Leu Ser Trp His Arg Thr Val Pro Thr Leu Al - #a Lys Arg Phe Lys Val 35 - # 40 - # 45 - - Ile Val Phe Asp Asn Arg Gly Val Gly Lys Se - #r Ser Lys Pro Glu Gln 50 - # 55 - # 60 - - Pro Tyr Ser Ile Glu Met Met Ala Glu Asp Al - #a Arg Ala Val Leu Asp 65 - # 70 - # 75 - # 80 - - Ala Val Ser Val Asp Ser Ala His Val Tyr Gl - #y Ile Ser Met Gly Gly 85 - # 90 - # 95 - - Met Ile Ala Gln Arg Leu Ala Ile Thr Tyr Pr - #o Glu Arg Val Arg Ser 100 - # 105 - # 110 - - Leu Val Leu Gly Cys Thr Thr Ala Gly Gly Th - #r Thr His Ile Gln Pro 115 - # 120 - # 125 - - Ser Pro Glu Ile Ser Thr Leu Met Val Ser Ar - #g Ala Ser Leu Thr Gly 130 - # 135 - # 140 - - Ser Pro Arg Asp Asn Ala Trp Leu Ala Ala Pr - #o Ile Val Tyr Ser Gln 145 1 - #50 1 - #55 1 -#60 - - Ala Phe Ile Glu Lys His Pro Glu Leu Ile Gl - #n Glu Asp Ile GlnLys 165 - # 170 - # 175 - - Arg Ile Glu Ile Ile Thr Pro Pro Ser Ala Ty - #r Leu Ser Gln Leu Gln 180 - # 185 - # 190 - - Ala Cys Leu Thr His Asp Thr Ser Asn Glu Le - #u Asp Lys Ile Asn Ile 195 - # 200 - # 205 - - Pro Thr Leu Ile Ile His Gly Asp Ala Asp As - #n Leu Val Pro Tyr Glu 210 - # 215 - # 220 - - Asn Gly Lys Met Leu Ala Glu Arg Ile Gln Gl - #y Ser Gln Phe His Thr 225 2 - #30 2 - #35 2 -#40 - - Val Ser Cys Ala Gly His Ile Tyr Leu Thr Gl - #u Ala Ala Lys GluAla 245 - # 250 - # 255 - - Asn Asp Lys Val Ile Gln Phe Leu Ala His Le - #u 260 - # 265 - - - - <210> SEQ ID NO 11 <211> LENGTH: 1197 <212> TYPE: DNA <213> ORGANISM: Kurthia sp. <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (1)..(1194) - - <400> SEQUENCE: 11 - - atg cac agt gaa aaa caa tta cct tgt tgg ga - #a gaa aaa att aag aaa 48 Met His Ser Glu Lys Gln Leu Pro Cys Trp Gl - #u Glu Lys Ile Lys Lys 1 5 - # 10 - # 15 - - gaa ctg gct tat tta gaa gag ata tcg caa aa - #a cgt gaa ctc gtt tca 96 Glu Leu Ala Tyr Leu Glu Glu Ile Ser Gln Ly - #s Arg Glu Leu Val Ser 20 - # 25 - # 30 - - acg gaa ttc gcc gag cag cca tgg ctt atg at - #c aac ggg tgc aag atg 144 Thr Glu Phe Ala Glu Gln Pro Trp Leu Met Il - #e Asn Gly Cys Lys Met 35 - # 40 - # 45 - - cta aat cta gct tct aat aac tat tta gga ta - #t gca ggg gat gag cgg 192 Leu Asn Leu Ala Ser Asn Asn Tyr Leu Gly Ty - #r Ala Gly Asp Glu Arg 50 - # 55 - # 60 - - ctg aaa aag gct atg gta gat gca gta cat ac - #a tat ggt gca gga gcg 240 Leu Lys Lys Ala Met Val Asp Ala Val His Th - #r Tyr Gly Ala Gly Ala 65 - # 70 - # 75 - # 80 - - acg gct tca cgt tta att att ggc aat cac cc - #t ctt tac gag caa gca 288 Thr Ala Ser Arg Leu Ile Ile Gly Asn His Pr - #o Leu Tyr Glu Gln Ala 85 - # 90 - # 95 - - gaa caa gct ctt gtc aat tgg aag aaa gcc ga - #a gca gga ctc att att 336 Glu Gln Ala Leu Val Asn Trp Lys Lys Ala Gl - #u Ala Gly Leu Ile Ile 100 - # 105 - # 110 - - aac agt gga tat aac gcg aac ctt gga att at - #c tcc acc ttg ctg tcc 384 Asn Ser Gly Tyr Asn Ala Asn Leu Gly Ile Il - #e Ser Thr Leu Leu Ser 115 - # 120 - # 125 - - cgt aac gat att att tat agc gat aaa ttg aa - #t cat gca agc att gtc 432 Arg Asn Asp Ile Ile Tyr Ser Asp Lys Leu As - #n His Ala Ser Ile Val 130 - # 135 - # 140 - - gat gga gct ctc tta agc cgt gca aag cat ct - #a cgc tat cgt cat aat 480 Asp Gly Ala Leu Leu Ser Arg Ala Lys His Le - #u Arg Tyr Arg His Asn 145 1 - #50 1 - #55 1 -#60 - - gat tta gat cat tta gaa gca tta ttg aaa aa - #a tca tcg atg gaagca 528 Asp Leu Asp His Leu Glu Ala Leu Leu Lys Ly - #s Ser Ser Met Glu Ala 165 - # 170 - # 175 - - cgt aaa tta att gtg acg gat acg gtc ttc ag - #c atg gac ggt gac ttt 576 Arg Lys Leu Ile Val Thr Asp Thr Val Phe Se - #r Met Asp Gly Asp Phe 180 - # 185 - # 190 - - gct tat ctt gaa gac ctt gtt cgg tta aaa ga - #a cgt tat aac gct atg 624 Ala Tyr Leu Glu Asp Leu Val Arg Leu Lys Gl - #u Arg Tyr Asn Ala Met 195 - # 200 - # 205 - - tta atg aca gat gaa gca cac gga agc ggc at - #c tac ggt aaa aac ggt 672 Leu Met Thr Asp Glu Ala His Gly Ser Gly Il - #e Tyr Gly Lys Asn Gly 210 - # 215 - # 220 - - gaa ggt tat gcc ggt cat ctc cat ctt caa aa - #t aaa ata gat atc caa 720 Glu Gly Tyr Ala Gly His Leu His Leu Gln As - #n Lys Ile Asp Ile Gln 225 2 - #30 2 - #35 2 -#40 - - atg gga aca ttc agt aaa gcg ctc ggt tca tt - #c ggg gcc tat gtcgtc 768 Met Gly Thr Phe Ser Lys Ala Leu Gly Ser Ph - #e Gly Ala Tyr Val Val 245 - # 250 - # 255 - - ggg aaa aaa tgg ctc atc gac tat tta aaa aa - #t cgc atg cgc gga ttc 816 Gly Lys Lys Trp Leu Ile Asp Tyr Leu Lys As - #n Arg Met Arg Gly Phe 260 - # 265 - # 270 - - ata tat tca act gca ctc ccc ccg gcc ata ct - #c ggt gct atg aaa aca 864 Ile Tyr Ser Thr Ala Leu Pro Pro Ala Ile Le - #u Gly Ala Met Lys Thr 275 - # 280 - # 285 - - gcg ata gaa ctt gta cag caa gaa cca gaa cg - #c cgc tca ctg ctc caa 912 Ala Ile Glu Leu Val Gln Gln Glu Pro Glu Ar - #g Arg Ser Leu Leu Gln 290 - # 295 - # 300 - - aca cat tca gaa cac ttt aga gaa gaa ctc ac - #a tat tac ggg ttt aat 960 Thr His Ser Glu His Phe Arg Glu Glu Leu Th - #r Tyr Tyr Gly Phe Asn 305 3 - #10 3 - #15 3 -#20 - - att tgt gga agt cga tca caa att gtt cct at - #c gtc atc ggg gaaaac 1008 Ile Cys Gly Ser Arg Ser Gln Ile Val Pro Il - #e Val Ile Gly Glu Asn 325 - # 330 - # 335 - - gaa aaa gcg atg gaa ttt gcc aca cgt ttg ca - #g aaa gaa gga att gca 1056 Glu Lys Ala Met Glu Phe Ala Thr Arg Leu Gl - #n Lys Glu Gly Ile Ala 340 - # 345 - # 350 - - gct att gct gtc agg ccg ccg acc gtt cct ga - #a aat gag gcg aga atc 1104 Ala Ile Ala Val Arg Pro Pro Thr Val Pro Gl - #u Asn Glu Ala Arg Ile 355 - # 360 - # 365 - - cgt ttt act gta aca gct ctc cac gat aaa aa - #a gat ctt gat tgg gca 1152 Arg Phe Thr Val Thr Ala Leu His Asp Lys Ly - #s Asp Leu Asp Trp Ala 370 - # 375 - # 380 - - gtt gaa aaa gtt tcg atc att gga aaa gaa at - #g ggt gtt att taa 1197 Val Glu Lys Val Ser Ile Ile Gly Lys Glu Me - #t Gly Val Ile 385 3 - #90 3 - #95 - - - - <210> SEQ ID NO 12 <211> LENGTH: 398 <212> TYPE: PRT <213> ORGANISM: Kurthia sp. - - <400> SEQUENCE: 12 - - Met His Ser Glu Lys Gln Leu Pro Cys Trp Gl - #u Glu Lys Ile Lys Lys 1 5 - # 10 - # 15 - - Glu Leu Ala Tyr Leu Glu Glu Ile Ser Gln Ly - #s Arg Glu Leu Val Ser 20 - # 25 - # 30 - - Thr Glu Phe Ala Glu Gln Pro Trp Leu Met Il - #e Asn Gly Cys Lys Met 35 - # 40 - # 45 - - Leu Asn Leu Ala Ser Asn Asn Tyr Leu Gly Ty - #r Ala Gly Asp Glu Arg 50 - # 55 - # 60 - - Leu Lys Lys Ala Met Val Asp Ala Val His Th - #r Tyr Gly Ala Gly Ala 65 - # 70 - # 75 - # 80 - - Thr Ala Ser Arg Leu Ile Ile Gly Asn His Pr - #o Leu Tyr Glu Gln Ala 85 - # 90 - # 95 - - Glu Gln Ala Leu Val Asn Trp Lys Lys Ala Gl - #u Ala Gly Leu Ile Ile 100 - # 105 - # 110 - - Asn Ser Gly Tyr Asn Ala Asn Leu Gly Ile Il - #e Ser Thr Leu Leu Ser 115 - # 120 - # 125 - - Arg Asn Asp Ile Ile Tyr Ser Asp Lys Leu As - #n His Ala Ser Ile Val 130 - # 135 - # 140 - - Asp Gly Ala Leu Leu Ser Arg Ala Lys His Le - #u Arg Tyr Arg His Asn 145 1 - #50 1 - #55 1 -#60 - - Asp Leu Asp His Leu Glu Ala Leu Leu Lys Ly - #s Ser Ser Met GluAla 165 - # 170 - # 175 - - Arg Lys Leu Ile Val Thr Asp Thr Val Phe Se - #r Met Asp Gly Asp Phe 180 - # 185 - # 190 - - Ala Tyr Leu Glu Asp Leu Val Arg Leu Lys Gl - #u Arg Tyr Asn Ala Met 195 - # 200 - # 205 - - Leu Met Thr Asp Glu Ala His Gly Ser Gly Il - #e Tyr Gly Lys Asn Gly 210 - # 215 - # 220 - - Glu Gly Tyr Ala Gly His Leu His Leu Gln As - #n Lys Ile Asp Ile Gln 225 2 - #30 2 - #35 2 -#40 - - Met Gly Thr Phe Ser Lys Ala Leu Gly Ser Ph - #e Gly Ala Tyr ValVal 245 - # 250 - # 255 - - Gly Lys Lys Trp Leu Ile Asp Tyr Leu Lys As - #n Arg Met Arg Gly Phe 260 - # 265 - # 270 - - Ile Tyr Ser Thr Ala Leu Pro Pro Ala Ile Le - #u Gly Ala Met Lys Thr 275 - # 280 - # 285 - - Ala Ile Glu Leu Val Gln Gln Glu Pro Glu Ar - #g Arg Ser Leu Leu Gln 290 - # 295 - # 300 - - Thr His Ser Glu His Phe Arg Glu Glu Leu Th - #r Tyr Tyr Gly Phe Asn 305 3 - #10 3 - #15 3 -#20 - - Ile Cys Gly Ser Arg Ser Gln Ile Val Pro Il - #e Val Ile Gly GluAsn 325 - # 330 - # 335 - - Glu Lys Ala Met Glu Phe Ala Thr Arg Leu Gl - #n Lys Glu Gly Ile Ala 340 - # 345 - # 350 - - Ala Ile Ala Val Arg Pro Pro Thr Val Pro Gl - #u Asn Glu Ala Arg Ile 355 - # 360 - # 365 - - Arg Phe Thr Val Thr Ala Leu His Asp Lys Ly - #s Asp Leu Asp Trp Ala 370 - # 375 - # 380 - - Val Glu Lys Val Ser Ile Ile Gly Lys Glu Me - #t Gly Val Ile 385 3 - #90 3 - #95 - - - - <210> SEQ ID NO 13 <211> LENGTH: 747 <212> TYPE: DNA <213> ORGANISM: Kurthia sp. <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (1)..(744) - - <400> SEQUENCE: 13 - - atg aaa cag ccg aat tta gtc atg ctt cct gg - #c tgg gga atg gaa aaa 48 Met Lys Gln Pro Asn Leu Val Met Leu Pro Gl - #y Trp Gly Met Glu Lys 1 5 - # 10 - # 15 - - gat gcg ttt caa ccg tta atc aaa ccg ctg tc - #a gaa gta ttt cac ctc 96 Asp Ala Phe Gln Pro Leu Ile Lys Pro Leu Se - #r Glu Val Phe His Leu 20 - # 25 - # 30 - - tca ttc ata gaa tgg aga gat atg aaa aca ct - #a aat gac ttt gaa gaa 144 Ser Phe Ile Glu Trp Arg Asp Met Lys Thr Le - #u Asn Asp Phe Glu Glu 35 - # 40 - # 45 - - cga gtc ata gac aca atc gct tct att gat gg - #t cct gtt ttt tta ctt 192 Arg Val Ile Asp Thr Ile Ala Ser Ile Asp Gl - #y Pro Val Phe Leu Leu 50 - # 55 - # 60 - - ggc tgg tca tta gga tct cta tta tca ctt ga - #a ctt gta agt tcg tat 240 Gly Trp Ser Leu Gly Ser Leu Leu Ser Leu Gl - #u Leu Val Ser Ser Tyr 65 - # 70 - # 75 - # 80 - - cga gaa aaa ata aaa ggt ttt ata cta att gg - #c gca aca agt cgt ttt 288 Arg Glu Lys Ile Lys Gly Phe Ile Leu Ile Gl - #y Ala Thr Ser Arg Phe 85 - # 90 - # 95 - - acc aca gga gat aat tat tca ttt ggc tgg ga - #t cca cga atg gtc gag 336 Thr Thr Gly Asp Asn Tyr Ser Phe Gly Trp As - #p Pro Arg Met Val Glu 100 - # 105 - # 110 - - cgc atg aag aaa caa ctg cag cgc aat aaa ga - #g aag act ttg act tct 384 Arg Met Lys Lys Gln Leu Gln Arg Asn Lys Gl - #u Lys Thr Leu Thr Ser 115 - # 120 - # 125 - - ttc tat gaa gca atg ttt tcc gaa gct gaa aa - #a gaa gaa ggt ttt tat 432 Phe Tyr Glu Ala Met Phe Ser Glu Ala Glu Ly - #s Glu Glu Gly Phe Tyr 130 - # 135 - # 140 - - cat caa ttc atc acg aca att caa agc gag tt - #t cat ggg gat gac gta 480 His Gln Phe Ile Thr Thr Ile Gln Ser Glu Ph - #e His Gly Asp Asp Val 145 1 - #50 1 - #55 1 -#60 - - ttt tcg ctt ctt ata ggt ttg gat tat tta ct - #t cag aaa gat gttaga 528 Phe Ser Leu Leu Ile Gly Leu Asp Tyr Leu Le - #u Gln Lys Asp Val Arg 165 - # 170 - # 175 - - gta aag ctc gac cag att gaa act ccc att tt - #a ttg atc cat ggg aga 576 Val Lys Leu Asp Gln Ile Glu Thr Pro Ile Le - #u Leu Ile His Gly Arg 180 - # 185 - # 190 - - gaa gac aaa att tgt cca ctc gaa gcc tca tc - #t ttc att aaa gaa aat 624 Glu Asp Lys Ile Cys Pro Leu Glu Ala Ser Se - #r Phe Ile Lys Glu Asn 195 - # 200 - # 205 - - ctg ggt ggg aaa gcc gag gtt cat att atc ga - #a ggc gct ggt cat att 672 Leu Gly Gly Lys Ala Glu Val His Ile Ile Gl - #u Gly Ala Gly His Ile 210 - # 215 - # 220 - - cca ttt ttc aca aaa cca cag gaa tgt gtg ca - #g ctt ata aaa aca ttt 720 Pro Phe Phe Thr Lys Pro Gln Glu Cys Val Gl - #n Leu Ile Lys Thr Phe 225 2 - #30 2 - #35 2 -#40 - - att caa aag gag tac att cat gat tga - # - # 747 Ile Gln Lys Glu Tyr Ile His Asp 245 - - - - <210> SEQ ID NO 14 <211> LENGTH: 248 <212> TYPE: PRT <213> ORGANISM: Kurthia sp. - - <400> SEQUENCE: 14 - - Met Lys Gln Pro Asn Leu Val Met Leu Pro Gl - #y Trp Gly Met Glu Lys 1 5 - # 10 - # 15 - - Asp Ala Phe Gln Pro Leu Ile Lys Pro Leu Se - #r Glu Val Phe His Leu 20 - # 25 - # 30 - - Ser Phe Ile Glu Trp Arg Asp Met Lys Thr Le - #u Asn Asp Phe Glu Glu 35 - # 40 - # 45 - - Arg Val Ile Asp Thr Ile Ala Ser Ile Asp Gl - #y Pro Val Phe Leu Leu 50 - # 55 - # 60 - - Gly Trp Ser Leu Gly Ser Leu Leu Ser Leu Gl - #u Leu Val Ser Ser Tyr 65 - # 70 - # 75 - # 80 - - Arg Glu Lys Ile Lys Gly Phe Ile Leu Ile Gl - #y Ala Thr Ser Arg Phe 85 - # 90 - # 95 - - Thr Thr Gly Asp Asn Tyr Ser Phe Gly Trp As - #p Pro Arg Met Val Glu 100 - # 105 - # 110 - - Arg Met Lys Lys Gln Leu Gln Arg Asn Lys Gl - #u Lys Thr Leu Thr Ser 115 - # 120 - # 125 - - Phe Tyr Glu Ala Met Phe Ser Glu Ala Glu Ly - #s Glu Glu Gly Phe Tyr 130 - # 135 - # 140 - - His Gln Phe Ile Thr Thr Ile Gln Ser Glu Ph - #e His Gly Asp Asp Val 145 1 - #50 1 - #55 1 -#60 - - Phe Ser Leu Leu Ile Gly Leu Asp Tyr Leu Le - #u Gln Lys Asp ValArg 165 - # 170 - # 175 - - Val Lys Leu Asp Gln Ile Glu Thr Pro Ile Le - #u Leu Ile His Gly Arg 180 - # 185 - # 190 - - Glu Asp Lys Ile Cys Pro Leu Glu Ala Ser Se - #r Phe Ile Lys Glu Asn 195 - # 200 - # 205 - - Leu Gly Gly Lys Ala Glu Val His Ile Ile Gl - #u Gly Ala Gly His Ile 210 - # 215 - # 220 - - Pro Phe Phe Thr Lys Pro Gln Glu Cys Val Gl - #n Leu Ile Lys Thr Phe 225 2 - #30 2 - #35 2 -#40 - - Ile Gln Lys Glu Tyr Ile His Asp 245 - - - - <210> SEQ ID NO 15 <211> LENGTH: 831 <212> TYPE: DNA <213> ORGANISM: Kurthia sp. <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (1)..(828) - - <400> SEQUENCE: 15 - - atg att gat aaa caa ttg tta agt aag cga tt - #c agt gaa cat gcgaaa 48 Met Ile Asp Lys Gln Leu Leu Ser Lys Arg Ph - #e Ser Glu His Ala Lys 1 5 - # 10 - # 15 - - aca tat gat gca tat gcc aat gtt caa aaa aa - #c atg gcg aaa caa tta 96 Thr Tyr Asp Ala Tyr Ala Asn Val Gln Lys As - #n Met Ala Lys Gln Leu 20 - # 25 - # 30 - - gtg gat ttg ctc cct caa aaa aac agc aaa ca - #a aga att aac atc ctt 144 Val Asp Leu Leu Pro Gln Lys Asn Ser Lys Gl - #n Arg Ile Asn Ile Leu 35 - # 40 - # 45 - - gaa att ggc tgc ggt act ggt tac tta acc ag - #g tta ctc gtt aat aca 192 Glu Ile Gly Cys Gly Thr Gly Tyr Leu Thr Ar - #g Leu Leu Val Asn Thr 50 - # 55 - # 60 - - ttt cct aat gct tct att acc gct gtt gat tt - #a gca cca ggg atg gtt 240 Phe Pro Asn Ala Ser Ile Thr Ala Val Asp Le - #u Ala Pro Gly Met Val 65 - # 70 - # 75 - # 80 - - gaa gtg gcg aaa gga ata aca atg gaa gac cg - #t gtt act ttt tta tgt 288 Glu Val Ala Lys Gly Ile Thr Met Glu Asp Ar - #g Val Thr Phe Leu Cys 85 - # 90 - # 95 - - gct gat atc gaa gaa atg acg ctt aat gaa aa - #t tac gac tta att att 336 Ala Asp Ile Glu Glu Met Thr Leu Asn Glu As - #n Tyr Asp Leu Ile Ile 100 - # 105 - # 110 - - tct aat gca acg ttt caa tgg ctg aat aat ct - #t cct gga acc att gaa 384 Ser Asn Ala Thr Phe Gln Trp Leu Asn Asn Le - #u Pro Gly Thr Ile Glu 115 - # 120 - # 125 - - caa ttg ttt aca cga tta acg cct gaa gga aa - #c ctg ata ttt tca acg 432 Gln Leu Phe Thr Arg Leu Thr Pro Glu Gly As - #n Leu Ile Phe Ser Thr 130 - # 135 - # 140 - - ttt gga att aaa acc ttt caa gag ctt cat at - #g tcc tat gaa cat gcg 480 Phe Gly Ile Lys Thr Phe Gln Glu Leu His Me - #t Ser Tyr Glu His Ala 145 1 - #50 1 - #55 1 -#60 - - aaa gaa aag ctt caa ctt tca att gat agt tc - #a cca ggc caa ctgttt 528 Lys Glu Lys Leu Gln Leu Ser Ile Asp Ser Se - #r Pro Gly Gln Leu Phe 165 - # 170 - # 175 - - tac gct cta gaa gaa tta tcc caa att tgt ga - #a gaa gca atc cct ttt 576 Tyr Ala Leu Glu Glu Leu Ser Gln Ile Cys Gl - #u Glu Ala Ile Pro Phe 180 - # 185 - # 190 - - tca tca gca ttt cca tta gag ata aca aaa at - #a gaa aag ctt gaa cta 624 Ser Ser Ala Phe Pro Leu Glu Ile Thr Lys Il - #e Glu Lys Leu Glu Leu 195 - # 200 - # 205 - - gag tac ttt cag aca gta cgt gaa ttt ttc ac - #t tca att aaa aag att 672 Glu Tyr Phe Gln Thr Val Arg Glu Phe Phe Th - #r Ser Ile Lys Lys Ile 210 - # 215 - # 220 - - ggt gca gct aac agc aac aaa gaa aac tac tg - #c cag cgc cct tct ttt 720 Gly Ala Ala Asn Ser Asn Lys Glu Asn Tyr Cy - #s Gln Arg Pro Ser Phe 225 2 - #30 2 - #35 2 -#40 - - ttt cga gag tta atc aac ata tac gaa aca aa - #a tac caa gat gaatca 768 Phe Arg Glu Leu Ile Asn Ile Tyr Glu Thr Ly - #s Tyr Gln Asp Glu Ser 245 - # 250 - # 255 - - ggt gtg aag gca acc tat cac tgt ttg ttt tt - #t aag ata ata aaa cat 816 Gly Val Lys Ala Thr Tyr His Cys Leu Phe Ph - #e Lys Ile Ile Lys His 260 - # 265 - # 270 - - gcc ccc cta ccc taa - # - # - # 831 Ala Pro Leu Pro 275 - - - - <210> SEQ ID NO 16 <211> LENGTH: 276 <212> TYPE: PRT <213> ORGANISM: Kurthia sp. - - <400> SEQUENCE: 16 - - Met Ile Asp Lys Gln Leu Leu Ser Lys Arg Ph - #e Ser Glu His Ala Lys 1 5 - # 10 - # 15 - - Thr Tyr Asp Ala Tyr Ala Asn Val Gln Lys As - #n Met Ala Lys Gln Leu 20 - # 25 - # 30 - - Val Asp Leu Leu Pro Gln Lys Asn Ser Lys Gl - #n Arg Ile Asn Ile Leu 35 - # 40 - # 45 - - Glu Ile Gly Cys Gly Thr Gly Tyr Leu Thr Ar - #g Leu Leu Val Asn Thr 50 - # 55 - # 60 - - Phe Pro Asn Ala Ser Ile Thr Ala Val Asp Le - #u Ala Pro Gly Met Val 65 - # 70 - # 75 - # 80 - - Glu Val Ala Lys Gly Ile Thr Met Glu Asp Ar - #g Val Thr Phe Leu Cys 85 - # 90 - # 95 - - Ala Asp Ile Glu Glu Met Thr Leu Asn Glu As - #n Tyr Asp Leu Ile Ile 100 - # 105 - # 110 - - Ser Asn Ala Thr Phe Gln Trp Leu Asn Asn Le - #u Pro Gly Thr Ile Glu 115 - # 120 - # 125 - - Gln Leu Phe Thr Arg Leu Thr Pro Glu Gly As - #n Leu Ile Phe Ser Thr 130 - # 135 - # 140 - - Phe Gly Ile Lys Thr Phe Gln Glu Leu His Me - #t Ser Tyr Glu His Ala 145 1 - #50 1 - #55 1 -#60 - - Lys Glu Lys Leu Gln Leu Ser Ile Asp Ser Se - #r Pro Gly Gln LeuPhe 165 - # 170 - # 175 - - Tyr Ala Leu Glu Glu Leu Ser Gln Ile Cys Gl - #u Glu Ala Ile Pro Phe 180 - # 185 - # 190 - - Ser Ser Ala Phe Pro Leu Glu Ile Thr Lys Il - #e Glu Lys Leu Glu Leu 195 - # 200 - # 205 - - Glu Tyr Phe Gln Thr Val Arg Glu Phe Phe Th - #r Ser Ile Lys Lys Ile 210 - # 215 - # 220 - - Gly Ala Ala Asn Ser Asn Lys Glu Asn Tyr Cy - #s Gln Arg Pro Ser Phe 225 2 - #30 2 - #35 2 -#40 - - Phe Arg Glu Leu Ile Asn Ile Tyr Glu Thr Ly - #s Tyr Gln Asp GluSer 245 - # 250 - # 255 - - Gly Val Lys Ala Thr Tyr His Cys Leu Phe Ph - #e Lys Ile Ile Lys His 260 - # 265 - # 270 - - Ala Pro Leu Pro 275 - - - - <210> SEQ ID NO 17 <211> LENGTH: 360 <212> TYPE: DNA <213> ORGANISM: Kurthia sp. <220> FEATURE: <221> NAME/KEY: RBS <222> LOCATION: Complement((67)..(76)) <220> FEATURE: <221> NAME/KEY: -35.sub.-- signal <222> LOCATION: (210)..(215) <220> FEATURE: <221> NAME/KEY: -10.sub.-- signal <222> LOCATION: (234)..(239) <220> FEATURE: <221> NAME/KEY: -10.sub.-- signal <222> LOCATION: Complement((235)..(240)) <220> FEATURE: <221> NAME/KEY: RBS <222> LOCATION: (289)..(293) <220> FEATURE: <221> NAME/KEY: misc.sub.-- feature <222> LOCATION: (302)..(358) <223> OTHER INFORMATION: Partial sequence of ORF2. <220> FEATURE: <221> NAME/KEY: misc.sub.-- feature <222> LOCATION: (125)..(164) <223> OTHER INFORMATION: BOX1 - inverted repeat <220> FEATURE: <221> NAME/KEY: misc.sub.-- feature <222> LOCATION: (244)..(283) <223> OTHER INFORMATION: BOX2 - inverted repeat <220> FEATURE: <221> NAME/KEY: misc.sub.-- feature <222> LOCATION: Complement((1)..(58)) <223> OTHER INFORMATION: Partial sequnce of ORF1. - - <400> SEQUENCE: 17 - - cgccaatggc agttaacgca gtaaataggg caacataaga gatcgtttta gc -#tttcaagt 60 - - tttcaacctc ctttttttaa aattggtagg taaaatgccc actattagtg tg -#catgattt 120 - - atattttata tgtcaaccat ttattatttt agttaacata taaagcgcaa tt -#aaaatgac 180 - - agacttagaa aaatattgaa aattagtaat tgaacaatat tttatttgtg tg -#ttattata 240 - - caatttatat gttaactatt ttaagatata gttaacatat aaaggcttgg ag -#ggaacaaa 300 - - tatgacagga gaaatgttaa tacaggatga actttccaga gaaacagcgg ta -#tttgtggc 360 - - - - <210> SEQ ID NO 18 <211> LENGTH: 20 <212> TYPE: PRT <213> ORGANISM: Kurthia sp. - - <400> SEQUENCE: 18 - - Leu Lys Ala Lys Thr Ile Ser Tyr Val Ala Le - #u Phe Thr Ala LeuThr 1 5 - # 10 - # 15 - - Ala Ile Gly Ala 20 - - - - <210> SEQ ID NO 19 <211> LENGTH: 20 <212> TYPE: PRT <213> ORGANISM: Kurthia sp. - - <400> SEQUENCE: 19 - - Met Thr Gly Glu Met Leu Ile Gln Asp Glu Le - #u Ser Arg Glu Thr Ala 1 5 - # 10 - # 15 - - Val Phe Val Ala 20 - - - - <210> SEQ ID NO 20 <211> LENGTH: 240 <212> TYPE: DNA <213> ORGANISM: Kurthia sp. <220> FEATURE: <221> NAME/KEY: -35.sub.-- signal <222> LOCATION: (65)..(70) <220> FEATURE: <221> NAME/KEY: -10.sub.-- signal <222> LOCATION: (91)..(96) <220> FEATURE: <221> NAME/KEY: RBS <222> LOCATION: (127)..(135) <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (142)..(240) <223> OTHER INFORMATION: bioH - - <400> SEQUENCE: 20 - - actaacccct atggtgtcca gattaagcgt aatgtagtat aggattttag tc -#aattagca 60 - - atttttgaaa tatttagtac gatcacataa tagaatcata tataatgatt aa -#aatattaa 120 - - ttacagaaaa gaggtatttt c atg cca ttc gta aat cat - #gac aat gaa agc 171 - # Met Pro Phe Val Asn His Asp - #Asn Glu Ser - # 1 - # 5 - # 10 - - ctt tac tat gag gtt cac gga caa ggt gat cc - #t tta ttg ttg att atg 219 Leu Tyr Tyr Glu Val His Gly Gln Gly Asp Pr - #o Leu Leu Leu Ile Met 15 - # 20 - # 25 - - ggg ctc ggc tat aac tct tta - # - # 240 Gly Leu Gly Tyr Asn Ser Leu 30 - - - - <210> SEQ ID NO 21 <211> LENGTH: 33 <212> TYPE: PRT <213> ORGANISM: Kurthia sp. - - <400> SEQUENCE: 21 - - Met Pro Phe Val Asn His Asp Asn Glu Ser Le - #u Tyr Tyr Glu Val His 1 5 - # 10 - # 15 - - Gly Gln Gly Asp Pro Leu Leu Leu Ile Met Gl - #y Leu Gly Tyr Asn Ser 20 - # 25 - # 30 - - Leu - - - - <210> SEQ ID NO 22 <211> LENGTH: 300 <212> TYPE: DNA <213> ORGANISM: Kurthia sp. <220> FEATURE: <221> NAME/KEY: -35.sub.-- signal <222> LOCATION: (83)..(88) <220> FEATURE: <221> NAME/KEY: -10.sub.-- signal <222> LOCATION: (107)..(112) <220> FEATURE: <221> NAME/KEY: misc.sub.-- feature <222> LOCATION: (115)..(154) <223> OTHER INFORMATION: Box 3 - inverted repea - #t <220> FEATURE: <221> NAME/KEY: RBS <222> LOCATION: (154)..(161) <220> FEATURE: <221> NAME/KEY: misc.sub.-- feature <222> LOCATION: (168)..(299) <223> OTHER INFORMATION: Partial sequence of bioFI - #I (full gene isSEQ ID NO: 11). - - <400> SEQUENCE: 22 - - ttatgataag tgtctttttt cgccccttga tttctcctag attaatggat aa -#tcaattta 60 - - ttatcatgtt ccttttcaaa gcttgacagt ttcattgagt catgattaga at -#gttttata 120 - - tgttaaccta tattattttt agttaacata taaaaaggag aatggctatg ca -#cagtgaaa 180 - - aacaattacc ttgttgggaa gaaaaaatta agaaagaact ggcttattta ga -#agagatat 240 - - cgcaaaaacg tgaactcgtt tcaacggaat tcgccgagca gccatggctt at -#gatcaacg 300 - - - - <210> SEQ ID NO 23 <211> LENGTH: 45 <212> TYPE: PRT <213> ORGANISM: Kurthia sp. - - <400> SEQUENCE: 23 - - Met His Ser Glu Lys Gln Leu Pro Cys Trp Gl - #u Glu Lys Ile LysLys 1 5 - # 10 - # 15 - - Glu Leu Ala Tyr Leu Glu Glu Ile Ser Gln Ly - #s Arg Glu Leu Val Ser 20 - # 25 - # 30 - - Thr Glu Phe Ala Glu Gln Pro Trp Leu Met Il - #e Asn Gly 35 - # 40 - # 45__________________________________________________________________________

Number	Date	Country
0 375 525	Jun 1990	EPX
635572	Jan 1995	EPX
747483	Dec 1996	EPX
0 799 895	Oct 1997	EPX
8701391	Mar 1987	WOX
9408023	Apr 1994	WOX

Biotin biosynthetic genes

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Abstract

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US Referenced Citations (1)

Foreign Referenced Citations (6)

Non-Patent Literature Citations (15)

Entry
Cleary et al., "Deletion and Complementation Analysis of the Biotin Gene Cluster of Escherichia Coli", J. Bacterial., 112:830-839 (1972).
Barker et al., "Use of bio-lac Fusion Strains to Study Regulation of Biotin Biosyntehsis in Escherichia Coli", J. Bacterial., 143:789-800 (1980).
Tanaka et al., "Site-Specific In vitro Binding of Plasmid pUb110 to Bacillus subtilis Membrane Fraction", J. Bacteriol., 154:1184-1194 (1983).
Otsuka et al., "The Escherichia coli Biotin Biosynthetic Enzyme Sequences Predicted from the Nucleotide Sequence of the bio Operon", J. Biol. Chem., 263:19577-19585 (1988).
M. Smith, "In vitro Mutagenesis", Ann. Rev. Genet 19:423-462 (1985).
Haima et al., "The effect of restriction on shotgun cloning and plasmid stability in Bacillus subtilis Marburg", Mol. Gen. Genet., 209:335-342 (1987).
Maniatis et al., "Transformation of Escherichia Coli by Plasmid DNA", Molecular Cloning, Cold Spring Harbor Laboratory Press, pp. 252-253, (1982).
Derwent Abstract No. 86-216622 of Japanese Patent Kokai 149091/1986.
Derwent Abstract No. 87-231579 of Japanese Patent Kokai 155081/1987.
Derwent Abstract No. 90-226023 of Japanese Patent Kokai 180174/1991.
Derwent Abstract No. 90-072971 of Japanese Patent Kokai 27980/1990.
Derwent Abstract No. 91-358298 of Japanese Patent Kokai 240489/1991.
U.S. application No. 08/833,411, Hoshino et al., filed Apr. 4, 1997, pending.
U.S. application No. 08/840,059, Hoshino et al., filed Apr. 24, 1997, pending.
Derwent Abstract of EP 375 525.