Method for producing nucleic acid substances

A nucleic acid substance is efficiently produced by cultivating a microorganism whose repressor protein for purine operon does not function in a normal manner, preferably a strain whose gene encoding the repressor protein on a chromosome (purR) is disrupted, in a culture medium so that the nucleic acid substance should accumulate in the culture medium, and collecting the substance from the culture medium.

TECHNICAL FIELD
 The present invention relates to a method for producing nucleic acid
 substances by fermentation. Nucleic acid substances are industrially
 useful as materials of seasonings and the like.
 BACKGROUND ART
 In the production of nucleic acid substances such as inosine, guanosine and
 bases thereof (hypoxanthine and guanine and the like) by fermentation,
 there have conventionally been used mutant strains imparted with adenine
 auxotropy and nucleic acid analogue resistance (Japanese Patent
 Publication (KOKOKU) Nos. Sho 55-2956 and Sho 55-45199) with a limited
 amount of adenine substances in their culture medium.
 Mutant strains obtained by usual mutagenesis procedures are often
 introduced with mutations in their genes other than the target gene. In
 addition, because complicated regulation mechanisms are involved in
 biosynthetic pathways of nucleic acid substances, it is difficult to
 obtain a microorganism that produces a significant amount of nucleic acid
 substance. Therefore, mutant strains obtained by conventional breeding
 methods have not necessarily been satisfactory ones.
 On the other hand, there has been disclosed a method for producing inosine
 or guanosine by utilizing a bacterium belonging to the genus Bacillus
 exhibiting enhanced purine operon expression obtained by modification of a
 gene sequence encoding enzymes involved in the purine biosynthesis (purine
 operon) (Japanese Patent Unexamined Publication (KOKAI) No. Hei 3-164185).
 This method is characterized in that a promoter or operator of the purine
 operon is modified to increase the expression level of the operon, thereby
 increasing the production of inosine or guanosine.
 Expression of the purine operon of Bacillus subtilis (purEKBC(ORF)OLFMNHD
 where ORF represents an open reading frame of unknown function) is
 suppressed by an excessive amount of adenine, and also regulated by
 attenuation caused by guanine. Further, a repressor protein binding to the
 5' flanking region of the purine operon and a gene encoding the protein
 (purR) have been isolated, and it has been reported that, in a Bacillus
 subtilis strain whose purR gene was disrupted, suppression by adenine as
 for the expression of purC-lacZ fusion gene integrated into the purine
 operon was reduced to about 1/10 (Proc. Natl. Acad. Sci. USA, 92,
 7455-7549 (1995)).
 In Bacillus subtilis, the repressor protein has been known to regulate the
 expression of, in addition to the genes of the purine operon, purA gene
 involved in the biosynthesis of adenine and genes of pyrimidine operon
 involved in the pyrimidine biosynthesis (1997, J. Bacteriol. 179,
 7394-7402, H. Zalkin).
 On the other hand, in Escherichia coli, it has been reported that the
 purine operon repressor also affects the expression of glyA gene involved
 in the biosynthesis of glycine, which is a substance of 5'-IMP (inosinic
 acid) biosynthesis (1990, J. Bacteriol. 172, 3799-3803, H. Zalkin et al.),
 and the expression of gcv operon genes involved in the production of
 C.sub.1 and CO.sub.2 supplied from glycine (1993, J. Bacteriol. 175,
 5129-5134, G. V. Stauffer et al.) in addition to the genes of the purine
 operon.
 As described above, several reports have been made about the relationship
 between the purine operon and inosine or guanosine production. However,
 there are various biosynthesis pathways involving the purR gene. Further,
 the purine operon of Bacillus subtilis encodes ten enzymes, and involved
 in many reactions. Thus, the biosynthesis pathways of nucleic acid
 substances are very complicated, and the relationship between the purR
 gene and accumulation of nucleic acid substances has scarcely been known.
 DESCRIPTION OF THE INVENTION
 The object of the present invention is to provide a method for more
 advantageously producing nucleic acid substances compared with
 conventional methods in view of the industrial importance of nucleic acid
 substances.
 The present inventors earnestly conducted studies about the function of
 purR gene in order to achieve the aforementioned object, and as a result
 they found that Bacillus subtilis strain whose purR gene had been
 disrupted accumulated a nucleic acid substance. Thus, the present
 invention has been accomplished.
 That is, the present invention provides:
 (1) a method for producing a nucleic acid substance comprising steps of:
 cultivating in a culture medium a microorganism whose repressor protein
 for purine operon does not function in a normal manner to produce and
 accumulate the nucleic acid substance in the culture medium, and
 collecting the substance from the culture medium;
 (2) the method defined in the above (1), whrerin the repressor protein does
 not function in a normal manner because a gene encoding the repressor
 protein on a chromosome of the microorganism is disrupted;
 (3) the method defined in the above (1), wherein the repressor protein has
 the amino acid sequence shown in SEQ ID NO: 6;
 (4) the method defined in the above (1), wherein the microorganism is a
 bacterium belonging to the genus Bacillus;
 (5) The method defined in the above (4), wherein the microorganism is
 Bacillus subtilis;
 (6) the method defined in the above (1), wherein the nucleic acid substance
 is a nucleic acid base, nucleoside or nucleotide; and
 (7) the method defined in the above (6), wherein the nucleic acid substance
 is selected from the group consisting of hypoxanthine, uracil, guanine and
 adenine.
 For the purpose of the present invention, the expression "repressor protein
 for purine operon does not function in a normal manner" means that the
 repressor protein binds to the 5' flanking region of the purine operon,
 and hence the function for suppressing transcription of the operon is
 reduced compared with the normal level, or it is substantially eliminated.
 In the present invention, the nucleic acid substance includes nucleic acid
 bases such as hypoxanthine, adenine, guanine, uracil, thymine and
 cytosine, nucleosides such as inosine, adenosine, guanosine, uridine,
 thymidine and cytidine, nucleotides such as inosinic acid, adenylic acid,
 guanylic acid, uridylic acid, thymidylic acid and cytidylic acid, and
 compounds composed of any one of those nucleosides or nucleotides whose
 ribose is replaced with deoxyribose. Among these, the nucleic acid bases
 are preferred. In the present invention, the nucleic acid substance
 includes both of purine compounds and pyrimidine compounds. Such purine
 compounds include purine base, and nucleosides and nucleotides having
 purine base. The pyrimidine compounds include pyrimidine base, and
 nucleosides and nucleotides having pyrimidine base. In the present
 invention, hypoxanthine, adenine and guanine are preferred as the purine
 compounds, and uracil is preferred as the pyrimidine compound

DETAILED DESCRIPTION OF THE INVENTION
 The present invention will be hereinafter explained in detail.
 The microorganism used for the method of the present invention is a
 microorganism whose repressor protein for purine operon (also referred to
 an merely "repressor" hereinafter) does not function in a normal manner.
 The microorganism is not particularly limited, so long as its genes for
 enzymes of biosynthesis of purine compounds form an operon, and it has a
 gene encoding a repressor involved in the regulation of the operon (purR).
 Examples of such a microorganism include bacteria belonging to the gene
 Bacillus and the like. Specific examples of the bacteria of the gene
 Bacillus include, for example, Bacillus subtilis, Bacillus
 amyloliquefaciens and the like.
 The microorganism whose repressor does not function in a normal manner can
 be obtained by modifying its purR gene so that the activity of the
 repressor should be reduced or eliminated, or the transcription of the
 purR gene should be reduced or eliminated. Such a microorganism can be
 obtained by, for example, replacing the chromosomal purR gene with a purR
 gene that does not function in a normal manner (occasionally referred to
 as "disrupted purR gene" hereinafter) through, for example, homologous
 recombination based on genetic recombination techniques (Experiments in
 Molecular Genetics, Cold Spring Harbor Laboratory Press (1972); Matsuyama,
 S. and Mizushima, S., J. Bacteriol., 162, 1196 (1985)).
 In the homologous recombination, when a plasmid carrying a sequence
 exhibiting homology with a chromosomal sequence or the like is introduced
 into a corresponding bacterial cell, recombination occurs at a site of the
 homologous sequence at a certain frequency, and thus the introduced
 plasmid as a whole is integrated into the chromosome. Then, by causing
 recombination again at the site of the homologous sequence in the
 chromosome, the plasmid may be removed from the chromosome. However,
 depending on the position at which the recombination is caused, the
 disrupted gene may remain on the chromosome, while the original normal
 gene may be removed from the chromosome together with the plasmid. By
 selecting such a bacterial strain, a bacterial strain in which the normal
 purR gene is replaced with a disrupted purR gene can be obtained.
 Such a gene disruption technique based on the homologous recombination has
 already been established, and a method utilizing a linear DNA, method
 utilizing temperature sensitive plasmid or the like can be used therefore.
 The purR gene can also be disrupted by using a plasmid that contains the
 purR gene inserted with a marker gene such as drug resistance gene, and
 cannot replicate in a target microbial cell. That is, in a transformant
 that has been transformed with such a plasmid and hence acquired drug
 resistance, the marker gene is integrated in a chromosome DNA. It is
 likely that this marker gene has been integrated by homologous
 recombination of the purR gene present at the both sides of the marker
 with the purR on the chromosome, and therefore a gene-disrupted strain can
 efficiently be selected.
 Specifically, a disrupted purR gene used for the gene disruption can be
 obtained by deletion of a certain region of purR gene by means of
 digestion with restriction enzyme(s) and the religation; by insertion of
 another DNA fragment (marker gene etc.) into the purR gene, by introducing
 substitution, deletion, insertion, addition or inversion of one or more
 nucleotides in a nucleotide sequence of coding region of purR gene, its
 promoter region or the like by means of site-specific mutagenesis (Kramer,
 W. and Frits, H. J., Methods in Enzymology, 154, 350 (1987)) or treatment
 with a chemical reagent such as sodium hyposulfite and hydroxylamine
 (Shortle, D. and Nathans, D., Proc. Natl. Acad. Sci. U.S.A., 75,
 270(1978)) or the like, so that the activity of the encoded repressor
 should be reduced or eliminated, or transcription of the purR gene should
 be reduced or eliminated. Among these embodiments, a method utilizing
 deletion of a certain region of the purR gene by digestion with a
 restriction exzyme and religation, or insertion of another DNA fragment
 into the purR gene is preferred in view of reliability and stability.
 The purR gene can be obtained from a chromosomal DNA of a microorganism
 which has a purine operon by PCR using oligonucleotides prepared based on
 known nucleotide sequences of the purR gene as primers. The purR gene can
 also be obtained from a chromosome DNA library of a microorganism which
 has a purine operon by a hybridization technique using an oligonucleotide
 prepared based on a known nucleotide sequence of the purR gene as a probe.
 A nucleotide sequence of the purR gene has been reported for Bacillus
 subtilis 168 Marburg strain (GenBank accession No. D26185 (the coding
 region corresponds to the nucleotide numbers 118041-118898), DDBB
 accession No. Z99104 (the coding region corresponds to the nucleotide
 numbers 54439-55296). The nucleotide sequence of purR gene and the amino
 acid sequence coded by the gene is shown in SEQ ID NO: 5 and 6 in Sequence
 Listing. For the purpose of the present invention, because the purR gene
 is used for preparing a disrupted purR gene, it is not necessarily
 required to contain the full length, and it may contain a length required
 to cause gene disruption.
 The microorganism used for obtaining the purR gene is not particularly
 limited, so long as its purR gene has homology of such a degree that
 allows homologous recombination with purR gene of a microorganism used for
 creation of gene-disrupted strain. However, it is normally preferable to
 use the same microorganism.
 The primers used for PCR may be any one allowing amplification of the purR
 gene, and specific examples thereof include oligonucleotides having a
 nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2.
 Examples of the marker gene include, for example, drug resistance genes
 such as spectinomycin resistance gene. A spectinomycin resistance gene can
 be obtained by preparing plasmid pDG1726 from Escherichia coli ECE101
 strain commercially available from Bacillus Genetic Stock Center (BGSC),
 and excising the gene from the plasmid as a cassette.
 When a drug resistance gene is used as the marker gene, a purR
 gene-disrupted strain can be obtained by inserting the drug resistance
 gene into a suitable site of the purR gene carried by a plasmid,
 transforming a microorganism with the plasmid, and selecting a drug
 resistant transformant. Disruption of purR gene on a chromosome can be
 confirmed by analyzing the purR gene or the marker gene on the chromosome
 by Southern blotting, PCR, or the like. Integration of the spectinomycin
 resistance gene into a chromosomal DNA can be confirmed by PCR using
 primers that allow amplification of the spectinomycin resistance gene
 (e.g., oligonucleotides having nucleotide sequences shown in SEQ ID NOS: 3
 and 4).
 By cultivating a microorganism whose repressor does not function in a
 normal manner obtained as described above in a suitable culture medium, a
 nucleic acid substance may be produced and accumulated in the culture
 medium.
 As the culture medium used for the present invention, an ordinary nutrient
 medium containing a carbon source, nitrogen source, mineral salt, and
 organic trace nutrient such as amino acids and vitamins as required can be
 used, and the cultivation ca be performed in a conventional manner. Either
 a synthetic culture medium or a natural medium can be used. The carbon
 source and the nitrogen source used for the culture medium may be of any
 kinds so long as they can be utilized by a bacterial strain to be
 cultured.
 As the carbon source, saccharides such as glucose, glycerol, fructose,
 sucrose, maltose, mannose, galactose, starch hydrolysates and molasses may
 be used, and organic acids such as acetic acid and citric acid may be used
 by themselves or in combination with another carbon source.
 As the nitrogen source, ammonia, ammonium salts such as ammonium sulfate,
 ammonium carbonate, ammonium chloride, ammonium phosphate, and ammonium
 acetate, nitrates and the like can be used.
 As the trace nutrient, amino acids, vitamins, aliphatic acids, nucleic
 acids, as well as peptone, casamino acid, yeast extract, soybean protein
 decomposition products and the like, which contain the foregoing
 substances, may be used. When an auxotroph mutant requiring amino acid or
 the like for its growth is used, it is necessary to supplement the
 required nutrient.
 As the mineral salt, phosphates, magnesium salts, calcium salts, iron
 salts, manganese salts and the like may used.
 The culture condition depends on the kind of microorganism to be used. For
 example, Bacillus subtilis is cultured by aeration culture while adjusting
 fermentation temperature to 20-50.degree. C., and pH to 4-9. When pH
 decreases during cultivation, the medium may be neutralized with an alkali
 such as ammonia gas. A nucleic acid substance is accumulated in the
 culture medium by performing cultivation as described above for around 40
 hours to 3 days.
 After the cultivation is completed, the nucleic acid substance accumulated
 in the culture medium may be recovered by a known method. For example, it
 can be isolated by precipitation, ion exchange chromatography or the like.
 If the microorganism used for the present invention is made defective for a
 gene encoding a nucleosidase or nucleotidase, a corresponding nucleoside
 or nucleotide can be accumulated. If it is imparted with adenine or
 guanine auxotrophy, precursors in biosynthesis pathways of these
 substances and related substances can be accumulated.
 Further, by treating a nucleic acid base produced by the method of the
 present invention with purine nucleoside phosphorylase or
 phsophoribosyltransferase, a nucleoside or nucleotide corresponding to the
 base can be obtained.
 BEST MODE FOR CARRYING OUT THE INVENTION
 The present invention will be explained more specifically with reference to
 the following examples.
 &lt;1&gt; Cloning of purR gene
 A DNA fragment containing purR gene of Bacillus subtilis 168 Marburg strain
 (ATCC 6051) was obtained by PCR using chromosomal DNA of the bacterium as
 a template.
 The chromosomal DNA was prepared as follows. The Bacillus subtilis ATCC
 6051 strain was inoculated in 50 ml of LB medium, cultured at 37.degree.
 C. overnight, then collected and lysed with a lysing solution containing 1
 mg/ml of lysozyme. The lysate was treated with phenol, and then DNA was
 precipitated by ethanol precipitation in a usual manner. The resulting
 precipitates of DNA were collected by winding them on a glass rod, washed,
 and used for PCR.
 As primers for the PCR, oligonucleotides having the nucleotide sequences
 shown in SEQ ID NOS: 1 and 2 (their synthesis was consigned to Japan
 Bioservice Co., Ltd), which has been designed based on the known
 nucleotide sequences of the purR gene of Bacillus subtilis 168 Marburg
 strain (GenBank accession No. D26185 (the coding region corresponds to the
 nucleotide numbers 118041-118898), DDJB accession No. Z99104 (the coding
 region corresponds to the nucleotide numbers 54439-55296). These primers
 had HindIII and PstI restriction enzyme recognition sequences near the 5'
 end and 3' end, respectively.
 The PCR was performed by repeating a reaction cycle of denaturation at
 94.degree. C. for 1 minute, annealing at 45.degree. C. for 1 minute and
 extension reaction at 72.degree. C. for 1 minute, for 30 cycles in 0.1 ml
 of PCR reaction solution containing 6 ng/.mu.l of the chromosome DNA and 3
 .mu.M of the primers.
 The reaction product and plasmid pHSG398 (Takara Shuzo) were digested with
 HindIII and PstI, and ligated by using a ligation kit (Takara Shuzo).
 Using the resulting recombinant plasmid, Escherichia coli JM109 was
 transformed. The transformants were cultured on LB agar medium containing
 30 .mu./ml of chloramphenicol (Cm) and X-Gal
 (5-bromo-4-chloro-3-indolyl-B-D-galactoside) to obtain white colonies of
 chloramphenicol resistant transformants. Plasmids were extracted from the
 transformants obtained as described above, and the plasmid inserted with
 the target DNA fragment was designated pHSG398BSPR (FIG. 1).
 &lt;2&gt; Construction of plasmid containing disrupted purR (.DELTA.purR)
 Plasmid pDG1726 was prepared from Escherichia coli ECE101 strain
 commercially available from Bacillus Genetic Stock Center (BGSC). From the
 plasmid, spectinomycin resistance (Sp.sup.r) gene can be taken out as a
 cassette.
 The plasmids pDG1726 and pHSG398BSPR obtained in &lt;1&gt; were completely (FIG.
 2) and partially digested with restriction enzymes HincII and EcoRV,
 respectively, and treated with phenol. Each DNA was ligated by using the
 ligation kit, and Escherichia coli JM109 was transformed by using the
 ligation solution, and cultured on LB agar medium containing 50 .mu.g/ml
 of chloramphenicol and 100 .mu.g/ml of spectinomycin. Plasmid was
 extracted from the resulting transformant to afford plasmid
 pHSG398.DELTA.BSPR:Sp.sup.r whose purR gene was disrupted by insertion of
 the spectinomycin resistance gene (FIG. 3).
 &lt;3&gt; Creation of purR gene disrupted strain
 Bacillus subtilis SB112 (BGSC 1A227) was transformed with the plasmid
 pHSG398.DELTA.BSPR:Sp.sup.r obtained in the aforementioned &lt;2&gt;. The
 transformation was performed by using competent cells prepared according
 to the method of Ishiwa (Ishiwa H. et al., 1986, Jpn. J. Genet. 61
 515-528). The plasmid pHSG398.DELTA.BSPR:Sp.sup.r cannot replicate DNA in
 Bacillus subtilis cells. However, because the plasmid has regions
 homologous to the chromosome purR gene at the 5' side and 3' side of the
 spectinomycin resistance gene, it can undergo gene substitution with the
 chromosomal purR gene by double crossover recombination.
 The aforementioned transformants were cultured on LB agar medium containing
 100 .mu.g/ml of spectinomycin, and grown strains were selected to obtain
 strains in which the chromosomal purR gene was replaced by the
 .DELTA.BSPR:Sp.sup.r gene. By performing PCR using chromosomal DNA of
 candidate strain as template, the primers for the purR gene (SEQ ID NO: 1
 and 2), and primers for the Sp.sup.r gene having nucleotide sequences
 shown in SEQ ID NOS: 3 and 4 (their synthesis was consigned to Japan
 Bioservice Co., Ltd), it was confirmed that the intended gene substitution
 had occurred based on the size of the .DELTA.BSPR:Sp.sup.r portion and
 insertion of spectinomycin resistance gene. One of gene substituted
 strains obtained as described above was designated Bacillus subtilis
 SB112K.
 &lt;4&gt; Production of nucleic acid by purR defective strain
 The Bacillus subtilis SB112K strain produced in the aforementioned &lt;3&gt;, and
 the parent strain, Bacillus subtilis SB112, were cultured overnight in LB
 and LB agar medium containing 100 .mu.g/ml of spectinomycin at 37.degree.
 C. overnight, and 3 loops of the cells were inoculated into 20 ml of
 production medium shown in Table 1, and cultured at 32.degree. C. for 72
 hours.
 TABLE 1
 Composition of fermentation medium
 Medium component Concentration
 Glucose 100 g/L
 NH.sub.4 Cl 20 g/L
 KH.sub.2 PO.sub.4 0.5 g/L
 MgSO.sub.4.7H.sub.2 O 0.4 g/L
 FeSO.sub.4.7H.sub.2 O 2 ppm
 MnSO.sub.4.5H.sub.2 O 2 ppm
 L-Tryptophan 300 mg/L
 L-Phenylalanine 300 mg/L
 L-glutamic acid 15.0 g/L
 DL-methionine 0.3 g/L
 Bean concentrate (T-N) 1.5 g/L
 Antifoamer (GD-113) 0.05 ml/L
 pH modifier (KOH) 7.2 g/L
 After the cultivation was completed, accumulated amounts of hypoxanthine,
 uracil, guanosine, guanine, adenosine, and adenine in the culture medium
 were measured by high performance liquid chromatography. The results are
 shown in Table 2. In the purR defective strain SB112K, a marked amount of
 hypoxanthine, i.e., about 600 mg/L, was accumulated, and thus it showed
 about 20-fold increase of the accumulation compared with the parent
 strain. As for uracil, the accumulation increased by 5 times compared with
 the parent strain. Further, accumulation of adenine and guanine, which was
 not recognized in the parent strain, was also recognized in the purR
 defective strain.
 TABLE 2
 Evaluation of fermentation
 Product (mg/L)
 Strain Hypoxanthine uracil guanine adenine
 SB112K 585 166 20 127
 SB112 30 30 0 0
 (parent strain)
 SEQUENCE LISTING
 &lt;100&gt; GENERAL INFORMATION:
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 &lt;400&gt; SEQUENCE: 1
 ctcaagcttg aagttgcgat gatcaaaa 28
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
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 ctcctgcaga catattgttg acgataat 28
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 &lt;400&gt; SEQUENCE: 3
 gtgaggagga tatatttgaa t 21
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
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 &lt;212&gt; TYPE: DNA
 &lt;213&gt; ORGANISM: Artificial Sequence
 &lt;220&gt; FEATURE:
 &lt;223&gt; OTHER INFORMATION: Description of Artificial Sequence primer for
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 &lt;400&gt; SEQUENCE: 4
 ttataatttt tttaatctgt t 21
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 5
 &lt;211&gt; LENGTH: 1200
 &lt;212&gt; TYPE: DNA
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;220&gt; FEATURE:
 &lt;221&gt; NAME/KEY: CDS
 &lt;222&gt; LOCATION: (259)..(1113)
 &lt;400&gt; SEQUENCE: 5
 atatgcatcc tgaagttgcg atgatcaaaa accagatgaa acgctttggt gcagatgccg 60
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 agagaattta taacgggtta agaggcttct gcgatcaagt ttatgcggtg agaatgatcg 180
 gcgaacagaa cgctcttgat taaatccgta tgttaagtta tattgatctt aaaatattcg 240
 gattttgggg gtgagttc atg aag ttt cgt cgc agc ggc aga ttg gtg gac 291
 Met Lys Phe Arg Arg Ser Gly Arg Leu Val Asp
 1 5 10
 tta aca aat tat ttg tta acc cat ccg cac gag tta ata ccg cta acc 339
 Leu Thr Asn Tyr Leu Leu Thr His Pro His Glu Leu Ile Pro Leu Thr
 15 20 25
 ttt ttc tct gag cgg tat gaa tct gca aaa tca tcg atc agt gaa gat 387
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 30 35 40
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 45 50 55
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 Leu Thr Val Pro Gly Ala Ala Gly Gly Val Lys Tyr Ile Pro Lys Met
 60 65 70 75
 aag cag gct gaa gct gaa gag ttt gtg cag aca ctt gga cag tcg ctg 531
 Lys Gln Ala Glu Ala Glu Glu Phe Val Gln Thr Leu Gly Gln Ser Leu
 80 85 90
 gca aat cct gag cgt atc ctt ccg ggc ggt tat gta tat tta acg gat 579
 Ala Asn Pro Glu Arg Ile Leu Pro Gly Gly Tyr Val Tyr Leu Thr Asp
 95 100 105
 atc tta gga aag cca tct gta ctc tcc aag gta ggg aag ctg ttt gct 627
 Ile Leu Gly Lys Pro Ser Val Leu Ser Lys Val Gly Lys Leu Phe Ala
 110 115 120
 tcc gtg ttt gca gag cgc gaa att gat gtt gtc atg acc gtt gcc acg 675
 Ser Val Phe Ala Glu Arg Glu Ile Asp Val Val Met Thr Val Ala Thr
 125 130 135
 aaa ggc atc cct ctt gcg tac gca gct gca agc tat ttg aat gtg cct 723
 Lys Gly Ile Pro Leu Ala Tyr Ala Ala Ala Ser Tyr Leu Asn Val Pro
 140 145 150 155
 gtt gtg atc gtt cgt aaa gac aat aag gta aca gag ggc tcc aca gtc 771
 Val Val Ile Val Arg Lys Asp Asn Lys Val Thr Glu Gly Ser Thr Val
 160 165 170
 agc att aat tac gtt tca ggc tcc tca aac cgc att caa aca atg tca 819
 Ser Ile Asn Tyr Val Ser Gly Ser Ser Asn Arg Ile Gln Thr Met Ser
 175 180 185
 ctt gcg aaa aga agc atg aaa acg ggt tca aac gta ctc att att gat 867
 Leu Ala Lys Arg Ser Met Lys Thr Gly Ser Asn Val Leu Ile Ile Asp
 190 195 200
 gac ttt atg aaa gca ggc ggc acc att aat ggt atg att aac ctg ttg 915
 Asp Phe Met Lys Ala Gly Gly Thr Ile Asn Gly Met Ile Asn Leu Leu
 205 210 215
 gat gag ttt aac gca aat gtg gcg gga atc ggc gtc tta gtt gaa gcc 963
 Asp Glu Phe Asn Ala Asn Val Ala Gly Ile Gly Val Leu Val Glu Ala
 220 225 230 235
 gaa gga gta gat gaa cgt ctt gtt gac gaa tat atg tca ctt ctt act 1011
 Glu Gly Val Asp Glu Arg Leu Val Asp Glu Tyr Met Ser Leu Leu Thr
 240 245 250
 ctt tca acc atc aac atg aaa gag aag tcc att gaa att cag aat ggc 1059
 Leu Ser Thr Ile Asn Met Lys Glu Lys Ser Ile Glu Ile Gln Asn Gly
 255 260 265
 aat ttt ctg cgt ttt ttt aaa gac aat ctt tta aag aat gga gag aca 1107
 Asn Phe Leu Arg Phe Phe Lys Asp Asn Leu Leu Lys Asn Gly Glu Thr
 270 275 280
 gaa tca tgacaaaagc agtccacaca aaacatgccc cagcggcaat cgggccttat 1163
 Glu Ser
 285
 tcacaaggga ttatcgtcaa caatatgttt tacagct 1200
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 6
 &lt;211&gt; LENGTH: 285
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Bacillus subtilis
 &lt;400&gt; SEQUENCE: 6
 Met Lys Phe Arg Arg Ser Gly Arg Leu Val Asp Leu Thr Asn Tyr Leu
 1 5 10 15
 Leu Thr His Pro His Glu Leu Ile Pro Leu Thr Phe Phe Ser Glu Arg
 20 25 30
 Tyr Glu Ser Ala Lys Ser Ser Ile Ser Glu Asp Leu Thr Ile Ile Lys
 35 40 45
 Gln Thr Phe Glu Gln Gln Gly Ile Gly Thr Leu Leu Thr Val Pro Gly
 50 55 60
 Ala Ala Gly Gly Val Lys Tyr Ile Pro Lys Met Lys Gln Ala Glu Ala
 65 70 75 80
 Glu Glu Phe Val Gln Thr Leu Gly Gln Ser Leu Ala Asn Pro Glu Arg
 85 90 95
 Ile Leu Pro Gly Gly Tyr Val Tyr Leu Thr Asp Ile Leu Gly Lys Pro
 100 105 110
 Ser Val Leu Ser Lys Val Gly Lys Leu Phe Ala Ser Val Phe Ala Glu
 115 120 125
 Arg Glu Ile Asp Val Val Met Thr Val Ala Thr Lys Gly Ile Pro Leu
 130 135 140
 Ala Tyr Ala Ala Ala Ser Tyr Leu Asn Val Pro Val Val Ile Val Arg
 145 150 155 160
 Lys Asp Asn Lys Val Thr Glu Gly Ser Thr Val Ser Ile Asn Tyr Val
 165 170 175
 Ser Gly Ser Ser Asn Arg Ile Gln Thr Met Ser Leu Ala Lys Arg Ser
 180 185 190
 Met Lys Thr Gly Ser Asn Val Leu Ile Ile Asp Asp Phe Met Lys Ala
 195 200 205
 Gly Gly Thr Ile Asn Gly Met Ile Asn Leu Leu Asp Glu Phe Asn Ala
 210 215 220
 Asn Val Ala Gly Ile Gly Val Leu Val Glu Ala Glu Gly Val Asp Glu
 225 230 235 240
 Arg Leu Val Asp Glu Tyr Met Ser Leu Leu Thr Leu Ser Thr Ile Asn
 245 250 255
 Met Lys Glu Lys Ser Ile Glu Ile Gln Asn Gly Asn Phe Leu Arg Phe
 260 265 270
 Phe Lys Asp Asn Leu Leu Lys Asn Gly Glu Thr Glu Ser
 275 280 285