JP2000287688A - Gene coding for enzyme constituting the pyrimidine- biosynthesizing system in coryneform bacterium - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new gene which codes for a protein having a specific amino acid sequence and an asparaginate carbamoyl transferase activity, and is useful for producing the protein, breeding a nucleic acid-producing microorganism, and so on. SOLUTION: This is a new gene which codes for an enzyme constituting the pyridinebiosynthesizing system having the amino acid sequence shown by the formula or an amino acid sequence obtained by substituting, deleting, inserting, adding, or reversing one or more amino acid(s) in, from, into, to, or in the amino acid sequence, and an asparaginate carbamoyl transferase activity, and is useful for producing an enzyme constituting the pyridine- biosynthesizing system such as asparaginate carbamoyl transferase, breeding a nucleic acid-producing microorganism, and so on. This DNA is obtained by preparing a gene library using chromosomal DNA of a coryneform bacterium Brevibacterium lactofermentum ATCC 13869, followed by screening the obtained library.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to pyrimidine biosynthesis enzymes of coryneform bacteria and genes thereof, and more particularly to aspartate carbamoyltransferase, dihydroorotase and orotidylate decarboxylase, and genes encoding these. These genes can be used for production of the above-mentioned enzymes, breeding of nucleic acid-producing bacteria, and the like.

[0002]

2. Description of the Related Art Pyrimidine compounds such as uridine-5'-monophosphate are biosynthesized from L-glutamic acid via carbamoyl phosphate, carbamoyl aspartate, dihydroorotic acid, orotic acid, orotidic acid, and uridylic acid. Is done. These intermediates are in turn carbamoyl phosphate synthetase, aspartate carbamoyl transferase, dihydroorotase,
It is formed by a reaction catalyzed by dihydroorotate dehydrogenase, orotidylate phosphoribosyltransferase, and orotidylate decarboxylase, respectively. Further, among the above intermediates, carbamoyl phosphate is a source of a carbamoyl group required for a reaction for producing citrulline from ornithine in the L-arginine biosynthesis pathway.

In Bacillus subtilis, a group of enzymes involved in pyrimidine biosynthesis is encoded by a pyrimidine operon, and studies on expression at a gene level have been conducted (1997, J. Bacteriol. 179, 7394-740).
2, H. Zalkin). It is also known that aspartate carbamoyltransferase, dihydroorotase, and orotidylate decarboxylase are encoded by pyrB, pyrC, and pyrF genes in Escherichia bacteria and Bacillus bacteria, respectively (derived from Bacillus bacteria). pyrB: J. Biol. Chem. (1986), 261 (24), 1156-1
165, pyrC from Escherichia coli: J. Biol. Chem. (1
986), 261 (13), 5917-5919, py from Escherichia coli
rF: Eur. J. Biochem. (1984), 140 (2), 343-352). Furthermore, carbamoyl phosphate synthetase is composed of two subunits, and it is known that in Escherichia bacteria and Bacillus bacteria, these subunits are encoded by both carA and carB genes (Mol. Gen.
Genet., 133, 299-316).

Further, the present inventors have studied carbamoyl phosphate synthetase and its gene, and have succeeded in obtaining the gene (Japanese Patent Application No. 11-24149). However, no other pyrimidine biosynthetic enzymes have been found in coryneform bacteria, and their genes have not been known.

[0005]

An object of the present invention is to provide pyrimidine biosynthetic enzymes of coryneform bacteria and genes encoding them.

[0006]

Means for Solving the Problems In the course of research on carbamoyl phosphate synthetase and its gene, the present inventors have found that aspartate carbamoyltransferase, dihydroorotase, orotidylate decarboxylase are contained in a DNA fragment encoding the enzyme. The present inventors have found that there is a gene encoding, and have completed the present invention.

That is, the present invention is as follows. (1) DNA encoding a protein shown in the following (A) or (B). (A) a protein having the amino acid sequence of SEQ ID NO: 2 in the sequence listing; (B) the amino acid sequence represented by SEQ ID NO: 2 in the sequence listing, comprising an amino acid sequence containing substitution, deletion, insertion, addition, or inversion of one or several amino acid residues, and aspartate carbamoyltransferase activity A protein having (2) a DNA represented by the following (a) or (b): (1)
DNA. (A) DNA comprising a base sequence consisting of base numbers 11 to 904 in the base sequence described in SEQ ID NO: 1 in the sequence listing. (B) encodes a protein that hybridizes under stringent conditions to the base sequence consisting of base numbers 11 to 904 among the base sequences described in SEQ ID NO: 1 of the sequence listing, and that has an aspartate carbamoyltransferase activity. DNA. (3) DNA encoding the protein shown in (C) or (D) below. (C) a protein having the amino acid sequence of SEQ ID NO: 3 in the sequence listing; (D) the amino acid sequence represented by SEQ ID NO: 3 in the sequence listing, comprising an amino acid sequence containing substitution, deletion, insertion, addition, or inversion of one or several amino acid residues, and a dihydroorotase activity A protein having (4) DNA shown in the following (c) or (d) (3)
DNA. (C) DN containing a base sequence consisting of base numbers 960 to 2150 among the base sequences described in SEQ ID NO: 1 in the sequence listing
A. (D) a DNA that hybridizes with a base sequence consisting of base numbers 960 to 2150 of the base sequence shown in SEQ ID NO: 1 in the sequence listing under stringent conditions and encodes a protein having dihydroorotase activity . (5) DNA encoding a protein shown in the following (E) or (F). (E) a protein having the amino acid sequence of SEQ ID NO: 6 in the sequence listing, (F) an amino acid sequence represented by SEQ ID NO: 6 in the sequence listing, comprising an amino acid sequence containing substitution, deletion, insertion, addition, or inversion of one or several amino acid residues, and orotidylate decarboxylase; A protein having activity. (6) a DNA represented by the following (e) or (f): (5)
DNA. (E) DN containing a base sequence consisting of base numbers 6969 to 7802 in the base sequence described in SEQ ID NO: 1 in the sequence listing
A. (F) encodes a protein that hybridizes with a base sequence consisting of base numbers 6969 to 7802 of the base sequence described in SEQ ID NO: 1 under stringent conditions and has orotidylate decarboxylase activity. DNA. (7) A DNA containing all the DNAs described in (1), (3) and (5). (8) Furthermore, DNA encoding a small subunit of carbamoyl phosphate synthetase having the amino acid sequence shown in SEQ ID NO: 4 in the sequence listing, and encoding a large subunit of carbamoyl phosphate synthetase having the amino acid sequence shown in SEQ ID NO: 5 (7) The DNA according to (7), which contains a DNA to be treated. (9) base numbers 11 to 78 in SEQ ID NO: 1 of the sequence listing
The DNA according to (8), which has a nucleotide sequence consisting of 02. (10) The DNA according to any one of the above (1) to (9)
Coryneform bacteria transformed with the fragment.

(11) A protein represented by the following (A) or (B): (A) a protein having the amino acid sequence of SEQ ID NO: 2 in the sequence listing; (B) the amino acid sequence represented by SEQ ID NO: 2 in the sequence listing, comprising an amino acid sequence containing substitution, deletion, insertion, addition, or inversion of one or several amino acid residues, and aspartate carbamoyltransferase activity A protein having (12) A protein represented by the following (C) or (D): (C) a protein having the amino acid sequence of SEQ ID NO: 3 in the sequence listing; (D) the amino acid sequence represented by SEQ ID NO: 3 in the sequence listing, comprising an amino acid sequence containing substitution, deletion, insertion, addition, or inversion of one or several amino acid residues, and a dihydroorotase activity A protein having (13) A protein represented by the following (E) or (F): (E) a protein having the amino acid sequence of SEQ ID NO: 6 in the sequence listing, (F) an amino acid sequence represented by SEQ ID NO: 6 in the sequence listing, comprising an amino acid sequence containing substitution, deletion, insertion, addition, or inversion of one or several amino acid residues, and orotidylate decarboxylase; A protein having activity.

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail.
As described above, the DNA of the present invention is obtained together with the gene of the carbamoyl phosphate synthetase in the process of obtaining the gene. The DNA of the present invention
From a chromosomal DNA library of a coryneform bacterium prepared using a vector such as a plasmid, a microorganism deficient in carA or carB, for example, Escherichia coli RC50 (carA
^{50, tsx - 273, λ -} , rpsL135 (str R), malT1 (λR), xylA7,
^{thi -.. 1, Mol Gen.} Genet 133, 299 (1974)), Escherichia coli ^{JEF8 (thr - 31, ΔcarB,} relA -, metB1, Mo
l. Gen. Genet., 133, 299 (1974)). Since the microorganism deficient in carA or carB shows L-arginine and uracil auxotrophy, the microorganism is transformed with a chromosomal DNA library, and a clone complementing the auxotrophy is selected.
If the recombinant vector is recovered from the obtained transformant,
A DNA fragment containing carA or carB is obtained.

The coryneform bacterium for preparing the chromosomal DNA library is not particularly limited, but a bacterium which has been conventionally classified into the genus Brevibacterium but is now integrated into the genus Corynebacterium (Int. J. Syst. . Bacterio
l., 41, 255 (1981)), and bacteria of the genus Brevibacterium very closely related to the genus Corynebacterium, for example, wild-type strains of Brevibacterium lactofermentum. The chromosomal DNA of a coryneform bacterium can be obtained, for example, by the method of Saito and Miura (Biochem. Biophys. Acta., 72, 619,
(1963)), the method of KS Kirby (Biochem. J., 64, 405, (19)
56)) and the like.

[0011] The chromosomal DNA library contains the chromosome DN.
A can be obtained by partially decomposing A with an appropriate restriction enzyme, ligating the resulting DNA fragment to a vector DNA capable of autonomous replication in Escherichia coli cells, preparing a recombinant DNA, and introducing it into Escherichia coli. The vector is
The vector is not particularly limited as long as it is a vector usually used for cloning a gene, and a plasmid vector such as pUC19, pUC18, pUC118, and puC119, and a phage vector such as λ phage DNA can be used. Alternatively, a vector that is capable of autonomous replication in both Escherichia coli and coryneform bacteria may be used. One such vector is Escherichia
It can be constructed by ligating an E. coli vector and pAM330 (see JP-A-58-67699), which is a cryptic plasmid of Brevibacterium lactofermentum.

[0012] Specifically, as a vector capable of autonomous replication in both Escherichia coli and coryneform bacteria,
pSAC4 (see Examples) and pHK4 (see JP-A-5-7491). Escherichia coli HB101 retaining pHK4 is named Escherichia coli AJ13136,
Deposited on August 1, 1995 with the Research Institute of Biotechnology and Industrial Technology of the Ministry of International Trade and Industry (Postal code: 305-8566 1-3-1 Higashi, Tsukuba, Ibaraki, Japan) under the accession number FERM BP-5186. I have.

The transformation of Escherichia coli is carried out by DAMo.
rrison's method (Methods in Enzymology, 68, 326, 197
9) Alternatively, treat recipient cells with calcium chloride
Methods to increase NA permeability (Mandel, M. and Higa, A., J. Mo
l., Biol., 53, 159 (1970)).
Preparation of chromosomal DNA library, plasmid D
For preparation of NA, cleavage and ligation of DNA, PCR described below, preparation of oligonucleotides, hybridization, and the like, ordinary methods well known to those skilled in the art can be employed. These methods are described in Sambrook, J., Frit.
sch, EF, andManiatis, T., "Molecular Cloning A
Laboratory Manual, Second Edition ", Cold Spring Ha
rbor Laboratory Press, (1989).

The nucleotide sequence of the DNA fragment obtained as described above is shown in SEQ ID NO: 1 in the sequence listing. In this sequence, there are five open reading frames (ORF1-5)
(Base numbers 11 to 904, 960 to 2150, 2434 to 3612, 3907 to 69
60, 6969-7802). The enzymes and gene names encoded by each ORF are shown below. ORF1: aspartate carbamoyltransferase (pyrB) ORF2: dihydroorotase (pyrC) ORF3: carbamoylphosphate synthetase small subunit (carA) ORF4: carbamoylphosphate synthetase large subunit (carB) ORF5: orotidylate decarboxylase (pyrF)

In SEQ ID NO: 1, the coding region of ORF3 (carA) is indicated by using GTG of base numbers 2434 to 2436 as the start codon, but GTG of base numbers 2566 to 2568 or base number 2
ATG 581-2583 may be the start codon. ORF1 (pyrB), ORF2 (pyrC), ORF3 (carA) and ORF4 (c
arB) describes the amino acid corresponding to GTG as the initiation codon as valine, which represents methionine, valine, or formylmethionine.

The amino acid sequence encoded by each gene is shown in SEQ ID NOs: 2 to 6. The aspartate carbamoyltransferase of the present invention has an amino acid sequence represented by SEQ ID NO: 2 as long as it has enzymatic activity.
It may have mutations such as substitution, deletion, insertion, addition, or inversion of one or several amino acid residues. In addition, the dihydroorotase of the present invention may be substituted, deleted, inserted, added, or inverted by one or several amino acid residues in the amino acid sequence shown in SEQ ID NO: 3 in the sequence listing, as long as it has enzymatic activity. May be mutated. Furthermore, as long as the orotidylate decarboxylase of the present invention has enzymatic activity, substitution, deletion, insertion, addition or inversion of one or several amino acid residues in the amino acid sequence shown in SEQ ID NO: 6 in the sequence listing is possible. And the like. Here, "several pieces" is preferably 1 to 20, more preferably 1 to 10.

DNA encoding a protein substantially the same as the above-described aspartate carbamoyltransferase, dihydroorotase or orotidylate decarboxylase can be obtained by substituting an amino acid residue at a specific site by, for example, site-directed mutagenesis. , Deletion, insertion, addition, or inversion, by modifying the nucleotide sequence of each gene. The modified DNA as described above can also be obtained by a conventionally known mutation treatment. Mutation treatment includes DN encoding each enzyme.
A method of in vitro treating A with hydroxylamine or the like,
Microorganisms carrying DNA encoding each gene, for example, bacteria belonging to the genus Escherichia, are used for ultraviolet irradiation or normal mutation treatment such as N-methyl-N′-nitro-N-nitrosoguanidine (NTG) or nitrite. A method of treating with a mutagen is included.

The substitution, deletion, insertion, addition, or inversion of the base as described above occurs naturally due to the difference in the species or strain of the coryneform bacterium holding each enzyme gene. Mutants (mutants or variants) are also included.

The DNA having the above mutation is expressed in a suitable cell, and the expression product is examined for each enzyme activity. As a result, substantially the same as aspartate carbamoyltransferase, dihydroorotase or orotidylate decarboxylase is obtained. Is obtained. In addition, from a DNA encoding a mutated aspartate carbamoyltransferase, dihydroorotase or orotidylate decarboxylase or a cell carrying the same, for example, base numbers 11 to 9 of the base sequence described in SEQ ID NO: 1 in the sequence listing
No. 04, a base sequence consisting of base numbers 960 to 2150, or a base sequence consisting of base numbers 6969 to 7802, which hybridizes under stringent conditions and has aspartate carbamoyltransferase activity, dihydroolo Isolation of a DNA encoding a protein having an enzyme activity or orotidylate decarboxylase activity also provides a DNA encoding a protein substantially identical to aspartate carbamoyltransferase, dihydroorotase or orotidylate decarboxylase. . The term "stringent conditions" as used herein refers to conditions under which a so-called specific hybrid is formed and a non-specific hybrid is not formed. Although it is difficult to quantify this condition clearly, as an example, DNAs having high homology, for example, DNAs having homology of 50% or more hybridize, and DNAs having lower homology These are the conditions under which they do not hybridize with each other, or the washing conditions for ordinary Southern hybridization.
0 ° C., 1 × SSC, 0.1% SDS, preferably 0.
Conditions for hybridization at a salt concentration corresponding to 1 × SSC and 0.1% SDS are exemplified.

Some of the genes that hybridize under such conditions include those in which a stop codon has been generated in the middle and those in which the activity has been lost due to mutation of the active center. Can be easily removed by measuring aspartate carbamoyltransferase activity, dihydroorotase activity or orotidylate decarboxylase activity.

Since the base sequences of the respective DNAs of the present invention have been clarified, the polymerase chain reaction method (PCR) using oligonucleotides prepared based on the base sequences as primers.
ion; White, TJ et al., Trends Genet., 5,185 (198
9)), from a coryneform bacterial chromosomal DNA library by amplification from a coryneform bacterial chromosomal DNA, or by hybridization using an oligonucleotide prepared based on the same base sequence as a probe,
It can be obtained by selecting. PCR
The base sequence of the primer used for the above may be appropriately selected as a sequence corresponding to a portion upstream of the coding region of each gene as the 5′-side primer and a portion downstream of the coding region as the 3′-side primer.

As a host for expressing the DNA of the present invention, various bacteria including coryneform bacteria such as Escherichia coli, Brevibacterium lactofermentum, and Brevibacterium flavum, and Saccharomyces cerevisiae ( Eukaryotic cells such as Saccharomyces cerevisiae). The DNA of the present invention is added to these hosts.
Can be introduced by transforming host cells with a recombinant vector obtained by inserting the DNA of the present invention into a vector corresponding to the type of host to be expressed. These operations can be performed by methods well known to those skilled in the genetic recombination technique. Specifically, for example,
Competent cells were prepared from cells at the growth stage by the method described in Bacillus subtilis, a method used for the transformation of Escherichia coli described above.
How to introduce NA (Duncan, CH, Wilson, GAand Yo
ung, FE, Gene, 1, 153 (1977)), as is known for Bacillus subtilis, actinomycetes and yeast,
A method of introducing a recombinant DNA into a DNA receiving bacterium by converting the cells of the DNA receiving bacterium into a protoplast or a spheroplast that easily takes up the recombinant DNA (Chang, S.
d Choen, SN, Molec. Gen. Genet., 168, 111 (1979); Bi
bb, MJ, Ward, JMand Hopwood, OA, Nature, 274,398
(1978); Hinnen, A., Hicks, JBand Fink, GR, Proc. Nat.
l. Acad. Sci. USA, 75 1929 (1978)), and the electric pulse method useful for coryneform bacteria (see Japanese Patent Application Laid-Open No. 2-207791).

The DNA to be introduced into the host as described above may be a DNA containing any of pyrB, pyrC and pyrF, or a DNA containing any two or all of these. Further, one or both of carA and carB may be further included. Specifically, as such DNA,
A DNA containing a base sequence consisting of base numbers 11 to 7802 in SEQ ID NO: 1 of the sequence listing is exemplified. In order to carry out the expression of these genes efficiently, lac, tr, which works in the host cell, upstream of the coding region of these genes.
A promoter such as p or P _L may be linked.

By culturing the above-mentioned transformant under conditions in which each gene is expressed, aspartate carbamoyltransferase, dihydroorotase, carbamoyl phosphate synthetase small subunit, carbamoyl phosphate synthetase large subunit or orotidyl. Acid decarboxylase or two or more of these enzymes can be produced. Further, the DNA of the present invention can be used for breeding L-arginine-producing bacteria or nucleic acid-producing bacteria such as uracil. That is, a transformant into which the DNA of the present invention has been introduced has an increased activity of each enzyme as compared with a non-transformant. As a result, the ability to produce pyrimidine such as uracil or L-arginine is improved.

[0025]

EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples.

<1> Preparation of chromosomal DNA of Brevibacterium lactofermentum ATCC13869 Brevibacterium lactofermentum ATCC13869
TY medium (Bacto-trypton (Difco) 1%, Bacto-yeast e
100 ml of xtract (Difco) 0.5%, NaCl 0.5% (pH 7.2) was inoculated and cultured at 31.5 ° C. for 8 hours to obtain a culture. This culture was centrifuged at 3,000 rpm for 15 minutes to obtain 0.5 g of wet cells, which were then subjected to the method of Saito and Miura (Bioc.
Chromosomal DNA was obtained by Chem. Biophys. Acta., 72, 619 (1963)). Next, 60 μg of this chromosomal DNA and 3 units of the restriction enzyme Sau3AI were mixed with 10 mM Tris-HCl buffer (containing 50 mM NaCl, 10 mM MgSO ₄ and 1 mM dithiothreitol (pH 7.4)), and reacted at 37 ° C. for 30 minutes. Was. The reaction solution was subjected to phenol extraction and ethanol precipitation by a conventional method to obtain 50 μg of a chromosomal DNA fragment of Brevibacterium lactofermentum ATCC13869 digested with Sau3AI.

<2> Brevibacterium lactofermentum ATCC138 using plasmid vector DNA
Construction of 69 gene libraries pSAC4 was used as a plasmid vector DNA capable of autonomous replication in both Escherichia coli and coryneform bacteria.
Was used. pSAC4 was produced as follows. In order to enable the Escherichia coli vector pHSG399 (Takara Shuzo Co., Ltd.) to replicate autonomously in coryneform bacteria, a plasmid pHM151 capable of autonomously replicating in coryneform bacteria already obtained.
9 (Miwa, k. Et al., Agric. Biol. Chem., 48 (1984) 2
901-2903) (Japanese Unexamined Patent Publication No. 5-7491). Specifically, pHM1519 is restricted to restriction enzymes BamHI and Kp.
After digestion with nI, a gene fragment containing the replication origin was obtained, and the obtained fragment was blunt-ended using a Blunting kit manufactured by Takara Shuzo Co., Ltd., followed by SalI linker (Takara Shuzo Co., Ltd.).
It was inserted into the SalI site of HSG399 to obtain pSAC4.

20 μg of pSAC4 and 200 units of BamHI restriction enzyme were mixed with 50 mM Tris-HCl buffer (containing 100 mM NaCl and 10 mM magnesium sulfate (pH 7.4)) and reacted at 37 ° C. for 2 hours to obtain a digested solution. The solution was subjected to phenol extraction and ethanol precipitation by a conventional method. Thereafter, in order to prevent recombination of the DNA fragment derived from the plasmid vector, Molecular Cloning 2nd editon (J. Sam
brook, EF Fritschand T. Maniatis, Cold Spring
Harbor Laboratory Press, p1.56 (1989)) and bacterial alkaline phosphatase (Bacterial Alkali).
ne Phosphatase), the DNA fragment was dephosphorylated, subjected to phenol extraction by a conventional method, and further subjected to ethanol precipitation.

1 μg of the pSAC4 digested with BamHI, 1 μg of the chromosomal DNA fragment of Brevibacterium lactofermentum ATCC13869 digested with Sau3AI obtained in Example 1, and 2 units of T4 DNA ligase (Takara Shuzo Co., Ltd. )) Was added to a 66 mM Tris-HCl buffer (pH 7.5) containing 66 mM magnesium chloride, 10 mM dithiothreitol and 10 mM ATP, and reacted at a temperature of 16 ° C. for 16 hours.
A was connected. Next, Escherichia coli DH5 was transformed with the DNA mixture by a conventional method, and the resulting mixture was added at 170 μg / m
spread on L agar medium containing 1 chloramphenicol,
About 20,000 colonies were obtained and used as a gene library.

<3> Escherichia coli carB deletion strain (JEF
8) Transformation Escherichia coli carB deletion strain JEF8 (thr ^- 31, Δcar
^{B, relA -.., MetB1} , Mol.Gen Genet, 133, 299 (197
4)), using the recombinant DN of the above gene library
Transformed with the A mixture. About 15,000 transformants were obtained as Cm resistant strains. These transformants were transformed into a minimal medium containing no arginine or uracil (glucose 5 g / L, Na ₂ HPO ₄
12.8 g / L, KH ₂ PO ₄ 3 g / L, NaCl 0.5 g / L NH ₄ Cl 1 g / L, L−
A strain in which arginine and uracil auxotrophy have been restored, or a strain in which arginine auxotrophy has been restored, which is replicated in a minimal medium containing only uracil 50 μg / ml without L-arginine (40 μg / ml of threonine and 40 μg / ml of L-methionine). Was screened. The strains with restored arginine auxotrophy have restored both arginine and uracil auxotrophy.

A plasmid is prepared from these strains according to a conventional method, and the nucleotide sequence of the gene fragment contained in the plasmid is determined using the Rohdamin Terminator Cycle Sequencing Kit (ABI
Was determined according to the method of Sanger. As a result, the clone fragments carried by the plasmids named p1, p19 and p25 contained the same region, and p1 and p25
When the cloned fragments contained in were combined, it was found that the cloned fragment in p19 was completely contained.

Escherichia coli J carrying p1 and p25
EF8 / p1 and JEF8 / p25 are Escherichia coli AJ, respectively.
13599, named AJ13600, and on April 6, 1999, the Ministry of International Trade and Industry
305-8566, 1-3-1 Higashi, Tsukuba City, Ibaraki Prefecture, Japan), and they have been sequentially assigned accession numbers FERM P-17360 and FERM P-17361.

<4> Analysis of Nucleotide Sequence of Clone Fragment The nucleotide sequence of the clone fragment contained in p1 and p25 is shown in SEQ ID NO: 1 in the Sequence Listing. In this nucleotide sequence, there are five open reading frames (ORFs). Amino acid sequences that can be encoded in each open reading frame are shown in SEQ ID NOs: 2 to 6. Database of known proteins for these amino acid sequences
When a homology search was performed on (GenBank CDS), high homology was recognized with the known pyrimidine biosynthesis enzyme and L-arginine biosynthesis enzyme, respectively. That is, the first ORF (base numbers 11 to 904) is a known aspartate carbamoyltransferase, the second ORF (base numbers 960 to 2150) is a known dihydroorotase, and the fifth ORF 8 (base number). Number 6969-
7802) is known to have a high homology (about 40%, about 38%, about 50% in sequence) to the known orotidylate decarboxylase, since the amino acid sequence encoded by each of these ORFs is very high. It is considered to be the gene encoding the corresponding enzyme (pyrB, pyrC, pyrF in order). The third ORF (base numbers 2434 to 3612)
Is a small subunit of carbamoyl phosphate synthetase such as Escherichia coli and Bacillus subtilis; and the fourth ORF (base numbers 3907 to 6960) is a known carbamoylphosphate such as Escherichia coli and Bacillus stearothermophilus. These ORFs are considered to be genes (carA, carB) encoding the corresponding enzymes, respectively, since they have high homology to the large subunit of acid synthetase (approximately 40%, approximately 40 to 50% in order).

[0034]

Industrial Applicability According to the present invention, pyrimidine biosynthetic enzymes derived from coryneform bacteria and genes encoding them are provided.

[0035]

[Sequence List] SEQUENCE LISTING <110> Ajinomoto Co., Inc. <120> Pyrimidine biosynthesis enzyme gene of coryneform bacterium <130> P-6310 <141> 1999-04-08 <160> 6 <170> PatentIn Ver. 2.0

<210> 1 <211> 9048 <212> DNA <213> Brevibacterium lactofermentum <220> <221> CDS <222> (11) .. (904) <220> <221> CDS <222> (960 ) .. (2150) <220> <221> CDS <222> (2434) .. (3612) <220> <221> CDS <222> (3907) .. (6960) <220> <221> CDS < 222> (6969) .. (7802) <400> 1 aagatccccg gtg acc gtg gcc gtc gat ttg gag gat cgc ttt aag gag 49 Val Thr Val Ala Val Asp Leu Glu Asp Arg Phe Lys Glu 1 5 10 gtg ctc gaa gga cgt gaa gta aag aag ctg ccc acg ctg cgt ggt cgc 97 Val Leu Glu Gly Arg Glu Val Lys Lys Leu Pro Thr Leu Arg Gly Arg 15 20 25 acc att ttt acc ttg ttc tat gag aac tcc acg cgc acc cgt tcg tcc 145 Thr Ile Phe Thr Leu Phe Tyr Glu Asn Ser Thr Arg Thr Arg Ser Ser 30 35 40 45 ttt gaa acc gca gga aag tgg atg agc gcc gat gtg att aac att tcg 193 Phe Glu Thr Ala Gly Lys Trp Met Ser Ala Asp Val Ile Asn Ile Ser 50 55 60 gcc tca tca tcc agc gtg aag aag ggc gag tcg ctg aaa gat acc ggc 241 Ala Ser Ser Ser Ser Val Lys Lys Gly Glu Ser Leu Lys Asp Thr Gly 65 70 75 ttg act ttg tcg gca atc ggc gcg gat gcg atc atc atg cgc cac cca 289 Leu Thr Leu Ser Ala Ile Gly Ala Asp Ala Ile Ile Met Arg His Pro 80 85 90 gcc tca ggc gcc gcg cag cag ctt gcg cag ttc gtc gca cca ggc ggc 337 Ala Ser Gly Ala Gln Leu Ala Gln Phe Val Ala Pro Gly Gly 95 100 105 aac ggc ccc agc gtg atc aac gcg ggt gac ggt tcg cac cag cac ccc 385 Asn Gly Pro Ser Val Ile Asn Ala Gly Asp Gly Ser His Gln His Pro 110 115 120 125 acc cag gcg ctt ctc gac gct tta acc atc cgg cag cgc acc ggc cgc 433 Thr Gln Ala Leu Leu Asp Ala Leu Thr Ile Arg Gln Arg Thr Gly Arg 130 135 140 att gag gga ctc aaa gtt gtc atc gtg ggc gac tgt tcc cgg 481 Ile Glu Gly Leu Lys Val Val Ile Val Gly Asp Cys Leu His Ser Arg 145 150 155 gtg gtg cgc tcc aat gtg gat ctg ctg tcc act ttg ggc gca gag gta 529 Val Val Arg Ser Asn Val Asp Leu Leu Ser Thr Leu Gly Ala Glu Val 160 165 170 gtg ctg gtt gct cct ccg aca ctg ctt cct att ggt gtg gag aac tgg 577 Val Leu Val Ala Pro Pro Thr Leu Leu Pro Ile Gly Val Glu Asn Trp 175 180 185 cca gtc cga ttc tcc tac gac atg gac gca gaa att gcc gac gcc gac 625 Pro Val Arg Phe Ser Tyr Asp Met Asp Ala Glu Ile Ala Asp Ala Asp 190 195 200 205 gta gtg atg atg ctg cgc gtt cag caa gaa cgc atg cag ggt ggt Met ttc 673 Val Met Leu Arg Val Gln Gln Glu Arg Met Gln Gly Gly Phe 210 215 220 ttc ccc tca cac cgt gag tac gca acg ctg tac ggc atg tcc aaa gag 721 Phe Pro Ser His Arg Glu Tyr Ala Thr Leu Tyr Gly Met Ser Lys Glu 225 230 235 cgc gaa gct cgc ctc aag gac tcc gcc atc atc atg cac ccc ggc ccc 769 Arg Glu Ala Arg Leu Lys Asp Ser Ala Ile Ile Met His Pro Gly Pro 240 245 250 atg ctt cgt ggc atg gaa atc aac ttccag gac gca cca cgc 817 Met Leu Arg Gly Met Glu Ile Asn Phe Gln Val Ala Asp Ala Pro Arg 255 260 265 acc gcg gta ctg cag cag gta agc aac ggt gtg cac atg cgc atg gcc 865 Thr Ala Val Leu Gln Gln Val Ser Asn Gly Val His Met Arg Met Ala 270 275 280 285 att ttg ttc gcc cta gtc gca ggc tct gac gcg act atc taatcgcgac 914 Ile Leu Phe Ala Leu Val Ala Gly Ser Asp Ala Thr Ile 290 295 catctgatc g cgaaaaggta taa cgagggatta aagaa gtg gtt gac 968 Val Val Asp 1 agc aac acc caa tat cca gaa acc ggc gca ctg gca ccg gct cct gca 1016 Ser Asn Thr Gln Tyr Pro Glu Thr Gly Ala Leu Ala Pro Ala Pro Ala 5 10 15 gac tca ctc cta atc agc aac gtt ttg gtc tac ggc gaa ggc gag cca 1064 Asp Ser Leu Leu Ile Ser Asn Val Leu Val Tyr Gly Glu Gly Glu Pro 20 25 30 35 acg aat gtg ttt gtt aaa gat ggt gtg atc gca gct atc ggc 1112 Thr Asn Val Phe Val Lys Asp Gly Val Ile Ala Ala Ile Gly Gly Thr 40 45 50 cat gag gct gac cgc acc atc gac ggc aat ggg gga gtt ctc ctt cca 1160 His Glu Ala Asp Arg Thr Ile Asp Gly Asn Gly Gly Val Leu Leu Pro 55 60 65 ggt ttc gtg gac atg cac gtt cac ctg cgt gag cca ggc cgc gaa gac 1208 Gly Phe Val Asp Met His Val His Leu Arg Glu Pro Gly Arg Glu Asp 70 75 80 act gaa acc att gcc act ggt tct gcc gcc gca gcc aag ggc gga ttc 1256 Thr Glu Thr Ile Ala Thr Gly Ser Ala Ala Ala Ala Lys Gly Gly Phe 85 90 95 acc gca gta ttc acc atg gcg aac acc act cca gtg atg gat cag ccg 1304 Thr Ala Val Phe Thr Met Al a Asn Thr Thr Pro Val Met Asp Gln Pro 100 105 110 115 gtt atc gcg gaa tcc gta tgg ttc aag ggc caa aac att ggc ctg tgc 1352 Val Ile Ala Glu Ser Val Trp Phe Lys Gly Gln Asn Ile Gly Leu Cys 120 125 130 gac gtg cat cca gtt gga tcc atc acc aag ggt ctt gag ggc aag gag 1400 Asp Val His Pro Val Gly Ser Ile Thr Lys Gly Leu Glu Gly Lys Glu 135 140 145 ctt act gag ttc ggc atg atg gct cgc tct gaa gcc aag gtg cgt atg 1448 Leu Thr Glu Phe Gly Met Met Ala Arg Ser Glu Ala Lys Val Arg Met 150 155 160 ttc tct gat gat ggt aag tgc gtc gat gat cct cag gtc atg cgc cgc 1496 Phe Ser Asp Asp Gly Lys Cys Val Asp Asp Pro Gln Val Met Arg Arg 165 170 175 gcg ctg gaa tac gcc aag ggc atg gac gtt ttg atc gcc cag cac gct 1544 Ala Leu Glu Tyr Ala Lys Gly Met Asp Val Leu Ile Ala Gln His Ala 180 185 190 195 gag gatcccg act gag ggc gct tca gca cac gag ggc gaa aac 1592 Glu Asp His Arg Leu Thr Glu Gly Ala Ser Ala His Glu Gly Glu Asn 200 205 210 gca gct cgt ctg ggt ctg cgc gga tgg cca cgt gtg gct gag gaa tcc 1640 Ala Ala Arg Leu Gly Leu Arg Gly Trp Pro Arg Val Ala Glu Glu Ser 215 220 225 atc gtg gtg cgt gat gcc atc atg gct cgt gac tac ggc aac cgc gtg 1688 Ile Val Val Arg Asp Ala Ile Met Ala Arg Asp Tyr Gn Asn Val 230 235 240 cac atc tgc cac gca tcc acc gaa ggc acc gtg gag ctg ctt cgt tgg 1736 His Ile Cys His Ala Ser Thr Glu Gly Thr Val Glu Leu Leu Arg Trp 245 250 255 gct aag tcc cag ggc att cca atc acc gcg gaa gcc acc ccg cac cac 1784 Ala Lys Ser Gln Gly Ile Pro Ile Thr Ala Glu Ala Thr Pro His His 260 265 270 270 275 ctc acc ttg acc gat gag cgc ctg gaa acc tac gac gcg gtc aac aaa 1832 Leu Thr Leu Thr Asp Glu Arg Leu Glu Thr Tyr Asp Ala Val Asn Lys 280 285 290 gtc aat ccg cca ctg cgc gaa agc cgc gat gcc gaa gcg ctc aag aag 1880 Val Asn Pro Pro Leu Arg Glu Ser Arg Asp Ala Glu Ala Leu Lys Lyc 295 ctt ctc gac ggc acc atc gat gtt gtt gca acc gac cac gct cct 1928 Ala Leu Leu Asp Gly Thr Ile Asp Val Val Ala Thr Asp His Ala Pro 310 315 320 cac ggt tcc gaa gat aag tgc tgt gaa ttc gaa aac gcc a ag cca ggc 1976 His Gly Ser Glu Asp Lys Cys Cys Glu Phe Glu Asn Ala Lys Pro Gly 325 330 335 atg ctc gga ttg gaa acc tca ctg tcc atc atc gtg gac acc ttc gtt 2024 Met Leu Gly Leu Glu Thr Ser Leu Ser Ile Ile Val Asp Thr Phe Val 340 345 350 355 gcc acc gga ctt gca gac tgg cgc ttt gtt gcg cgc gtg atg agt gaa 2072 Ala Thr Gly Leu Ala Asp Trp Arg Phe Val Ala Arg Val Met Ser Glu 360 365 370 cgc cca gca gaa atc acc cgt cta cca ggc cag ggt cgc cca atc gca 2120 Arg Pro Ala Glu Ile Thr Arg Leu Pro Gly Gln Gly Arg Pro Ile Ala 375 380 385 gaa ggt gag cca gca aac ctc gcg att tgt tgatccagga Gaaaaccgly G170 Asn Leu Ala Ile Cys 390 395 cagcatccgg tgcagacttt gcgtccaagg ctgaaaacac cccatttgag ggccaggaat 2230 tcagcgctaa ggtcacacac accgtgcttc gtggcaaggt gacttgtgca gacggagttg 2290 cgcaagacgc ttaacgggtg ggtgcatagt atgcacgcgc cgcattgcat ataatgcaat 2350 gaattgaata aactacattc agggttatca accagccaat ttcttttaaa aagacagaca 2410 cacgaaaggc gacaacagtc acc gtg agt aaa gac acc acc acc tac cag 2460 Val Ser Lys Asp Thr Thr Thr Tyr Gln 1 5 gga gtc acc gag atc gga tcc gtt ccg gca tac ctg gtt ctt gca gac 2508 Gly Val Thr Glu Ile Gly Ser Val Pro Ala Tyr Leu Val Leu Ala Asp 10 15 20 25 gga cgt acc ttc acc gga ttt ggc ttt gga gct atc ggc acc acc ctt 2556 Gly Arg Thr Phe Thr Gly Phe Gly Phe Gly Ala Ile Gly Thr Thr Leu 30 35 40 ggt gag gca gtg ttc acc acc gcc atg acc ggt tac caa gaa acc atg 2604 Gly Glu Ala Val Phe Thr Thr Ala Met Thr Gly Tyr Gln Glu Thr Met 45 50 55 acc gat cct tcc tat cac cgc cag att gtt gtg gct acc gca cca cag 2652 Thr Asp Pro Ser Tyr His Arg Gln Ile Val Val Ala Thr Ala Pro Gln 60 65 70 atc ggt aac acc ggc tgg aac gat gag gac aac gag tcc cgc gac ggc 2700 Ile Gly Asn Thr Gly Trp Asn Asp Glu Asp Asn Glu Ser Arg Asp Gly 75 80 85 aag att tgg gtt gca ggc ctt gtt atc cgc gac ctc gca gca cgt gtg 2748 Lys Ile Trp Val Ala Gly Leu Val Ile Arg Asp Leu Ala Ala Arg Val 90 95 100 105 tcc aac tgg cgc gcc acc acc tcc ttg cag cag gaa atg gca gac caa 2796 Ser Asn Trp Arg Ala Thr Thr Ser Le u Gln Gln Glu Met Ala Asp Gln 110 115 120 ggc atc gtc ggc atc ggc gga atc gac acc cgc gca ctg gtt cgc cac 2844 Gly Ile Val Gly Ile Gly Gly Ile Asp Thr Arg Ala Leu Val Arg His 125 130 135 ctg cgc aac gaa ggt tcc atc gca gcg ggc atc ttc tcc ggc gct gac 2892 Leu Arg Asn Glu Gly Ser Ile Ala Ala Gly Ile Phe Ser Gly Ala Asp 140 145 150 gca cag cgc cca gtt gaa gaa ctc gta gag atc gtcag ag ag ac ag ccagca Ala Gln Arg Pro Val Glu Glu Leu Val Glu Ile Val Lys Asn Gln Pro 155 160 165 gca atg acc ggc gca aac ctc tcc gtt gag gtc tct gct gat gaa acc 2988 Ala Met Thr Gly Ala Asn Leu Ser Val Glu Val Ser Ala Asp Glu Thr 170 175 180 185 tac gtc atc gaa gct gag ggc gaa gag cgc cac acc gtc gtg gcc tac 3036 Tyr Val Ile Glu Ala Glu Gly Glu Glu Arg His Thr Val Val Ala Tyr 190 195 200 gac ctg ggc att aag caa aac acc cca cgt cgt ttc tct gca cgc ggt 3084 Asp Leu Gly Ile Lys Gln Asn Thr Pro Arg Arg Phe Ser Ala Arg Gly 205 210 215 gtt cgc acc gtc atc gtg cct gct gaa acc cca ttg gag gac atc aag 3132 Val Arg Thr Val Ile Val Pro Ala Glu Thr Pro Leu Glu Asp Ile Lys 220 225 230 cag tac aac cca tca ggc gtg ttt atc tcc aat ggc cct ggc gac cct 3180 Gln Tyr Asn Pro Ser Gly Val Phe Ile Ser Asn Gly Pro Gly Asp Pro 235 240 245 gca gca gca gac gtc atg gtt gat atc gtc cgc gaa gtt ctg gaa gcc 3228 Ala Ala Ala Asp Val Met Val Asp Ile Val Arg Glu Val Leu Glu Ala 250 255 260 265 gac att cca ttc ttt ggc atc ttgc ttc atc ctc ggc cgc 3276 Asp Ile Pro Phe Phe Gly Ile Cys Phe Gly Asn Gln Ile Leu Gly Arg 270 275 280 gca ttc ggc atg gag acc tac aag ctg aag ttc ggc cac cgc ggc atc 3324 Ala Plu Gly Lyr Lys Phe Gly His Arg Gly Ile 285 290 295 295 aac gtt cca gtg aag aac cac atc acc ggc aag atc gac atc acc gcc 3372 Asn Val Pro Val Lys Asn His Ile Thr Gly Lys Ile Asp Ile Thr Ala 300 305 310 cag aac cac ggc ttc gca ctc aag ggt gaa gca ggc cag gaa ttc gag 3420 Gln Asn His Gly Phe Ala Leu Lys Gly Glu Ala Gly Gln Glu Phe Glu 315 320 325 aca gat ttc ggc act gcg att gtc acc cac acc tgc ctt ac 468 Thr Asp Phe Gly Thr Ala Ile Val Thr His Thr Cys Leu Asn Asp Gly 330 335 340 345 gtc gtt gaa ggt gtt gcg ctg aag tcc gga cgc gca tac tcc gtt cag 3516 Val Val Glu Gly Val Ala Leu Lys Ser Gly Arg Ala Tyr Ser Val Gln 350 355 360 tac cac cca gag gcc gct gcc ggc cca aat gat gca agc ccc ctg ttt 3564 Tyr His Pro Glu Ala Ala Ala Gly Pro Asn Asp Ala Ser Pro Leu Phe 365 370 375 gac cag ttt gtt gag ctg atg gat gca gac gct cag aag aaa ggc gca 3612 Asp Gln Phe Val Glu Leu Met Asp Ala Asp Ala Gln Lys Lys Gly Ala 380 385 390 taaataacat gccaaagcgt tcagatatta accacgtcct cgtcatcggt tccggcccca 3672 tcgtcattgg ccaggcatgt gaattcgact actccggcac ccaggcttgc cgcgtgctga 3732 aggaagaggg actgcgcgtc accctcatca actccaaccc agcaacgatc atgaccgacc 3792 cagaaatggc tgaccacacc tacgtggagc caatcgagcc ggaatacatc gacaagattt 3852 tcgctaagga gatcgagcag ggccacccaa tcgacgccgt cctggcaacc cttg gtg 3909 Val 1 gcc aga ctg cac tta acg cag cta tg ag Tcg cc tc gc tc gc cc tc gcc cc tc gcc he Gly Ile 5 10 15 ctg gaa aag tac ggc gtt gaa ctc atc ggt gca gac atc gat gcc att 4005 Leu Glu Lys Tyr Gly Val Glu Leu Ile Gly Ala Asp Ile Asp Ala Ile 20 25 30 gag cgc ggc gaa gag cg cag ttc aag gat att gtc acc acc atc 4053 Glu Arg Gly Glu Asp Arg Gln Lys Phe Lys Asp Ile Val Thr Thr Ile 35 40 45 ggt ggc gaa tcc gcg cgt tcc cgc gtc tgc cac aac atg gac gaa gtc 4101 Gly Gly Glu Glu Arg Ser Arg Val Cys His Asn Met Asp Glu Val 50 55 60 65 cat gag act gtc gca gaa ctt ggc ctt cca gta gtc gtg cgt cca tcc 4149 His Glu Thr Val Ala Glu Leu Gly Leu Pro Val Val Val Arg Pro Ser 70 75 80 ttc act atg ggt ggc ctg ggc tcc ggt ctt gca tac aac acc gaa gac 4197 Phe Thr Met Gly Gly Leu Gly Ser Gly Leu Ala Tyr Asn Thr Glu Asp 85 90 95 ctt gag cgc atc gca ggt ggc gga ctt gct gca tct gaa gca aac 4245 Leu Glu Arg Ile Ala Gly Gly Gly Leu Ala Ala Ser Pro Glu Ala Asn 100 105 110 gtc ttg atc gaa gaa tcc atc ctt ggt tgg aag gaa ttc gag ctc gag 4293 Val Leu Ile Glu Gly Ser Ileu Lys Glu Phe Gl u Leu Glu 115 120 125 ctc atg cgc gat acc gca gac aac gtt gtg gtt atc tgc tcc att gaa 4341 Leu Met Arg Asp Thr Ala Asp Asn Val Val Val Ile Cys Ser Ile Glu 130 135 140 145 aac gtc gac gca ctg ggc gtg cac acc ggc gac tct gtc acc gtg gca 4389 Asn Val Asp Ala Leu Gly Val His Thr Gly Asp Ser Val Thr Val Ala 150 155 160 cct gcc ctg acc ctg act gac cgt gaa ttc cag aag atg cgc gat cag 4437 Pro Ala Leu Thr Leu Thr Asp Arg Glu Phe Gln Lys Met Arg Asp Gln 165 170 175 ggt atc gcc atc atc cgc gag gtc ggc gtg gac acc ggt gga tgt aac 4485 Gly Ile Ala Ile Ile Arg Glu Val Gly Val Asp Thr Gly Gly Cys Asn 180 185 190 atc cag ttc gct atc aac cca gtt gat ggc cgc atc atc acc att gag 4533 Ile Gln Phe Ala Ile Asn Pro Val Asp Gly Arg Ile Ile Thr Ile Glu 195 200 205 atg aac cca cgt gtg tct cgt tcc tcc gcg ccg gca aag gca acg 4581 Met Asn Pro Arg Val Ser Arg Ser Ser Ala Leu Ala Ser Lys Ala Thr 210 215 220 225 ggc ttc cca att gcc aag atg gct gcc aag ctg gct atc gga tac acc 4629 Gly Phe Pro Ile Ala Lys Met Ala Ala Lys Leu Ala Ile Gly Tyr Thr 230 235 240 ctg gat gag atc acc aac gac atc act ggt gaa acc cca gct gcg ttt 4677 Leu Asp Glu Ile Thr Asn Asp Ile Thr Gly Glu Thr Pro Ala Ala Phe 245 250 255 gag ccc acc atc gac tac gtc gtg gtc aag gcc cca cgc ttt gct ttc 4725 Glu Pro Thr Ile Asp Tyr Val Val Val Lys Ala Pro Arg Phe Ala Phe 260 265 270 gag aag ttt gtc ggc gct gat gac act ttg acc acc acc atg aag tcc 4773 Glu Lys Phe Val Gly Ala Asp Asp Thr Leu Thr Thr Thr Met Lys Ser 275 280 285 gtc ggt gag gtc atg tcc ctg ggc cgt aac tac att gca gca ctg aac 4821 Val Gly Glu Val Met Ser Leu Gly Arg Asn Tyr Ile Ala Ala Leu Asn 290 295 300 305 aag gca ctg cgt tcc ctg gaa acc aag cag cag ggt ttc tgg acc aag 4869 Lys Ala Leu Arg Ser Leu Glu Thr Lys Gln Gln Gly Phe Trp Thr Lys 310 315 320 cct gat gag ttc ttc gca ggg cgc gct acc gat aag gca gct gtt 4917 Pro Asp Glu Phe Phe Ala Gly Glu Arg Ala Thr Asp Lys Ala Ala Val 325 330 335 ctg gaa gat ctc aag cgc cca acc gaa ggc cgc ctc tac gac gtt gag 4965 Leu Glu Leu Lys Arg Pro Thr Glu Gly Arg Leu Tyr Asp Val Glu 340 345 350 ctg gca atg cgc ctt ggc gca agc gtg gaa gaa ctc tac gaa gca tct 5013 Leu Ala Met Arg Leu Gly Ala Ser Val Glu Glu Leu Tyr Glu 360 365 tct att gat cct tgg ttc ctc gcc gag ctt gaa gct ctc gtg cag ttc 5061 Ser Ile Asp Pro Trp Phe Leu Ala Glu Leu Glu Ala Leu Val Gln Phe 370 375 380 385 cgc cag aag ctc gtt gac gcacca ctt gaa gat ctc ctg cgc 5109 Arg Gln Lys Leu Val Asp Ala Pro Phe Leu Asn Glu Asp Leu Leu Arg 390 395 400 gaa gca aag ttc atg ggt ctg tcc gac ctg cag atc gca gcc ctt cgc 5157 Glu Ala Lys Phe Asp Leu Gln Ile Ala Ala Leu Arg 405 410 415 cca gag ttc gct ggc gaa gac ggc gta cgc acc ttg cgt ctg tcc cta 5205 Pro Glu Phe Ala Gly Glu Asp Gly Val Arg Thr Leu Arg Leu Ser Leu 420 425 430c ggc cca gta ttc aag act gtg gat acc tgt gca gca gag ttt 5253 Gly Ile Arg Pro Val Phe Lys Thr Val Asp Thr Cys Ala Ala Glu Phe 435 440 445 gaa gct aag act ccg tac cac tac tcc gca tac gag ctg gat cca gc a 5301 Glu Ala Lys Thr Pro Tyr His Tyr Ser Ala Tyr Glu Leu Asp Pro Ala 450 455 460 465 gct gag tct gag gtc gca cca cag act gag cgt gaa aag gtc ctg atc 5349 Ala Glu Ser Glu Val Ala Pro Gln Thr Glu Arg Glu Lys Val Leu Ile 470 475 480 ttg ggc tcc ggt cca aac cgc atc ggc cag ggc atc gag ttc gac tat 5397 Leu Gly Ser Gly Pro Asn Arg Ile Gly Gln Gly Ile Glu Phe Asp Tyr 485 490 490 tcc tgt gtt ctt gag ctc tcc cgc gtc ggc tac gaa act 5445 Ser Cys Val His Ala Ala Leu Glu Leu Ser Arg Val Gly Tyr Glu Thr 500 505 510 gtc atg gtc aac tgc aac cca gag acc gtg tcc acc gac tac gac acc 5493 Val Met Val Asn Cys Asn Pro Glu Thr Val Ser Thr Asp Tyr Asp Thr 515 520 525 gct gac cgc ctg tac ttc gag cca ctg acc ttc gaa gac gtc atg gag 5541 Ala Asp Arg Leu Tyr Phe Glu Pro Leu Thr Phe Glu Asp Val Met Glu 530 535 540 545 gtc tac cac gct gag gcg cag tcc ggc acc gtc gca ggt gtt atc gtc 5589 Val Tyr His Ala Glu Ala Gln Ser Gly Thr Val Val Ala Gly Val Ile Val 550 555 560 cag ctt ggt ggc cag act cct ctg ggctt gca gat cgt ttg aag aag 5637 Gln Leu Gly Gly Gln Thr Pro Leu Gly Leu Ala Asp Arg Leu Lys Lys 565 570 575 gct ggc gtc cct gtc att ggt acc tcc cca gag gca atc gac atg gct 5685 Ala Gly Val Pro Val Ile Thr Ser Pro Glu Ala Ile Asp Met Ala 580 585 590 gag gac cgt ggc gag ttc ggt gca ctg ctg aac cgc gag cag ctt cct 5733 Glu Asp Arg Gly Glu Phe Gly Ala Leu Leu Asn Arg Glu Gln Leu Pro 595ca 605 g gca ttc ggc acc gca acc tct ttc gaa gag gct cgc aca gta 5781 Ala Pro Ala Phe Gly Thr Ala Thr Ser Phe Glu Glu Ala Arg Thr Val 610 615 620 625 gcc gat gag atc agc tac cca gtg ctg gtt cgc cct tcc ttg 5829 Ala Asp Glu Ile Ser Tyr Pro Val Leu Val Arg Pro Ser Tyr Val Leu 630 635 640 ggt ggc cgt ggc atg gag att gtc tac gat gag gct tcc ctc gag gat 5877 Gly Gly Arg Gly Met Glu Ile Val Tyr Asp Glu Ala Ser Leu Glu Asp 645 650 655 tac atc aac cgc gca act gag ttg tct tct gac cac cca gtg ctg gtt 5925 Tyr Ile Asn Arg Ala Thr Glu Leu Ser Ser Asp His Pro Val Leu Val 660 665 670 gac cgc ttc ctg gac aac gct att gag atc gac gtc gac gca ctg tgc 5973 Asp Arg Phe Leu Asp Asn Ala Ile Glu Ile Asp Val Asp Ala Leu Cys 675 680 685 gac ggc gac gaa gtc tac ctg gcg ggc gtc atg gac gac c Asp Glu Val Tyr Leu Ala Gly Val Met Glu His Ile Glu Glu 690 695 700 705 gcc ggc att cac tcc ggt gac tcc gca tgt gca ctt cct cca atg act 6069 Ala Gly Ile His Ser Gly Asp Ser Ala Cys Ala Leu Pro Pro Met Thr 710 715 720 ttg ggc gca cag gac atc gag aag gtc cgc gaa gca acc aag aag ctg 6117 Leu Gly Ala Gln Asp Ile Glu Lys Val Arg Glu Ala Thr Lys Lys Leu 725 730 735 gct ctg ggc atc ggc gta cag aac gtc cag tac gca ctc 6165 Ala Leu Gly Ile Gly Val Gln Gly Leu Met Asn Val Gln Tyr Ala Leu 740 745 750 aag gac gac atc ctc tac gtc atc gag gca aac cca cgt gca tcc cgc 6213 Lys Asp Tap Val Ile Glu Ala Asn Pro Arg Ala Ser Arg 755 760 765 acc gtg ccg ttc gtc tcc aag gca acg ggc gtc aac ctg gcc aag gca 6261 Thr Val Pro Phe Val Ser Lys Ala Thr Gly Val Asn Leu Ala Lys Ala 770 775 780 7 85 gca tcc cgt atc gca gtg ggc gcc acc atc aag gat ctc caa gat gag 6309 Ala Ser Arg Ile Ala Val Gly Ala Thr Ile Lys Asp Leu Gln Asp Glu 790 795 800 ggc atg att cct acc gag tac gac ggc ggc tcc ttg ctg gac gct 6357 Gly Met Ile Pro Thr Glu Tyr Asp Gly Gly Ser Leu Pro Leu Asp Ala 805 810 815 cca atc gct gtg aag gaa gca gtg ttg ccg ttc aac cgc ttc cgt cgc 6tt Pro Ile Ala Val Les Glu Alu Phe Asn Arg Phe Arg Arg 820 825 830 cca gat gga aag acc ctg gac acc ctg ctt tcc cca gag atg aag tcc 6453 Pro Asp Gly Lys Thr Leu Asp Thr Leu Leu Ser Pro Glu Met Lys Ser 835 840 845 845 act ggc gag gtc atg ggc ttg gcc aac aac ttc ggc gct gca tat gca 6501 Thr Gly Glu Val Met Gly Leu Ala Asn Asn Phe Gly Ala Ala Tyr Ala 850 855 860 865 aag gct gaa gct ggc gcg ttt ggt gca ttg cca acc gaa Ala Glu Ala Gly Ala Phe Gly Ala Leu Pro Thr Glu Gly Thr Val 870 875 880 ttc gtg acc gtg gct aac cgc gac aag cgc acc ctg atc ctg cca atc 6597 Phe Val Thr Val Ala Asn Arg Asp Lys Arg Thr Leu Ile Le u Pro Ile 885 890 895 cag cgc ctg gcg tcg atg ggc tac aag atc ctc gcc acc gaa ggc acc 6645 Gln Arg Leu Ala Ser Met Gly Tyr Lys Ile Leu Ala Thr Glu Gly Thr 900 905 910 910 gca ggc atg ctg cgc cgcgccgccgccgccgc att gat tgt gaa gtt gtg ctc aag 6693 Ala Gly Met Leu Arg Arg Asn Gly Ile Asp Cys Glu Val Val Leu Lys 915 920 925 gct tcc gac atc cgc gaa ggt gta gag ggc aag tcc atc gtg gat Ascpt 671 Ala Serg Glu Gly Val Glu Gly Lys Ser Ile Val Asp Arg 930 935 940 945 atc cgc gaa ggc gaa gtt gac ctc atc ctc aac acc cca gct ggt tct 6789 Ile Arg Glu Gly Glu Val Asp Leu Ile Leu Asn Thr Pro Ala Gly Ser 950 960 gct ggc gct cgc cac gat ggc tac gat atc cgc gca gca gca gtg acc 6837 Ala Gly Ala Arg His Asp Gly Tyr Asp Ile Arg Ala Ala Ala Val Thr 965 970 975 gtg ggt gtt cca ctg atc acc act gtc cag gca gct gtc 6885 Val Gly Val Pro Leu Ile Thr Thr Val Gln Gly Val Thr Ala Ala Val 980 985 990 cag ggc att gag gcc ctg cgt gag ggc gtt gtc agc gtc cgc gcg ctg 6933 Gln Gly Ile Glu Ala Leu Arg Glu Val Val Ser Val Arg Ala Leu 995 1000 1005 cag gaa ctc gac cac gca gtc aag gct taagccct atg aca ttc ggc gag 6983 Gln Glu Leu Asp His Ala Val Lys Ala Met Thr Phe Gly Glu 1010 1015 1 5 aag ctt ctg aac gcc tcc acc cgt ggc agg cta tgt gtg ggc att 7031 Lys Leu Leu Asn Ala Ala Ser Thr Arg Gly Arg Leu Cys Val Gly Ile 10 15 20 gat ccc cac gaa agc ctg ctg acg tcc tgg ggg ctg ccg gta aac Gta 70p Glu Ser Leu Leu Thr Ser Trp Gly Leu Pro Val Asn Val 25 30 35 gac gga ctt gcg gag ttc tcc cgc gcc tgc gtg gag gct ttc gcc gac 7127 Asp Gly Leu Ala Glu Phe Ser Arg Ala Cys Val Glu Ala Phe Ala Asp 40 45 50 acc gtg gca ttg gtg aag cct cag gtg gcg ttc tat gag cgt ttc ggt 7175 Thr Val Ala Leu Val Lys Pro Gln Val Ala Phe Tyr Glu Arg Phe Gly 55 60 65 tcc gct ggc ttt gcc atc ttg gaa ga acc att acg ctg cgt gag 7223 Ser Ala Gly Phe Ala Ile Leu Glu Glu Thr Ile Gln Thr Leu Arg Glu 70 75 80 85 cgt ggc tgt ttg gtg gtc tct gac gcc aaa cgc ggc gat att ggc tcc 7271 Arg Gly Cys Leu Val Ser As Ala Lys Arg Gly Asp Ile Gly Ser 90 95 100 acc atg gct ggc tat gcc tca gcg tgg tta gat cca gcg tca ccg ctg 7319 Thr Met Ala Gly Tyr Ala Ser Ala Trp Leu Asp Pro Ala Ser Pro Leu 105 110 115 tct agc gac gct gtg acg gtc tct ccc tac ctt ggt ttt cat tcc ttg 7367 Ser Ser Asp Ala Val Thr Val Ser Pro Tyr Leu Gly Phe His Ser Leu 120 125 130 gac cca gtg ttc gaa ctt gcc gag caa cac ggc agg gga gtg ttt gtc 7415 Asp Pro Val Phe Glu Leu Ala Glu Gln His Gly Arg Gly Val Phe Val 135 140 145 ttg gcc gcg acc tca aac cct gag gcc cgc gaa ctc cag gac cag caa 7463 Leu Ala Ala Thr Ser Sern Pro Glu Ala Arg Glu Leu Gln Asp Gln Gln 150 155 160 165 aac gct gac ggc gtg agc att tcc cag cag atc gtg gat cag gca gcg 7511 Asn Ala Asp Gly Val Ser Ile Ser Gln Gln Ile Val Asp Gln Ala Ala 170 175 180 gcg ctt aac gcg cct tat atg gcc ggc aag gct ggc aac att ggc 7559 Ala Leu Asn Ala Pro Tyr Met Ala Gln Gly Lys Ala Gly Asn Ile Gly 185 190 195 gtc gtc atc ggc gcc acc ttg tcc aaa cca cca cgc tta tcg acg ctc G7 Al Val a Thr Leu Ser Lys Pro Pro Arg Leu Ser Thr Leu 200 205 210 ggg ggc gcc att ttg atg ccc ggc gtc ggc gcc cag ggc ggc acg gca 7655 Gly Gly Ala Ile Leu Met Pro Gly Val Gly Ala Gln Gly Gly Thr Ala 215 220 225 agc gac gtt gat gag att gcg gga gac atg gct cat ctt gcg ttc cca 7703 Ser Asp Val Asp Glu Ile Ala Gly Asp Met Ala His Leu Ala Phe Pro 230 235 240 245 aat gtc tct aga agt att ttg gcg aca ggc atc gct gaa atg 7751 Asn Val Ser Arg Ser Ile Leu Ala Thr Gly Pro Asp Ile Ala Glu Met 250 255 260 aag aat tct gtg gca aaa act gct gca gac ttt cct ggt ttc ccc agg 7799 Lys Asn Ser Val Ala Lys Thr Ala Ala Asp Phe Pro Gly Phe Pro Arg 265 270 275 tca tagtcgcgga aacggccctt gattgcaagc ctgtgacctg caggtttagc 7852 Ser ttttaggggt tctgccacct ttgtgtttgt gaaagtagtg ctagactatt cgaaaatcgc 7912 aggctgacat ccttggtatt aaccaggtgt accctcgatt tctggatact ttggtattca 7972 ttttgtcact aaaaaccaca cgataacgga ggaaccccgt ggcccttcca cagttgactg 8032 atgagcagcg caaggcagct cttgctaagg cagcagaggc acgcaaggca cgcgcagagc 8092 tcaaag agaa cctgaagcgc ggcaacacta acctcaggga agttctggac aaggctgagt 8152 ctgacgagat catcggcaag accaaggtct ccgctctcct cgaggctctc cctaaggttg 8212 gcaaggtcaa ggcaaaggag attatggacg agctgggcat tgctcagacc cgtcgtcttc 8272 gtggactggg tgaccgtcag cgtcgcgcac ttctcgagcg tttcggcttc gaggattaat 8332 tcttcagtgt cgggcgataa tcaactagga cggctcgtaa ttcttgcggg cccctcagcg 8392 gtcggtaaat cgactgtggt tgatcgcctc cgcaatgacg ttccaaacct gtatttcagt 8452 gtgtcgatga ccaccagggc acctcgtcct ggtgaagtcg atggacgtga ctacttctat 8512 gtcactgcac aggaatttca ggacaaaatc gactgtggag agatgcttga atgggcagat 8572 atccacggcg gtttgcagcg ttcaggcact ccagcaggtc ccgtcaatga ggctcgccaa 8632 aatggtcggc cagtattggt tgaggttgat cttgcaggag cccgaaacat cgctagctta 8692 attccagatg cagaaaccat cttccttgct ccaccttcat gggaagtttt ggttgaacgc 8752 ctcactggac gtggcaccga aagcgaagac gttattgctc gcaggctcga gaccgcacgc 8812 gaagaattgg ctgctcagag cgaatttaag cacgtcatta tcaatgatga tgtggataca 8872 gccgtcaagg ctattgagga tgttctcctc ggcgcttagc caaaacatag agcggtaggg 8932 tatgcttttc c gattgagca acctttcccg ctcttaacac tactgtccat atacttttga 8992 aaaggtgtca gtgaccaacg tgagcaacga gaccaacgcc accaaggccg tcttcg 9048

<210> 2 <211> 298 <212> PRT <213> Brevibacterium lactofermentum <400> 2 Val Thr Val Ala Val Asp Leu Glu Asp Arg Phe Lys Glu Val Leu Glu 1 5 10 15 Gly Arg Glu Val Lys Lys Leu Pro Thr Leu Arg Gly Arg Thr Ile Phe 20 25 30 Thr Leu Phe Tyr Glu Asn Ser Thr Arg Thr Arg Ser Ser Phe Glu Thr 35 40 45 Ala Gly Lys Trp Met Ser Ala Asp Val Ile Asn Ile Ser Ala Ser Ser 50 55 60 Ser Ser Val Lys Lys Gly Glu Ser Leu Lys Asp Thr Gly Leu Thr Leu 65 70 75 80 Ser Ala Ile Gly Ala Asp Ala Ile Ile Met Arg His Pro Ala Ser Gly 85 90 95 Ala Ala Gln Gln Leu Ala Gln Phe Val Ala Pro Gly Gly Asn Gly Pro 100 105 110 Ser Val Ile Asn Ala Gly Asp Gly Ser His Gln His Pro Thr Gln Ala 115 120 125 Leu Leu Asp Ala Leu Thr Ile Arg Gln Arg Thr Gly Arg Ile Glu Gly 130 135 140 Leu Lys Val Val Ile Val Gly Asp Cys Leu His Ser Arg Val Val Arg 145 150 155 160 Ser Asn Val Asp Leu Leu Ser Thr Leu Gly Ala Glu Val Val Leu Val 165 170 175 Ala Pro Pro Thr Leu Leu Pro Ile Gly Val Glu Asn Trp Pro Val Arg 180 185 190 Phe Ser Tyr Asp Met Asp Ala Glu Ile Ala Asp Ala Asp Val Val Met 195 200 205 Met Leu Arg Val Gln Gln Glu Arg Met Gln Gly Gly Phe Phe Pro Ser 210 215 220 His Arg Glu Tyr Ala Thr Leu Tyr Gly Met Ser Lys Glu Arg Glu Ala 225 230 235 240 Arg Leu Lys Asp Ser Ala Ile Ile Met His Pro Gly Pro Met Leu Arg 245 250 255 Gly Met Glu Ile Asn Phe Gln Val Ala Asp Ala Pro Arg Thr Ala Val 260 265 270 Leu Gln Gln Val Ser Asn Gly Val His Met Arg Met Ala Ile Leu Phe 275 280 285 Ala Leu Val Ala Gly Ser Asp Ala Thr Ile 290 295

<210> 3 <211> 397 <212> PRT <213> Brevibacterium lactofermentum <400> 3 Val Val Asp Ser Asn Thr Gln Tyr Pro Glu Thr Gly Ala Leu Ala Pro 1 5 10 15 Ala Pro Ala Asp Ser Leu Leu Ile Ser Asn Val Leu Val Tyr Gly Glu 20 25 30 Gly Glu Pro Thr Asn Val Phe Val Lys Asp Gly Val Ile Ala Ala Ile 35 40 45 Gly Gly Thr His Glu Ala Asp Arg Thr Ile Asp Gly Asn Gly Gly Val 50 55 60 Leu Leu Pro Gly Phe Val Asp Met His Val His Leu Arg Glu Pro Gly 65 70 75 80 Arg Glu Asp Thr Glu Thr Ile Ala Thr Gly Ser Ala Ala Ala Ala Lys 85 90 95 Gly Gly Phe Thr Ala Val Phe Thr Met Ala Asn Thr Thr Pro Val Met 100 105 110 Asp Gln Pro Val Ile Ala Glu Ser Val Trp Phe Lys Gly Gln Asn Ile 115 120 125 Gly Leu Cys Asp Val His Pro Val Gly Ser Ile Thr Lys Gly Leu Glu 130 135 140 Gly Lys Glu Leu Thr Glu Phe Gly Met Met Ala Arg Ser Glu Ala Lys 145 150 155 160 Val Arg Met Phe Ser Asp Asp Gly Lys Cys Val Asp Asp Pro Gln Val 165 170 175 Met Arg Arg Ala Leu Glu Tyr Ala Lys Gly Met Asp Val Leu Ile Ala 180 185 190 Gln His Ala Glu Asp His Arg Leu Thr Glu Gly Ala Ser Ala His Glu 195 200 205 Gly Glu Asn Ala Ala Arg Leu Gly Leu Arg Gly Trp Pro Arg Val Ala 210 215 220 Glu Glu Ser Ile Val Val Arg Asp Ala Ile Met Ala Arg Asp Tyr Gly 225 230 235 240 Asn Arg Val His Ile Cys His Ala Ser Thr Glu Gly Thr Val Glu Leu 245 250 255 Leu Arg Trp Ala Lys Ser Gln Gly Ile Pro Ile Thr Ala Glu Ala Thr 260 265 270 Pro His His Leu Thr Leu Thr Asp Glu Arg Leu Glu Thr Tyr Asp Ala 275 280 285 Val Asn Lys Val Asn Pro Pro Leu Arg Glu Ser Arg Asp Ala Glu Ala 290 295 300 Leu Lys Lys Ala Leu Leu Asp Gly Thr Ile Asp Val Val Ala Thr Asp 305 310 315 320 His Ala Pro His Gly Ser Glu Asp Lys Cys Cys Glu Phe Glu Asn Ala 325 330 335 Lys Pro Gly Met Leu Gly Leu Glu Thr Ser Leu Ser Ile Ile Val Asp 340 345 350 Thr Phe Val Ala Thr Gly Leu Ala Asp Trp Arg Phe Val Ala Arg Val 355 360 365 Met Ser Glu Arg Pro Ala Glu Ile Thr Arg Leu Pro Gly Gln Gly Arg 370 375 380 Pro Ile Ala Glu Gly Glu Pro Ala Asn Leu Ala Ile Cys 385 390 395

<210> 4 <211> 393 <212> PRT <213> Brevibacterium lactofermentum <400> 4 Val Ser Lys Asp Thr Thr Thr Tyr Gln Gly Val Thr Glu Ile Gly Ser 1 5 10 15 Val Pro Ala Tyr Leu Val Leu Ala Asp Gly Arg Thr Phe Thr Gly Phe 20 25 30 Gly Phe Gly Ala Ile Gly Thr Thr Leu Gly Glu Ala Val Phe Thr Thr 35 40 45 Ala Met Thr Gly Tyr Gln Glu Thr Met Thr Asp Pro Ser Tyr His Arg 50 55 60 Gln Ile Val Val Ala Thr Ala Pro Gln Ile Gly Asn Thr Gly Trp Asn 65 70 75 80 Asp Glu Asp Asn Glu Ser Arg Asp Gly Lys Ile Trp Val Ala Gly Leu 85 90 95 Val Ile Arg Asp Leu Ala Ala Arg Val Ser Asn Trp Arg Ala Thr Thr 100 105 110 Ser Leu Gln Gln Glu Met Ala Asp Gln Gly Ile Val Gly Ile Gly Gly 115 120 125 Ile Asp Thr Arg Ala Leu Val Arg His Leu Arg Asn Glu Gly Ser Ile 130 135 140 Ala Ala Gly Ile Phe Ser Gly Ala Asp Ala Gln Arg Pro Val Glu Glu 145 150 155 160 Leu Val Glu Ile Val Lys Asn Gln Pro Ala Met Thr Gly Ala Asn Leu 165 170 175 Ser Val Glu Val Ser Ala Asp Glu Thr Tyr Val Ile Glu Ala Glu Gly 180 185 190 Glu Glu Arg His Thr Val Val Ala Tyr Asp Leu Gly Ile Lys Gln Asn 195 200 205 Thr Pro Arg Arg Phe Ser Ala Arg Gly Val Arg Thr Val Ile Val Pro 210 215 220 Ala Glu Thr Pro Leu Glu Asp Ile Lys Gln Tyr Asn Pro Ser Gly Val 225 230 235 240 Phe Ile Ser Asn Gly Pro Gly Asp Pro Ala Ala Ala Asp Val Met Val 245 250 255 Asp Ile Val Arg Glu Val Leu Glu Ala Asp Ile Pro Phe Phe Gly Ile 260 265 270 Cys Phe Gly Asn Gln Ile Leu Gly Arg Ala Phe Gly Met Glu Thr Tyr 275 280 285 Lys Leu Lys Phe Gly His Arg Gly Ile Asn Val Pro Val Lys Asn His 290 295 300 Ile Thr Gly Lys Ile Asp Ile Thr Ala Gln Asn His Gly Phe Ala Leu 305 310 315 320 Lys Gly Glu Ala Gly Gln Glu Phe Glu Thr Asp Phe Gly Thr Ala Ile 325 330 335 Val Thr His Thr Cys Leu Asn Asp Gly Val Val Glu Gly Val Ala Leu 340 345 350 Lys Ser Gly Arg Ala Tyr Ser Val Gln Tyr His Pro Glu Ala Ala Ala 355 360 365 Gly Pro Asn Asp Ala Ser Pro Leu Phe Asp Gln Phe Val Glu Leu Met 370 375 380 Asp Ala Asp Ala Gln Lys Lys Gly Ala 385 390

<210> 5 <211> 1018 <212> PRT <213> Brevibacterium lactofermentum <400> 5 Val Ala Arg Leu His Leu Thr Gln Leu Ser Ser Trp Ile Ala Phe Gly 1 5 10 15 Ile Leu Glu Lys Tyr Gly Val Glu Leu Ile Gly Ala Asp Ile Asp Ala 20 25 30 Ile Glu Arg Gly Glu Asp Arg Gln Lys Phe Lys Asp Ile Val Thr Thr 35 40 45 Ile Gly Gly Glu Ser Ala Arg Ser Arg Val Cys His Asn Met Asp Glu 50 55 60 Val His Glu Thr Val Ala Glu Leu Gly Leu Pro Val Val Val Arg Pro 65 70 75 80 Ser Phe Thr Met Gly Gly Leu Gly Ser Gly Leu Ala Tyr Asn Thr Glu 85 90 95 Asp Leu Glu Arg Ile Ala Gly Gly Gly Leu Ala Ala Ser Pro Glu Ala 100 105 110 Asn Val Leu Ile Glu Glu Ser Ile Leu Gly Trp Lys Glu Phe Glu Leu 115 120 125 Glu Leu Met Arg Asp Thr Ala Asp Asn Val Val Val Ile Cys Ser Ile 130 135 140 Glu Asn Val Asp Ala Leu Gly Val His Thr Gly Asp Ser Val Thr Val 145 150 155 160 Ala Pro Ala Leu Thr Leu Thr Asp Arg Glu Phe Gln Lys Met Arg Asp 165 170 175 Gln Gly Ile Ala Ile Ile Arg Glu Val Gly Val Asp Thr Gly Gly Cys 180 185 190 Asn Il e Gln Phe Ala Ile Asn Pro Val Asp Gly Arg Ile Ile Thr Ile 195 200 205 Glu Met Asn Pro Arg Val Ser Arg Ser Ser Ala Leu Ala Ser Lys Ala 210 215 220 Thr Gly Phe Pro Ile Ala Lys Met Ala Ala Lys Leu Ala Ile Gly Tyr 225 230 235 240 Thr Leu Asp Glu Ile Thr Asn Asp Ile Thr Gly Glu Thr Pro Ala Ala 245 250 255 Phe Glu Pro Thr Ile Asp Tyr Val Val Val Lys Ala Pro Arg Phe Ala 260 265 270 Phe Glu Lys Phe Val Gly Ala Asp Asp Thr Leu Thr Thr Thr Met Lys 275 280 285 Ser Val Gly Glu Val Met Ser Leu Gly Arg Asn Tyr Ile Ala Ala Leu 290 295 300 Asn Lys Ala Leu Arg Ser Leu Glu Thr Lys Gln Gln Gly Phe Trp Thr 305 310 315 320 Lys Pro Asp Glu Phe Phe Ala Gly Glu Arg Ala Thr Asp Lys Ala Ala 325 330 335 Val Leu Glu Asp Leu Lys Arg Pro Thr Glu Gly Arg Leu Tyr Asp Val 340 345 350 Glu Leu Ala Met Arg Leu Gly Ala Ser Val Glu Glu Leu Tyr Glu Ala 355 360 365 Ser Ser Ile Asp Pro Trp Phe Leu Ala Glu Leu Glu Ala Leu Val Gln 370 375 380 Phe Arg Gln Lys Leu Val Asp Ala Pro Phe Leu Asn Glu Asp Leu Leu 385 390 395 395 400 Arg Gl u Ala Lys Phe Met Gly Leu Ser Asp Leu Gln Ile Ala Ala Leu 405 410 415 Arg Pro Glu Phe Ala Gly Glu Asp Gly Val Arg Thr Leu Arg Leu Ser 420 425 430 Leu Gly Ile Arg Pro Val Phe Lys Thr Val Asp Thr Cys Ala Ala Glu 435 440 445 Phe Glu Ala Lys Thr Pro Tyr His Tyr Ser Ala Tyr Glu Leu Asp Pro 450 455 460 Ala Ala Glu Ser Glu Val Ala Pro Gln Thr Glu Arg Glu Lys Val Leu 465 470 470 475 480 Ile Leu Gly Ser Gly Pro Asn Arg Ile Gly Gln Gly Ile Glu Phe Asp 485 490 495 Tyr Ser Cys Val His Ala Ala Leu Glu Leu Ser Arg Val Gly Tyr Glu 500 505 510 Thr Val Met Val Asn Cys Asn Pro Glu Thr Val Ser Thr Asp Tyr Asp 515 520 525 Thr Ala Asp Arg Leu Tyr Phe Glu Pro Leu Thr Phe Glu Asp Val Met 530 535 540 Glu Val Tyr His Ala Glu Ala Gln Ser Gly Thr Val Val Ala Gly Val Ile 545 550 555 560 Val Gln Leu Gly Gly Gln Thr Pro Leu Gly Leu Ala Asp Arg Leu Lys 565 570 575 Lys Ala Gly Val Pro Val Ile Gly Thr Ser Pro Glu Ala Ile Asp Met 580 585 590 Ala Glu Asp Arg Gly Glu Phe Gly Ala Leu Leu Asn Arg Glu Gln Leu 595 600 605 Pro Ala Pr o Ala Phe Gly Thr Ala Thr Ser Phe Glu Glu Ala Arg Thr 610 615 620 Val Ala Asp Glu Ile Ser Tyr Pro Val Leu Val Arg Pro Ser Tyr Val 625 630 635 640 Leu Gly Gly Arg Gly Met Glu Ile Val Tyr Asp Glu Ala Ser Leu Glu 645 650 655 Asp Tyr Ile Asn Arg Ala Thr Glu Leu Ser Ser Asp His Pro Val Leu 660 665 670 Val Asp Arg Phe Leu Asp Asn Ala Ile Glu Ile Asp Val Asp Ala Leu 675 680 685 Cys Asp Gly Asp Glu Val Tyr Leu Ala Gly Val Met Glu His Ile Glu 690 695 700 Glu Ala Gly Ile His Ser Gly Asp Ser Ala Cys Ala Leu Pro Pro Met 705 710 710 720 Thr Leu Gly Ala Gln Asp Ile Glu Lys Val Arg Glu Ala Thr Lys Lys 725 730 735 Leu Ala Leu Gly Ile Gly Val Gln Gly Leu Met Asn Val Gln Tyr Ala 740 745 750 Leu Lys Asp Asp Ile Leu Tyr Val Ile Glu Ala Asn Pro Arg Ala Ser 755 760 760 765 Arg Thr Val Pro Phe Val Ser Lys Ala Thr Gly Val Asn Leu Ala Lys 770 775 780 Ala Ala Ser Arg Ile Ala Val Gly Ala Thr Ile Lys Asp Leu Gln Asp 785 790 795 800 Glu Gly Met Ile Pro Thr Glu Tyr Asp Gly Gly Ser Leu Pro Leu Asp 805 810 815 815 Ala Pro Il e Ala Val Lys Glu Ala Val Leu Pro Phe Asn Arg Phe Arg 820 825 830 Arg Pro Asp Gly Lys Thr Leu Asp Thr Leu Leu Ser Pro Glu Met Lys 835 840 845 Ser Thr Gly Glu Val Met Gly Leu Ala Asn Asn Phe Gly Ala Ala Tyr 850 855 860 Ala Lys Ala Glu Ala Gly Ala Phe Gly Ala Leu Pro Thr Glu Gly Thr 865 870 875 880 Val Phe Val Thr Val Ala Asn Arg Asp Lys Arg Thr Leu Ile Leu Pro 885 890 895 895 Ile Gln Arg Leu Ala Ser Met Gly Tyr Lys Ile Leu Ala Thr Glu Gly 900 905 910 Thr Ala Gly Met Leu Arg Arg Asn Gly Ile Asp Cys Glu Val Val Leu 915 920 925 Lys Ala Ser Asp Ile Arg Glu Gly Val Glu Gly Lys Ser Ile Val Asp 930 935 940 Arg Ile Arg Glu Gly Glu Val Asp Leu Ile Leu Asn Thr Pro Ala Gly 945 950 955 960 Ser Ala Gly Ala Arg His Asp Gly Tyr Asp Ile Arg Ala Ala Ala Val 965 970 975 Thr Thr Val Gly Val Pro Leu Ile Thr Thr Val Gln Gly Val Thr Ala Ala 980 985 990 Val Gln Gly Ile Glu Ala Leu Arg Glu Gly Val Val Ser Val Arg Ala 995 1000 1005 Leu Gln Glu Leu Asp His Ala Val Lys Ala 1010 1015

<210> 6 <211> 278 <212> PRT <213> Brevibacterium lactofermentum <400> 6 Met Thr Phe Gly Glu Lys Leu Leu Asn Ala Ala Ser Thr Arg Gly Arg 1 5 10 15 Leu Cys Val Gly Ile Asp Pro His Glu Ser Leu Leu Thr Ser Trp Gly 20 25 30 Leu Pro Val Asn Val Asp Gly Leu Ala Glu Phe Ser Arg Ala Cys Val 35 40 45 Glu Ala Phe Ala Asp Thr Val Ala Leu Val Lys Pro Gln Val Ala Phe 50 55 60 Tyr Glu Arg Phe Gly Ser Ala Gly Phe Ala Ile Leu Glu Glu Thr Ile 65 70 75 80 Gln Thr Leu Arg Glu Arg Gly Cys Leu Val Val Ser Asp Ala Lys Arg 85 90 95 Gly Asp Ile Gly Ser Thr Met Ala Gly Tyr Ala Ser Ala Trp Leu Asp 100 105 110 Pro Ala Ser Pro Leu Ser Ser Asp Ala Val Thr Val Ser Pro Tyr Leu 115 120 125 Gly Phe His Ser Leu Asp Pro Val Phe Glu Leu Ala Glu Gln His Gly 130 135 140 Arg Gly Val Phe Val Leu Ala Ala Thr Ser Asn Pro Glu Ala Arg Glu 145 150 155 160 Leu Gln Asp Gln Gln Asn Ala Asp Gly Val Ser Ile Ser Gln Gln Ile 165 170 175 Val Asp Gln Ala Ala Ala Leu Asn Ala Pro Tyr Met Ala Gln Gly Lys 180 185 190 Ala Gly Asn Ile Gly Val Val Ile Gly Ala Thr Leu Ser Lys Pro Pro 195 200 205 Arg Leu Ser Thr Leu Gly Gly Ala Ile Leu Met Pro Gly Val Gly Ala 210 215 220 Gln Gly Gly Thr Ala Ser Asp Val Asp Glu Ile Ala Gly Asp Met Ala 225 230 235 240 His Leu Ala Phe Pro Asn Val Ser Arg Ser Ile Leu Ala Thr Gly Pro 245 250 255 Asp Ile Ala Glu Met Lys Asn Ser Val Ala Lys Thr Ala Ala Asp Phe 260 265 270 Pro Gly Phe Pro Arg Ser 275

Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat II (reference) C12N 9/18 C12N 9/18 9/54 9/54 C12P 21/02 C12P 21/02 C // (C12N 15/09 ZNA C12R 1:13) (C12N 1/21 C12R 1:13) (C12N 9/00 C12R 1:13) (C12P 21/02 C12R 1:13) (72) Inventor Wataru Nakamatsu Suzuki, Kawasaki-ku, Kawasaki-shi, Kawasaki, Kanagawa Prefecture 1-1 Ajinomoto Town Fermentation Research Institute Co., Ltd. (72) Inventor Osamu Kurahashi 1-1 Suzukicho, Kawasaki-ku, Kawasaki City, Kanagawa Prefecture Ajinomoto Co., Ltd. Fermentation Research Laboratories F-term (reference) 4B024 AA03 BA07 BA10 BA11 DA05 EA04 GA11 HA01 4B050 CC03 DD02 LL05 4B064 AG01 CA02 CA19 CC24 DA16 4B065 AA22X AA22Y AA26X AB01 BA02 CA27 CA29 CA31 4H045 AA10 BA10 CA11 DA89 EA01

Claims (13)

[Claims] 1. A DNA encoding a protein represented by the following (A) or (B): (A) a protein having the amino acid sequence of SEQ ID NO: 2 in the sequence listing; (B) the amino acid sequence represented by SEQ ID NO: 2 in the sequence listing, comprising an amino acid sequence containing substitution, deletion, insertion, addition, or inversion of one or several amino acid residues, and aspartate carbamoyltransferase activity A protein having 2. The DNA according to claim 1, which is a DNA shown in (a) or (b) below. (A) DNA comprising a base sequence consisting of base numbers 11 to 904 in the base sequence described in SEQ ID NO: 1 in the sequence listing. (B) encodes a protein that hybridizes under stringent conditions to the base sequence consisting of base numbers 11 to 904 among the base sequences described in SEQ ID NO: 1 of the sequence listing, and that has an aspartate carbamoyltransferase activity. DNA. 3. A DNA encoding a protein represented by the following (C) or (D): (C) a protein having the amino acid sequence of SEQ ID NO: 3 in the sequence listing; (D) the amino acid sequence represented by SEQ ID NO: 3 in the sequence listing, comprising an amino acid sequence containing substitution, deletion, insertion, addition, or inversion of one or several amino acid residues, and a dihydroorotase activity A protein having 4. The DNA according to claim 3, which is a DNA shown in (c) or (d) below. (C) DN containing a base sequence consisting of base numbers 960 to 2150 among the base sequences described in SEQ ID NO: 1 in the sequence listing
A. (D) a DNA that hybridizes with a base sequence consisting of base numbers 960 to 2150 of the base sequence shown in SEQ ID NO: 1 in the sequence listing under stringent conditions and encodes a protein having dihydroorotase activity . 5. A DNA encoding a protein represented by the following (E) or (F): (E) a protein having the amino acid sequence of SEQ ID NO: 6 in the sequence listing, (F) an amino acid sequence represented by SEQ ID NO: 6 in the sequence listing, comprising an amino acid sequence containing substitution, deletion, insertion, addition, or inversion of one or several amino acid residues, and orotidylate decarboxylase; A protein having activity. 6. The DNA according to claim 5, which is a DNA shown in (e) or (f) below. (E) DN containing a base sequence consisting of base numbers 6969 to 7802 in the base sequence described in SEQ ID NO: 1 in the sequence listing
A. (F) encodes a protein that hybridizes with a base sequence consisting of base numbers 6969 to 7802 of the base sequence described in SEQ ID NO: 1 under stringent conditions and has orotidylate decarboxylase activity. DNA. 7. A DNA comprising all of the DNAs according to claim 1, 3 and 5. 8. A DNA encoding a small subunit of carbamoyl phosphate synthetase having the amino acid sequence shown in SEQ ID NO: 4 of the sequence listing, and a large subunit of carbamoyl phosphate synthetase having the amino acid sequence shown in SEQ ID NO: 5. 7. A DNA comprising DNA encoding
The described DNA. 9. The base number 1 in SEQ ID NO: 1 in the sequence listing
The DNA according to claim 8, which has a base sequence of 1 to 7802. 10. A coryneform bacterium transformed with the DNA fragment according to any one of claims 1 to 9. 11. A protein represented by the following (A) or (B): (A) a protein having the amino acid sequence of SEQ ID NO: 2 in the sequence listing; (B) the amino acid sequence represented by SEQ ID NO: 2 in the sequence listing, comprising an amino acid sequence containing substitution, deletion, insertion, addition, or inversion of one or several amino acid residues, and aspartate carbamoyltransferase activity A protein having 12. A protein represented by the following (C) or (D): (C) a protein having the amino acid sequence of SEQ ID NO: 3 in the sequence listing; (D) the amino acid sequence represented by SEQ ID NO: 3 in the sequence listing, comprising an amino acid sequence containing substitution, deletion, insertion, addition, or inversion of one or several amino acid residues, and a dihydroorotase activity A protein having 13. A protein represented by the following (E) or (F): (E) a protein having the amino acid sequence of SEQ ID NO: 6 in the sequence listing, (F) an amino acid sequence represented by SEQ ID NO: 6 in the sequence listing, comprising an amino acid sequence containing substitution, deletion, insertion, addition, or inversion of one or several amino acid residues, and orotidylate decarboxylase; A protein having activity.