# Patent

## Title
Method for producing alcohols free of enantiomers

## Abstract

This application is a Continuation-in-part (CIP) of application Ser. No. 09/494,359 filed on Jan. 31, 2000.

## Background
1. Field of the Invention

The present invention relates to carbamoyl-phosphate synthetase of coryneform bacteria, and a gene therefore. The gene can be utilized for production of carbamoyl-phosphate synthetase and subunits thereof, breeding of L-arginine-producing bacteria and nucleic acid-producing bacteria and so forth.

2. Description of the Related Art

Carbamoyl-phosphate synthetase is an enzyme that catalyzes the reactions producing carbamoyl phosphate from carbonic acid, ATP and glutamine. Carbamoyl phosphate produced by these reactions serves as a source of carbamoyl group required for the reaction producing citrulline from ornithine in the L-arginine biosynthetic pathway. Furthermore, carbamoyl aspartate produced from aspartic acid and carbamoyl phosphate is one of the intermnediates of the pyrimidine biosynthesis system including uridine 5'-monophosphate.

Carbamoyl-phosphate synthetase consists of two subunits, and it has been known for bacteria belonging to the genus Escherichia or Bacillus that those subunits are encoded by carA and carB genes.

However, as for coryneform bacteria, there have been no findings about the carbamoyl-phosphate synthetase activity and enzymes therefor, and any genes therefor have not been elucidated.

Incidentally, it has been reported that when a transformant of Escherichia coli to which introduced a plasmid harboring the genes carA, carB, argI and arg box was cultured in the medium added with glutamine which is substrate of carbamoyl-phosphate synthetase, the concentration of intracellular L-arginine was the same as that of a control strain to which only the vector was introduced. However, when the transformant was cultured in a medium added with glutamine accompanied with ornithine which is a substrate of ArgI together with carbamoyl phosphate, the concentration of intracellular L-arginine was higher than that of the control strain (Malamy M. et al., Applied Environmental Microbiology, 63(1), 33 (1997)). From these result, it was suggested that the rate-determining step of synthesis of L-arginine is supply of ornithine.

There was thought to be a possibility that the rate-determining step of supply of ornithine is N-acetylglutamine synthetase (ArgA). ArgA suffers feedback inhibition by the final product, L-arginine, in the biosynthesis pathway of Escherichia coli.

AS for the strain in which argA gene coding for feedback inhibition-desensitized ArgA was amplified by plasmid, the concentration of intracellular L-arginine was increased even in a medium added with only glutamine as well as in a medium added with both glutamine and ornithine. However, farther increase of concentration of intracellular L-arginine was not observed in the case that the strain was cultured with addition of glutamine, or glutamine and ornithin, also in the case that the both of carA and carB genes were further amplified in the strain (Malamy M. et al., Applied Environmental Microbiology, 64(5), 1805 (1998)).

On the other hand, any attempts have not been reported to enhance L-arginine productivity of microorganisms by utilizing a gene coding for carbamoyl-phosphate synthetase derived from coryneform bacterium.

An object of the present invention is to provide carbamoyl-phosphate synthetase of coryneform bacteria, a gene coding for it, and a method for producing L-arginine with a microorganism utilizing the gene.

The inventors of the present invention eagerly studied in order to achieve the aforementioned object. As a result, the inventors successfully obtained a DNA fragment containing the carA gene and the carB gene from a wild strain of Brevibacterium lactofermentum by utilizing a carB-deficient strain of Escherichia coli, and thus accomplished the present invention.

That is, the present invention provides the followings. or (B): amino acid numbers 50 to 393 of the amino acid sequence of SEQ ID NO: 2, amino acid numbers 50 to 393 of the amino acid sequence of SED ID NO: 2 including substitution, deletion, insertion, addition, or inversion of one or several amino acids, and can constitute a protein having a carbamoyl-phosphate synthetase activity with a large subunit of carbamoyl-phosphate synthetase having an amino acid sequence comprising at least the amino acid numbers 55 to 1113 of SEQ ID NO: 3. or (D): amino acid numbers 55 to 1113 of SEQ ID NO: 3, amino acid numbers 55 to 1113 of SEQ ID NO: 3 including substitution, deletion, insertion, addition, or inversion of one or several amino acids, and can constitute a protein having a carbamoyl-phosphate synthetase activity with a small subunit of carbamoyl-phosphate synthetase having an amino acid sequence comprises at least the amino acid numbers 50 to 393 of the amino acid sequence of SEQ ID No: 2. comprising at least the amino acid numbers 55 to 1113 of SEQ ID NO: 3 including substitution, deletion, insertion, addition, or inversion of one or several amino acids, and can constitute a protein having a carbamoyl-phosphate synthetase activity. or (b), and a polypeptide defined in the following (c) or (d): amino acid numbers 50 to 393 in SEQ ID NO: 2, amino acid numbers 50 to 393 in SEQ ID NO: 2 including substitution, deletion, insertion, addition, or inversion of one or several amino acids, and can constitute a protein having a carbamoyl-phosphate synthetase activity with a large subunit of carbamoyl-phosphate synthetase having an amino acid sequence comprising at least the amino acid numbers 55 to 1113 of SEQ ID NO; 3, amino acid numbers 55 to 1113 of SEQ ID NO. 3, amino acid numbers 55 to 1113 of SEQ ID NO: 3 including substitution, deletion, insertion, addition, or inversion of one or several amino acids, and can constitute a protein having a carbamoyl-phosphate synthetase activity with a small subunit of carbamoyl-phosphate synthetase having an amino acid sequence comprising the amino acid numbers 50 to 393 in SEQ ID NO: 2. comprising at least the nucleotide numbers 430 to 1461 in the nucleotide sequence of SEQ ID NO: 1. comprising at least the nucleotide numbers 1756 to 4808 in the nucleotide sequence of SEQ ID NO: 1. comprising at least the nucleotide numbers 430 to 4808 in the nucleotide sequence of SEQ ID NO: 1. (b), and a polypeptide defined in the following (c) or (d): amino acid numbers 50 to 393 in SEQ ID NO: 2, amino acid numbers 50 to 393 in SEQ ID NO: 2 including substitution, deletion, insertion, addition, or inversion of one or several amino acids, and can constitute a protein having a carbamoyl-phosphate synthetase activity with a large subunit of carbamoyl-phosphate synthetase having an amino acid sequence comprising at least the amino acid numbers 55 to 1113 of SEQ ID NO: 3, amino acid numbers 55 to 1113 of SEQ ID NO: 3, amino acid numbers 5 5 to 1113 of SEQ ID NO: 3 including substitution, deletion, insertion, addition, or inversion of one or several amino acids, and can constitute a protein having a carbamoyl-phosphate synthetase activity with a small subunit of carbamoyl-phosphate synthetase having an amino acid sequence comprising at least the amino acid numbers 50 to 393 in SEQ ID NO: 2. according to any one of (1) to (7). synthetase activity, and has L-arginine productivity. intracellular carbamoyl-phosphate synthetase activity is obtained by increasing copy number of DNA encoding carbamoyl-phosphate synthetase of the microorganism, or by modifying an expression regulation sequence so that expression of the gene encoding carbamoyl-phosphate synthetase in the cell should be enhanced. according to any one of (1) to (7). culturing a coryneform bacterium according to any one of (10) to (13) in a medium to produce and accumulate L-arginine in the medium, and collecting the L-arginine from the medium.

The present invention provides genes coding for the subunits that constitute carbamoyl-phosphate synthetase. The gene can be utilized for production of carbamoyl-phosphate synthetase and subunits thereof, breeding of L-arginine producing bacteria and nucleic acid-producing bacteria and so forth. Additionally, L-arginine can be produced efficiently according to the present invention.

FIG. 1 shows the structure of plasmid p19 containing the carA gene and carB gene.

FIG. 2 shows a construction process of plasmid pK1.

FIG. 3 shows a construction process of plasmid pSFK6.

Hereafter, the present invention will be explained in detail.

<1> DNA of the Present Invention

The DNA of the present invention can be obtained from a chromosome DNA library of coryneform bacteria prepared with vectors such as plasmids by selection of the DNA using a microorganism which is deficient in carA or carB, for example, Escherichia coli RC50 (carA50, tsx-273, .lambda.-, rpsL135 (str.sup.R), malT1 (.lambda.R), xylA7, thi.sup.- 1; Mol. Gen. Genet. , 133, 299 (1974)), Escherichia coli JEF8 (thr.sup.- 31, .DELTA.carB, relA.sup.-, metBl, Mol. Gen. Genet., 133, 299 (1974)) and so forth. Because a microorganism which is deficient in carA or carB exhibits L-arginine and uracil auxotrophy, a DNA fragment can be obtained by transforming such a microorganism with a chromosome DNA library, selecting clones in which the auxotrophy is complemented, and recovering a recombinant vector from the selected transformants.

The coryneform bacteria used for preparing a chromosome DNA library are not particularly limited, and examples thereof include bacteria having been hitherto classified into the genus Brevibacterium but united into the genus Corynebacterium at present (Ins. J. Syst. Bacteriol., 41, 255 (1981)), and include bacteria belonging to the genus Brevibacterium closely relative to the genus Corynebacterium, more specifically, wild strains of Brevibacterium lactofermentum and so forth. Chromosome DNA of coryneform bacteria can be prepared by, for example, the method of Saito and Miura (Biochem. Biophys. Acta., 72, 619, (1963)), the method of K. S. Kirby (Biochem. J., 64, 405, (1956)) and so forth.

A chromosome DNA library can be obtained by partially digesting chromosome DNA with suitable restriction enzymes, ligating each of the obtained DNA fragments to a vector DNA autonomously replicable in Escherichia coli cells to prepare a recombinant DNA, and introducing the DNA into Escherichia coli. The vector is not particularly limited so long as it is a vector usually used for genetic cloning, and plasmid vectors such as pUC19, pUC18, pUC118, and pUC119, phage vectors such as .lambda. phage DNA and so forth can be used. Further, a vector autonomously replicable in both of Escherichia coli cells and coryneform bacterium cells may also be used. Such a vector can be constructed by ligating a vector for Escherichia coli and pAM330, which is a cryptic plasmid of Brevibacterium lactofermentum (see Japanese Patent Laid-open No. 58-67699).

Specific examples of the vector autonomously replicable within both of Escherichia coli and coryneform bacterium cells include pSAC4 (see the examples mentioned below), pHK4 (see Japanese Patent Laid-open No. 5-7491) and so forth. Escherichia coli HB101 harboring pHK4 was designated as Escherichia coli AJ13136, and it was deposited at the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry (postal code 305-8566, 1-3 Higashi 1-chome, Tsukuba-shi, Ibaraki-ken, Japan) on Aug. 1, 1995, and received an accession number of FERM BP-5186.

The transformation of Escherichia coli cells can be performed by, for example, the method of D. A. Morrison (Methods in Enzymology, 68, 326, 1979), the method of treating recipient cells with calcium chloride so as to increase the permeability of DNA (Mandel, M. and Higa, A., J. Mol. Biol., 53, 159 (1970)) and so forth. As for methods for preparation of chromosome DNA library, preparation of plasmid DNA, and digestion and ligation of DNA, as well as methods for PCR, preparation of oligonucleotides and hybridization mentioned hereinafter, conventional methods well known to those skilled in the art can be used. Such methods are described in Sambrook, J., Fritsch, E. F. and Maniatis, T., "Molecular Cloning, A Laboratory Manual, Second Edition", Cold Spring Harbor Laboratory Press, (1989) and so forth.

A nucleotide sequence of a DNA fragment containing carA and carB obtained as described above is represented as SEQ ID NO: 1 in the Sequence Listing. This sequence contains two open reading frames (ORF, nucleotide numbers 283 to 1461 and nucleotide numbers 1756 to 4808). The upstream ORF is carA, and the downstream ORF is carB. The amino acid sequences encoded by these ORFs are shown in SEQ ID NOS: 2 and 3, respectively. According to the present invention, a peptide encoded by carA is referred to as a small subunit, and a peptide encoded by carB is referred to as a large subunit.

As for the coding region of carA, GTG of the nucleotide numbers 283 to 285 is indicated as the initiation codon in Sequence Listing. However, GTG of the nucleotide numbers 415 to 417 or ATG of the nucleotide numbers 430 to 432 may possibly be the initiation codon. In any case, an active small subunit can be obtained by using a longer open reading frame for the upstream region for the expression of carA. Similarly, as for the coding region of carB, ATG of the nucleotide numbers 1470 to 1472 is indicated as the initiation codon in the Sequence Listing. However, GTG of the nucleotide numbers 1575 to 1577 or ATG of the nucleotide numbers 1632 to 1634 may possibly be the initiation codon. In any case, an active large subunit can be obtained by using a longer open reading frame for the upstream region for the expression of carB.

The amino acid corresponding to the GTG which is a possible initiation codon is indicated as valine for each subunit, but it may be methionine, valine or formylmethionine.

The small subunit of the carbamoyl-phosphate synthetase of the present invention is, for example, a polypeptide having the amino acid sequence of the amino acid numbers 50 to 393 in SEQ ID NO: 2, polypeptide having the amino acid sequence of the amino acid numbers 45 to 393 in SEQ ID NO: 2, polypeptide having the amino acid sequence of the amino acid numbers 1 to 393 in SEQ ID NO: 2 or the like.

The large subunit of the carbamoyl-phosphate synthetase of the present invention is, for example, a polypeptide having the amino acid sequence of the amino acid numbers 55 to 1113 in SEQ ID NO: 3, a polypeptide having the amino acid sequence of the amino acid numbers 36 to 1113 in SEQ ID NO: 3, a polypeptide having the amino acid sequence of the amino acid numbers 1 to 1113 in SEQ ID NO: 3, or the like.

According to the present invention, the DNA coding for the small subunit may be one coding for an amino acid sequence comprising at least the amino acid numbers 50 to 393 in SEQ ID NO: 2 including substitution, deletion, insertion, addition, or inversion of one or several amino acids, or one coding for a polypeptide which can constitute a protein having a carbamoyl-phosphate synthetase activity with the large subunit.

According to the present invention, the DNA coding for the large subunit may be one coding for an amino acid sequence comprising at least the amino acid numbers 55 to 1113 in SEQ ID NO: 3 including substitution, deletion, insertion, addition, or inversion of one or several amino acids, or one coding for a polypeptide which can constitute a protein having a carbamoyl-phosphate synthetase activity with the small subunit. Alternatively, it may be one coding for a protein which has the amino acid sequence comprising at least the amino acid numbers 55 to 1113 in SEQ ID NO: 3 including substitution, deletion, insertion, addition, or inversion of one or several amino acids, and has a carbamoyl-phosphate synthetase activity.

Furthermore, a DNA that encodes carbamoyl-phosphate synthetase containing a mutation or mutations in the small subunit or the large subunit, or both of them, also falls within the scope of the DNA of the present invention.

The term "several amino acids" preferably means 1 to 20 amino acids, more preferably 1 to 10 amino acids.

DNA, which encodes the substantially same peptide as the small subunit or the large subunit as described above, is obtained, for example, by modifying the nucleotide sequence of the DNA encoding the small subunit or the large subunit, for example, by means of the site-directed mutagenesis method so that one or more amino acid residues at a specified site of the gene involve substitution, deletion, insertion, addition, or inversion. DNA modified as described above may be obtained by the conventionally known mutation treatment. The mutation treatment includes a method for treating DNA coding for the small subunit or the large subunit in vitro, for example, with hydroxylamine, and a method for treating a microorganism, for example, a bacterium belonging to the genus Escherichia harboring DNA coding for the small subunit and the large subunit with ultraviolet irradiation or a mutating agent such as N-methyl-N'-nitro-N-nitrosoguanidine (NTG) and nitrous acid usually used for the mutation treatment.

The substitution, deletion, insertion, addition, or inversion of nucleotide as described above also includes mutation (mutant or variant) which naturally occurs, for example, the difference in strains, species or genera of the microorganism having the small subunit and/or the large subunit.

The DNA, which encodes substantially the same protein as carbamoyl-phosphate synthetase, is obtained by expressing DNA having mutation as described above in an appropriate cell, and investigating the carbamoyl-phosphate synthetase activity of an expressed product. The carbamoyl-phosphate synthetase activity can be measured by the known method (Journal of General Microbiology, 136, 1177-1183 (1990)). The DNA, which encodes substantially the same protein as carbamoyl-phosphate synthetase, is also obtained by isolating DNA which is hybridizable with DNA having, for example, a nucleotide sequence corresponding to nucleotide numbers of 283 to 1461 or 1756 to 4808 of the nucleotide sequence of SEQ ID NO: 2, under stringent condition, and which encodes a protein having the carbamoyl-phosphate synthetase activity, from DNA coding for carbamoyl-phosphate synthetase having mutation or from a cell harboring it. The "stringent condition" referred to herein is a condition under which so-called specific hybrid is formed, and non-specific hybrid is not formed. It is difficult to clearly express this condition by using any numerical value. However, for example, the stringent condition includes a condition under which DNA's having high homology, for example, DNA's having homology of not less than 70%, preferably not less than 80%, more preferably not less than 90% are hybridized with each other, and DNA's having homology lower than the above are not hybridized with each other. Alternatively, the stringent condition is exemplified by a condition under which DNA's are hybridized with each other at a salt concentration corresponding to an ordinary condition of washing in Southern hybridization, i.e., 60.degree. C., 1.times.SSC, 0.1% SDS, preferably 0.1.times.SSC, 0.1% SDS.

As a probe, a partial sequence of the nucleotide sequence of SEQ ID NO: 1 can also be used. Such a probe may be prepared by PCR using oligonucleotides produced based on the nucleotide sequence of SEQ ID NO: 1 as primers, and a DNA fragment containing the nucleotide sequence of SEQ ID NO: 1 as a template. When a DNA fragment in a length of about 300 bp is used as the probe, the conditions of washing for the hybridization consist of, for example, 50.degree. C., 2.times.SSC, and 0.1% SDS.

Because the nucleotide sequence of the DNA of the present invention has been elucidated, the DNA of the present invention can be obtained by amplifying it from coryneform bacterial chromosome DNA through polymerase chain reaction (PCR: polymerase chain reaction; see White, T. J. et al., Trends Genet., 5, 185 (1989)) utilizing oligonucleotides prepared based on that nucleotide sequence as primers, or by selecting it from a coryneform bacterial chromosome DNA library by hybridization utilizing an oligonucleotide prepared based on that nucleotide sequence as a probe. As nucleotide sequences of the primers used for PCR, a region upstream from the nucleotide number 283, preferably a region upstream from the nucleotide number 185 of SEQ ID NO: 1 can suitably be selected as the 5' primer, and a region downstream from the nucleotide number 4808 of SEQ ID NO: 1 can suitably be selected as the 3' primer.

Examples of the host for the expression of the DNA of the present invention include various bacteria such as Escherichia coli and coryneform bacteria including Brevibacterium lactofermentum and Brevibacterium flavum, eukaryotic cells such as those of Saccharomyces cerevisiae and so forth. In order to introduce the DNA of the present invention into these hosts, the host cells can be transformed with a recombinant vector obtained by inserting the DNA of the present invention into a vector selected according to the nature of the host in which the DNA is to be expressed. This procedure can be performed by a method well known to those skilled in the art. Specific examples of the method include the methods used for transformation of Escherichia coli mentioned above, the method in which competent cells are prepared from cells at the proliferating stage to introduce DNA, as reported for Bacillus subtilis (Duncan, C. H., Wilson, G. A. and Young, F. E., Gene, 1, 153 (1977)), the method in which DNA recipient cells are allowed to be in a state of protoplasts or spheroplasts capable of incorporating recombinant DNA with ease to introduce recombinant DNA into the DNA recipient cells, as known for Bacillus subtilis, actinomycetes, and yeasts (Chang, S. and Choen, S. N., Molec. Gen. Genet., 168, 111 (1979); Bibb, M. J., Ward, J. M. and Hopwood, O. A., Nature, 274, 398 (1978); Hinnen, A., Hicks, J. B. and Fink, G. R., Proc. Natl. Acad. Sci. USA, 75, 1929 (1978)), the electric pulse method useful for coryneform bacteria (refer to Japanese Patent Publication Laid-Open No. 2-207791) and so forth.

The DNA to be introduced into the host such as those mentioned above may be DNA containing either carA or carB, or DNA containing both of them. Further, in order to attain efficient expression of these genes, a promoter functioning in the host cells such as lac, trp and P.sub.L may be ligated at a position upstream from carA or carB.

Carbamoyl-phosphate synthetase or its subunits can be produced by culturing a transformant such as those mentioned above under a condition that allows the expression of carA or carB. The DNA of the present invention can also be utilized for breeding of L-arginine-producing bacteria or nucleic acid-producing bacteria such as uracil-producing bacteria. That is, a transformant introduced with the DNA of the present invention, in particular, one introduced with either carA or carB or both of them, should have increased carbamoyl-phosphate synthetase activity compared with non-transformants. Consequently, its productivity for L-arginine or nucleic acid such as uracil is improved.

<2> Method for Producing L-arginine According to the Present Invention

L-Arginine can efficiently be produced by culturing a microorganism that has enhanced intracellular carbamoyl-phosphate synthetase activity, and has L-arginine productivity in a medium to produce and accumulate L-arginine in the medium, and collecting the L-arginine from the medium.

Specific examples of the microorganism having L-arginine productivity include coryneform bacteria, bacteria belonging to the genera Bacillus, Serratia and Escherichia, yeast species belonging to the genus Saccharomyces or Candida. Of these, coryneform bacteria are preferred.

Exemplary specific species include Bacillus subtilis as a bacterium belonging to the genus Bacillus, Serratia marcescens as a bacterium belonging to the genus Serratia, Escherichia coli as a bacterium belonging to the genus Escherichia, Saccharomyces cerevisiae as a yeast species belonging to the genus Saccharomyces, Candida tropicalis as a yeast species belonging to the genus Candida and so forth.

Exemplary microorganisms having L-arginine productivity include Bacillus subtilis resistant to 5-azauracil, 6-azauracil, 2-thiouracil, 5-fluorouracil, 5-bromouracil, 5-azacytosine and so forth, Bacillus subtilis is resistant to arginine hydroxamate and 2-thiouracil, Bacillus subtilis resistant to arginine hydroxamate and 6-azauracil (see Japanese Patent Laid. open No. 49-1268191), (see Japanese Patent Laid-open No. 52-114092), methionine, histidine, threonine, praline, isoleucine' lysine, adenine, guanine and uracil (or uracil precursor) (see Japanese Patent Laid-open NO. 52-99289), Publication No. 51-6754), nucleic acid base analogies (Japanese Patent Laid-open No. 58-9692), exhibiting resistance to arginine antagonists and canavanine and auxotorophy for lysine (see Japanese Patent Laid-open No. 52-8729), Laid-open No. 57-5693), homoarginine, D-arginine and canavanine, or resistant to arginine hydroxamate and 6-azauracil (see Japanese Patent Laid-open No. 53-143288), No. 53-3586) and so forth.

Coryneform bacteria include those bacteria having been hitherto classified into the genus Brevibacterium but united into the genus Corynebacterium at present (Ins. J. Syst. Bacteriol., 41, 255 (1981)), and include bacteria belonging to the genus Brevibacterium closely relative to the genus Corynebacterium. Examples of such coryneform bacteria are listed below.

The corynefonm bacteria that have the L-arginine productivity are not particularly limited so long as they have the L-arginine productivity. They include, for example, wild-type strains of coryneforn bacteria; coryneform bacteria resistant to certain agents including sulfa drugs, 2-thiazolealanine, .alpha.-amino-.beta.-hydroxyvaleric acid and the like; coryneform bacteria exhibiting L-histidine, L-proline, L-threonine, Lisoleucine, L-methionine, or L-tryptophan auxotrophy in addition to the resistance to 2-thiazolealanine (Japanese Patent Laid-open No. 54-44096); coryneform bacteria resistant to ketomalonic acid, fluoromalonic acid, of monofluoroacetic acid (Japanese Patent Laid-open No. 57-18989); coryneform bacteria resistant to argininol (Japanese Patent Laid-open No. 62-24075); coryneform bacteria resistant to X-guanidine (X represents a derivative of fatty acid or aliphatic chain, Japanese Patent Laid-open No. 2-186995) and so forth. Specifically, the following bacterial strains can be exemplified.

The AJ11169 strain and the AJ12092 strain are the 2-thiazolealanine resistant strains mentioned in Japanese Patent Laid-open No. 54-44096, the AJ11336 strain is the strain having argininol resistance and sulfadiazine resistance mentioned in Japanese Patent Publication No. 62-24075, the AJ11345 strain is the strain having argininol resistance, 2-thiazolealanine resistance, sulfaguanidine resistance, and exhibiting histidine auxotrophy mentioned in Japanese Patent Publication No. 62-24075, and the AJ12430 strain is the strain having octylguanidine resistance and 2-thiazolealanine resistance mentioned in Japanese Patent Laid-open No. 2-186995.

The intracellular carbamoyl-phosphate synthetase activity of such microorganisms having the L-arginine productivity as mentioned above can be enhanced by, for example, increasing copy number of a gene coding for the carbamoyl-phosphate synthetase in the cells of the aforementioned microorganisms. The enhancement of the carbamoyl-phosphate synthetase activity can also be achieved by, in addition to the aforementioned gene amplification, modifying an expression regulation sequence for the DNA coding for carbamoyl-phosphate synthetase so that expression of the DNA gene coding for carbamoyl-phosphate synthetase should be enhanced. Specifically, an expression regulation sequence such as a promoter for a gene coding for carbamoyl-phosphate synthetase on the chromosomal DNA or a plasmid can be replaced with a stronger one (see Japanese Patent Laid-open No. 1-215280). Strong promoters, which function in cells of coryneform bacteria, include lac promoter, tac promoter, trp promoter, of Escherichia coli (Y. Morinaga, M. Tsuchiya, R. Miwa and K. Sano, J. Biotech., 5, 305-312 (1987)) and the like. In addition, trp promoter of Corynebacterium bacteria is also a preferable promoter (Japanese Patent Laid-open No. 62-195294). By the replacement with these promoters the carbamoyl-phosphate synthetase activity is enhanced. The modification of expression regulation sequence may be combined with the increasing of the copy number of DNA coding for carbamoyl-phosphate synthetase. Further, the intracellular carbamoyl-phosphate synthetase activity can be enhanced by introducing one or more mutations into the enzyme protein of carbamoyl-phosphate synthetase so that the specific activity of the enzyme should be increased.

Examples of the DNA coding for carbamoyl-phosphate synthetase include the aforementioned carA and carB genes of Brevibacterium lactofermentum and one containing both of them.

Examples of the vector for introducing DNA coding for carbamoyl-phosphate synthetase into a microorganism include vectors autonomously replicable in cells of the microorganism. Specifically, the aforementioned vectors autonomously replicable in Escherichia coli cells, and the vectors autonomously replicable in both of Escherichia coli cells and coryneform bacterium cells.

The medium used for culturing a microorganism having enhanced intracellular carbamoyl-phosphate synthetase activity and L-arginine productivity obtained as described above may be a well-known medium conventionally used for the production of amino acids by fermentation. That is, it is a usual medium that contains a carbon source, nitrogen source, inorganic ions, and other organic components as required.

As the carbon source, it is possible to use sugars such as glucose, sucrose, lactose, galactose, fructose and starch hydrolysates; alcohols such as glycerol and sorbitol; or organic acids such as fumaric acid, citric acid and succinic acid and so forth.

As the nitrogen source, it is possible to use inorganic ammonium salts such as ammonium sulfate, ammonium chloride and ammonium phosphate, organic nitrogen such as soybean hydrolysates, ammonia gas, aqueous ammonia and so forth.

The medium preferably contains a suitable amount of required substance such as vitamin B.sub.1 and L-homoserine, yeast extract and so forth as trace amount organic nutrients. Other than those substances, a small amount of potassium phosphate, magnesium sulfate, iron ions, manganese ions and so forth may be added to the medium.

The cultivation is preferably performed under an aerobic condition for 1-7 days. Cultivation temperature is preferably 24-37.degree. C., and pH of the medium during the cultivation is preferably 5-9. Inorganic or organic acidic or alkaline substances, ammonia gas and so forth may be used for adjusting pH. L-Arginine can usually be recovered from the fermentation medium by a combination of known techniques such as ion exchange resin method.

Hereafter, the present invention will be explained more specifically with reference to the following examples.

<1> Preparation of Chromosome DNA of Brevibacterium lactofermentum ATCC13869

Brevibacterium lactofermentum ATCC13869 was inoculated to 100 ml of T-Y culture medium (1% of BactoTrypton Difco), 0.5% of Bacto-Yeast Extract (Difco) 0.5% of NaCl (pH 7.2)), and cultured at a temperature of 31.5.degree. C. for 8 hours to obtain a culture. The culture was centrifuged at 3,000 r.p.m. for 15 minutes to obtain 0.5 g of wet bacterial cells, and chromosome DNA was obtained from the bacterial cells according to the method of Saito and Miura (Biochem. Biophys. Acta., 72, 619 (1963)). Then, 60 .mu.g of the chromosome DNA and 3 units of restriction enzyme Sau3AI were each mixed in 10 mM Tris-HCl buffer (containing 50 mM NaCl, 10 mM MgSO.sub.4 and 1 mM dithiothreitol (pH 7.4)), and allowed to react at a temperature of 37.degree. C. for 30 minutes. The reaction mixture was subjected to phenol extraction and ethanol precipitation in a conventional manner to obtain 50 .mu.g of chromosome DNA fragments of Brevibacterium lactofermentum ATCC13869 digested with Sau3AI.

<2> Preparation of Gene Library of Brevibacterium lactofermentum ATCC13869 Using Plasmid Vector DNA

As a plasmid vector DNA autonomously replicable in both of Escherichia coli cells and coryneform bacterium cells, pSAC4 was used. pSAC4 was prepared as follows. In order to make a vector pHSG399 for Escherichia coli (Takara Shuzo) autonomously replicable in coryneform bacterium cells, a replication origin of the previously obtained plasmid pHM1519 autonomously replicable in coryneform bacterium cells (Miwa, K. et al., Agric. Biol. Chem., 48 (1984) 2901-2903) was introduced into the vector (Japanese Patent Laid-open No. 5-7491). Specifically, pHM1519 was digested with restriction enzymes BamHI and KpnI to obtain a gene fragment containing the replication origin, and the obtained fragment was blunt-ended by using Blunting Lit produced by Takara Shuzo, and inserted into the SalI site of pHSG399 using a SalI linker (produced by Takara Shuzo) to obtain pSAC4.

In 50 mM Tris-HCl buffer (containing 100 mM NaCl and 10 mM magnesium sulfate (pH 7.4)), 20 .mu.g of pSAC4 and 200 units of a restriction enzyme BamHI were mixed, and allowed to react at a temperature of 37.degree. C. for 2 hours to obtain a digestion solution. This solution was subjected to phenol extraction and ethanol precipitation in a conventional manner. Then, in order to inhibit re-ligation of the DNA fragments derived from the plasmid vector, the DNA fragments were dephosphorylated with bacterial alkaline phosphatase according to the method described in Molecular Cloning, 2nd Edition (J. Sambrook, E. F. Fritsch and T. Maniatis, Cold Spring Harbor Laboratory Press, p1.56 (1989)), and subjected to phenol extraction and ethanol precipitation in a conventional manner.

To 66 mM Tris-HCl buffer {pH 7.5) containing 66 mM magnesium chloride, 10 mM dithiothreitol and 10 mM ATP, 1 .mu.g of the pSAC4 digested with BamHI, 1 .mu.g of the chromosome DNA fragments of Brevibacterium lactofermentum ATCC13869 digested with Sau3AI obtained in Example 1, and 2 units of T4 DNA ligase (produced by Takara Shuzo) were added, and allowed to react at a temperature of 16.degree. C. for 16 hours to ligate the DNA. Then, Escherichia coli DH5 was transformed with this DNA mixture in a conventional manner, and plated on an L agar medium containing 170 .mu.g/ml of chloramphenicol to obtain about 20,000 colonies, which were used as a gene library.

<3> Transformation of carB-deficient Strain of Escherichia coli (JEF8)

The carB-deficient strain of Escherichia coli, JEF8 (thr.sup.- 31, .DELTA.carB, relA.sup.-, metBl ; Mol. Gen. Genet., 133, 299 (1974)) was transformed with a recombinant DNA mixture of the aforementioned gene library in a conventional manner. Transformants of about 15000 strains were obtained as Cm resistant strains. These transformants were replicated on a minimum medium (5 g/L of glucose, 12.8 g/L of Na.sub.2 HPO.sub.4, 3 g/L of KH.sub.2 PO.sub.4, 0.5 g/L of NaCl, 1 g/L of NH.sub.4 Cl, 40 .mu.g/ml of L-threonine, 40 .mu.g/ml of L-methionine) not containing arginine and uracil, and the minimum medium not containing L-arginine, but containing only 50 .mu.g/ml of uracil, and screened for a strain in which arginine auxotrophy and uracil auxotrophy were restored, or a strain in which arginine auxotrophy was restored. Strains in which arginine auxotrophy was restored recovered both of arginine auxotrophy and uracil auxotrophy. A plasmid harbored in one of such strains was designated as p19, and the strain harboring it was designated as JEF8/p19. The structure of p19 is shown in FIG. 1.

The Escherichia coli JEF8/p19 was designated as Escherichia coli AJ13574, and it was deposited at the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry (postal code 305-8566, 1-3 Higashi 1-chome, Tsukuba-shi, Ibaraki-ken, Japan) on Jan. 28, 1999, and received an accession number of FERM P-17180, and transferred from the original deposit to international deposit based on Budapest Treaty on Jan. 6, 2000, and has been deposited as deposition number of FERM BP-6989.

<4> Acquisition of Plasmid Complementing Arginine and Uracil Auxotrophy

A plasmid was prepared from JEF8/p19, in a conventional manner, and used for re-transformation of the JEF8 strain. The obtained transformants could grow in the minimum culture medium not containing L-arginine and uracil, and its auxotrophy for both of L-arginine and uracil was restored. Therefore, it was found that the plasmid contained a gene complementing the auxotrophy for both of L-arginine and uracil caused by deletion of carB in the Escherichia coli strain.

Further, this plasmid was introduced into the carA mutant of Escherichia coli, RC50 (carA50, tsx.sup.- 273, .lambda..sup.-, rpsL135 (str.sup.R), malT1 (.lambda.R), xylA7, thi.sup.- 1; Mol. Gen. Genet., 133, 299 (1974)). Since the strain introduced with the plasmid was able to grow in the minimum culture medium not containing arginine and uracil, the plasmid was also found to have a gene complementing the auxotrophy for both of L-arginine and uracil caused by carA mutation of the Escherichia coli strain.

<5> Nucleotide Sequence Analysis of p19

Among the DNA sequence of p19, the nucleotide sequence of about 4.8 kb from the HindIII side of the multi-cloning site of the vector to the HindIII site contained in the insertion DNA fragment was determined. The nucleotide sequencing was performed by using Rohdamin Terminator Cycle Sequencing Kit (produced by ABI) according to the method of singer. The obtained nucleotide sequence is shown as SEQ ID NO: 1 in Sequence Listing. From analysis of a consensus sequence which located in the upstream region of this gene, it was estimated that two open reading frames (open reading frame from 283rd G to 1461st A and open reading frame from 1756th G to 4808th T) were contained in this sequence. The nucleotides of the 162nd (TGCATA) to 194th (TATAAT), the 185th (TGCATA} to 213rd (TAAACT), the 203rd (TTGAAT) 230th (TATCAA), or the 224th (TTATCA to 251st (TAAAAA) can be estimated to be a promoter region for regulating the transcription.

The amino acid sequences encoded by these open reading frames are represented with the nucleotide sequences. The amino acid sequences were also shown in SEQ ID NOS; 2 and 3. A protein database (GenBank CDS) was searched for sequences exhibiting homology with these amino acid sequences. As a result, it was found that the 5' open reading frame showed high homology (about 40%) with carA gene products of Escherichia coli, Bacillus subtillis and so forth, and the 3' open reading frame showed high homology with known carB gene products of Escherichia coli, Bacillus stearothermophilus and so forth (about 40 to 50%). Therefore, it was suggested that these open reading frames coded for carA and carB, respectively.

<6> Introduction of carA and carB into Wild-type Strain of Coryneform Bacteria

p19 was introduced into the Brevibacterimn flavum wild strain 2247 (AJ14067) by the electric pulse method (Japanese Patent Laid-open NO. 2-207791). The transformants were selected as chloramphenicol resistant strains on a CM2G plate medium (containing 10 g of polypeptone, 10 g of yeast extract, 5 g of glucose, 5 g of NaCl, 15 g of agar in 1 L of pure water, pH 7.2) containing 5 .mu.g/ml of chloramphenicol to obtain 2247/p19. carB

<1> Preparation of Shuttle Vector

First, a plasmid vector autonomously replicable in both of Escherichia coli cells and coryneforn bacterium cells was newly produced as a plasmid used for introducing the carA and carB genes into coryneform bacteria.

A vector containing a drug resistance gene of Streptococcus faecalis was constructed first. The kanamycin resistant gene of Streptococcus faecalis was amplified by PCR from a known plasmid containing that gene. The nucleotide sequence of the kanamycin resistant gene of Streptococcus faecalis has already been clarified (Trieu-Cuot, P. and Courvalin, P., Gene, 23(3), 331-341 (1983)). The primers shown as SEQ ID NOS: 4 and 5 were synthesized based on that sequence, and PCR was performed by using pDG783 (Anne-Marie Guerout-Fleury et at., Gene, 167, 335-337 (1995)) as a template to amplify a DNA fragment containing the kanamycin resistant gene and its promoter.

The obtained DNA fragment was purified by SUPRECO2 produced by the Takara Shuzo, then fully digested with restriction enzymes HindIII and HincII, and blunt-ended. The blunt-ending was attained by using Blunting Kit produced by Takara Shuzo. This DNA fragment was mixed with and ligated to a DNA fragment, which had been obtained by performing PCR using the primers shown as SEQ ID NOS: 6 and 7 and pHSG399 (see S. Takeshita et al., Gene, 61, 63-74 (1987)) as a template, purifying and blunt-ending the resulted amplification product. The ligation reaction was performed by DNA Ligation Kit ver. 2 produced by Takara Shuzo. Competent cells of Escherichia coli JM109 (produced by Takara Shuzo) were transformed with the ligated DNA, plated on L medium (10 g/L of Bacto-trypton, 5 g/L of Bacto-yeast extract, 5 g/L of NaCl, 15 g/L of agar, pH 7.2) containing 10 .mu.g/ml of IPTGH (isopropyl-.beta.-D-thiogalactopyranoside), 40 .mu.g/ml of X-Gal (5-bromo-4-chloro-3-indolyl-.beta.-D-galactoside) and 25 .mu.g/ml of kanamycin, and cultured overnight. The emerged blue colonies were picked up, and separated into single colonies to obtain transformant strains.

Plasmids were prepared from the transformant strains by the alkali method (Text for Bioengineering Experiments, Edited by the society for Bioscience and Bioengineering, Japan, p.105, Baifukan, 1992), and restriction maps were prepared. One having a restriction map equivalent to that of FIG. 2 was designated as pK1. This plasmid is stably retained in Escherichia coli, and imparts kanamycin resistance to a host. Moreover, since it contains the lacZ' gene, it is suitably used as a cloning vector.

The plasmid pAM330 extracted from Brevibacterium lactofermentum ATCC13869 (see Japanese Patent Laid-open No. 58-67699) was fully digested with a restriction enzyme HindIII, and blunt-ended. This fragment was ligated to a fragment obtained by fully digesting the aforementioned pK1 with a restriction enzyme BsaAI. Brevibacterium lactofermentum ATCC13869 was transformed with the ligated DNA. The transformation was performed by the electric pulse method (see Japanese Patent Laidopen No. 2-207791). Transformants were selected on a M-CM2B plate (10 g/L of polypeptone, 10 g/L of yeast extract, 5 g/L of NaCl, 10 .mu.g/L of biotin, 15 g/L of agar, pH 7.2) containing 25 .mu.g/ml of kanamycin. After cultivation for 2 days, colonies were picked up, and separated into single colonies to obtain the transfonmants. Plasmid DNA was prepared from the transformants, and restriction maps were prepared. One having the same restriction map as that of FIG. 3 was designated as pSFK6. This plasmid can autonomously replicate in both of Escherichia coli and coryneform bacteria, and imparts kanamycin resistance to a host.

<2> Introduction of carA and carB Genes into Coryneform Bacteria and Production of L-arginine

The aforementioned pSFK6 wan digested with SmaI and HindIII. The product was ligated to carA and carB gene fragments, which had been obtained by digesting the plasmid p19 prepared from JEF8/p19F in a conventional manner with a restriction enzyme XbaI, blunt-ending the product by using Blunting Kit produced by Takara Shuzo, and further digesting the product with a restriction enzyme HindIII, to obtain a plasmid pcarAB, which contained the carA and carB genes and could autonomously replicate in coryneform bacteria.

pcarAB was introduced into Brevibacterium flavam AJ11345 and AJ11336 by the electric pulse method (Japanese Patent Laid-open No. 2-207791). Transformants were selected on a M-CM2B plate (10 g/L of polypeptone, 10 g/L of yeast extract, 5 g/L of glucose, 5 g/L of NaCl, 15 g/L of agar, pH 7.2) containing 25 .mu.g/ml of kanamycin as kanamycin resistant strains. As control, transformants were obtained by similarly introducing pSFK6 into AJ11345 and AJ11336.

Each of the aforementioned transformants was plated on an agar medium containing 0.5 g/dl of glucose, 1 g/dl of polypeptone, 1 g of yeast extract, 0.5 g/dl of NaCl and 5 .mu.g/l of chloramphenicol, and cultured at 31.5.degree. C. for 20 hours. One inoculating loop of the obtained cells were inoculated to a medium containing 4 g/dl of glucose, 6.5 g/dl of ammonium sulfate, 0.1 g/dl of KH.sub.2 PO.sub.4, 0.04 g/dl of MgSO.sub.4, 0.001 g/dl of FeSO.sub.4, 0.01 g/dl of MnSO.sub.4, 5 .mu.g/dl of VB.sub.1, 5 .mu.g/dl of biotin, 45 mg/dl of soybean hydrolysates (as an amount of N), and cultured in a flask at 31.5.degree. C. for 50 hours with shaking. The amounts of L-arginine produced by each strain were shown in Table 1.

The strains introduced with the carA and carB gene showed improved L-arginine productivity compared with the strains introduced only with the vector. Strain/plasmid          L-arginine (g/dl) AJ11345/pSFR6                  1.33 AJ11345/pcarAB                 1.39 AJ11336/pSFK6                  0.71 AJ11336/pcarAB                 0.79

Each reference, patent application or patent publication cited by or referred to in this disclosure is incorporated by reference in its entirety. Any patent document to which this application claims priority is also incorporated by reference in its entirety. Specifically, priority document Japan 11-24149, filed Feb. 1, 1999 is hereby incorporated by reference.

Various modifications and variations of the described products and concept of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed is not intended to be limited to such specific embodiments. Various modifications of the described modes for carrying out the invention which are obvious to those skilled in the biological or chemical arts or related fields are intended to be within the scope of the following claims. SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS:  7 <210> SEQ ID NO 1 <211> LENGTH: 4837 <212> TYPE: DNA <213> ORGANISM: Brevibacterium lactofermentum <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (283)..(1461) <221> NAME/KEY: CDS <222> LOCATION: (1470)..(4808) <400> SEQUENCE: 1 gatccaggaa aaacctggac agcatccggt gcagactttg cgtccaaggc tgaaaacacc     60 ccatttgagg gccaggaatt cagcgctaag gtcacacaca ccgtgcttcg tggcaaggtg    120 acttgtgcag acggagttgc gcaagacgct taacgggtgg gtgcatagta tgcacgcgcc    180 gcattgcata taatgcaatg aattgaataa actacattca gggttatcaa ccagccaatt    240 tcttttaaaa agacagacac acgaaaggcg acaacagtca cc gtg agt aaa gac       294 Val Ser Lys Asp 1 acc acc acc tac cag gga gtc acc gag atc gga tcc gtt ccg gca tac      342 Thr Thr Thr Tyr Gln Gly Val Thr Glu Ile Gly Ser Val Pro Ala Tyr 5                  10                  15                  20 ctg gtt ctt gca gac gga cgt acc ttc acc gga ttt ggc ttt gga gct      390 Leu Val Leu Ala Asp Gly Arg Thr Phe Thr Gly Phe Gly Phe Gly Ala 25                  30                  35 atc ggc acc acc ctt ggt gag gca gtg ttc acc acc gcc atg acc ggt      438 Ile Gly Thr Thr Leu Gly Glu Ala Val Phe Thr Thr Ala Met Thr Gly 40                  45                  50 tac caa gaa acc atg acc gat cct tcc tat cac cgc cag att gtt gtg      486 Tyr Gln Glu Thr Met Thr Asp Pro Ser Tyr His Arg Gln Ile Val Val 55                  60                  65 gct acc gca cca cag atc ggt aac acc ggc tgg aac gat gag gac aac      534 Ala Thr Ala Pro Gln Ile Gly Asn Thr Gly Trp Asn Asp Glu Asp Asn 70                  75                  80 gag tcc cgc gac ggc aag att tgg gtt gca ggc ctt gtt atc cgc gac      582 Glu Ser Arg Asp Gly Lys Ile Trp Val Ala Gly Leu Val Ile Arg Asp 85                  90                  95                 100 ctc gca gca cgt gtg tcc aac tgg cgc gcc acc acc tcc ttg cag cag      630 Leu Ala Ala Arg Val Ser Asn Trp Arg Ala Thr Thr Ser Leu Gln Gln 105                 110                 115 gaa atg gca gac caa ggc atc gtc ggc atc ggc gga atc gac acc cgc      678 Glu Met Ala Asp Gln Gly Ile Val Gly Ile Gly Gly Ile Asp Thr Arg 120                 125                 130 gca ctg gtt cgc cac ctg cgc aac gaa ggt tcc atc gca gcg ggc atc      726 Ala Leu Val Arg His Leu Arg Asn Glu Gly Ser Ile Ala Ala Gly Ile 135                 140                 145 ttc tcc ggc gct gac gca cag cgc cca gtt gaa gaa ctc gta gag atc      774 Phe Ser Gly Ala Asp Ala Gln Arg Pro Val Glu Glu Leu Val Glu Ile 150                 155                 160 gtc aag aat cag cca gca atg acc ggc gca aac ctc tcc gtt gag gtc      822 Val Lys Asn Gln Pro Ala Met Thr Gly Ala Asn Leu Ser Val Glu Val 165                 170                 175                 180 tct gct gat gaa acc tac gtc atc gaa gct gag ggc gaa gag cgc cac      870 Ser Ala Asp Glu Thr Tyr Val Ile Glu Ala Glu Gly Glu Glu Arg His 185                 190                 195 acc gtc gtg gcc tac gac ctg ggc att aag caa aac acc cca cgt cgt      918 Thr Val Val Ala Tyr Asp Leu Gly Ile Lys Gln Asn Thr Pro Arg Arg 200                 205                 210 ttc tct gca cgc ggt gtt cgc acc gtc atc gtg cct gct gaa acc cca      966 Phe Ser Ala Arg Gly Val Arg Thr Val Ile Val Pro Ala Glu Thr Pro 215                 220                 225 ttg gag gac atc aag cag tac aac cca tca ggc gtg ttt atc tcc aat     1014 Leu Glu Asp Ile Lys Gln Tyr Asn Pro Ser Gly Val Phe Ile Ser Asn 230                 235                 240 ggc cct ggc gac cct gca gca gca gac gtc atg gtt gat atc gtc cgc     1062 Gly Pro Gly Asp Pro Ala Ala Ala Asp Val Met Val Asp Ile Val Arg 245                 250                 255                 260 gaa gtt ctg gaa gcc gac att cca ttc ttt ggc atc tgc ttc ggc aac     1110 Glu Val Leu Glu Ala Asp Ile Pro Phe Phe Gly Ile Cys Phe Gly Asn 265                 270                 275 cag atc ctc ggc cgc gca ttc ggc atg gag acc tac aag ctg aag ttc     1158 Gln Ile Leu Gly Arg Ala Phe Gly Met Glu Thr Tyr Lys Leu Lys Phe 280                 285                 290 ggc cac cgc ggc atc aac gtt cca gtg aag aac cac atc acc ggc aag     1206 Gly His Arg Gly Ile Asn Val Pro Val Lys Asn His Ile Thr Gly Lys 295                 300                 305 atc gac atc acc gcc cag aac cac ggc ttc gca ctc aag ggt gaa gca     1254 Ile Asp Ile Thr Ala Gln Asn His Gly Phe Ala Leu Lys Gly Glu Ala 310                 315                 320 ggc cag gaa ttc gag aca gat ttc ggc act gcg att gtc acc cac acc     1302 Gly Gln Glu Phe Glu Thr Asp Phe Gly Thr Ala Ile Val Thr His Thr 325                 330                 335                 340 tgc ctt aac gac ggc gtc gtt gaa ggt gtt gcg ctg aag tcc gga cgc     1350 Cys Leu Asn Asp Gly Val Val Glu Gly Val Ala Leu Lys Ser Gly Arg 345                 350                 355 gca tac tcc gtt cag tac cac cca gag gcc gct gcc ggc cca aat gat     1398 Ala Tyr Ser Val Gln Tyr His Pro Glu Ala Ala Ala Gly Pro Asn Asp 360                 365                 370 gca agc ccc ctg ttt gac cag ttt gtt gag ctg atg gat gca gac gct     1446 Ala Ser Pro Leu Phe Asp Gln Phe Val Glu Leu Met Asp Ala Asp Ala 375                 380                 385 cag aag aaa ggc gca taaataac atg cca aag cgt tca gat att aac cac    1496 Gln Lys Lys Gly Ala          Met Pro Lys Arg Ser Asp Ile Asn His 390                        1               5 gtc ctc gtc atc ggt tcc ggc ccc atc gtc att ggc cag gca tgt gaa     1544 Val Leu Val Ile Gly Ser Gly Pro Ile Val Ile Gly Gln Ala Cys Glu 10                  15                  20                  25 ttc gac tac tcc ggc acc cag gct tgc cgc gtg ctg aag gaa gag gga     1592 Phe Asp Tyr Ser Gly Thr Gln Ala Cys Arg Val Leu Lys Glu Glu Gly 30                  35                  40 ctg cgc gtc acc ctc atc aac tcc aac cca gca acg atc atg acc gac     1640 Leu Arg Val Thr Leu Ile Asn Ser Asn Pro Ala Thr Ile Met Thr Asp 45                  50                  55 cca gaa atg gct gac cac acc tac gtg gag cca atc gag ccg gaa tac     1688 Pro Glu Met Ala Asp His Thr Tyr Val Glu Pro Ile Glu Pro Glu Tyr 60                  65                  70 atc gac aag att ttc gct aag gag atc gag cag ggc cac cca atc gac     1736 Ile Asp Lys Ile Phe Ala Lys Glu Ile Glu Gln Gly His Pro Ile Asp 75                  80                  85 gcc gtc ctg gca acc ctt ggt ggc cag act gca ctt aac gca gct atc     1784 Ala Val Leu Ala Thr Leu Gly Gly Gln Thr Ala Leu Asn Ala Ala Ile 90                  95                 100                 105 cag ctg gat cgc ctc ggc atc ctg gaa aag tac ggc gtt gaa ctc atc     1832 Gln Leu Asp Arg Leu Gly Ile Leu Glu Lys Tyr Gly Val Glu Leu Ile 110                 115                 120 ggt gca gac atc gat gcc att gag cgc ggc gaa gat cgc cag aag ttc     1880 Gly Ala Asp Ile Asp Ala Ile Glu Arg Gly Glu Asp Arg Gln Lys Phe 125                 130                 135 aag gat att gtc acc acc atc ggt ggc gaa tcc gcg cgt tcc cgc gtc     1928 Lys Asp Ile Val Thr Thr Ile Gly Gly Glu Ser Ala Arg Ser Arg Val 140                 145                 150 tgc cac aac atg gac gaa gtc cat gag act gtc gca gaa ctt ggc ctt     1976 Cys His Asn Met Asp Glu Val His Glu Thr Val Ala Glu Leu Gly Leu 155                 160                 165 cca gta gtc gtg cgt cca tcc ttc act atg ggt ggc ctg ggc tcc ggt     2024 Pro Val Val Val Arg Pro Ser Phe Thr Met Gly Gly Leu Gly Ser Gly 170                 175                 180                 185 ctt gca tac aac acc gaa gac ctt gag cgc atc gca ggt ggc gga ctt     2072 Leu Ala Tyr Asn Thr Glu Asp Leu Glu Arg Ile Ala Gly Gly Gly Leu 190                 195                 200 gct gca tct cct gaa gca aac gtc ttg atc gaa gaa tcc atc ctt ggt     2120 Ala Ala Ser Pro Glu Ala Asn Val Leu Ile Glu Glu Ser Ile Leu Gly 205                 210                 215 tgg aag gaa ttc gag ctc gag ctc atg cgc gat acc gca gac aac gtt     2168 Trp Lys Glu Phe Glu Leu Glu Leu Met Arg Asp Thr Ala Asp Asn Val 220                 225                 230 gtg gtt atc tgc tcc att gaa aac gtc gac gca ctg ggc gtg cac acc     2216 Val Val Ile Cys Ser Ile Glu Asn Val Asp Ala Leu Gly Val His Thr 235                 240                 245 ggc gac tct gtc acc gtg gca cct gcc ctg acc ctg act gac cgt gaa     2264 Gly Asp Ser Val Thr Val Ala Pro Ala Leu Thr Leu Thr Asp Arg Glu 250                 255                 260                 265 ttc cag aag atg cgc gat cag ggt atc gcc atc atc cgc gag gtc ggc     2312 Phe Gln Lys Met Arg Asp Gln Gly Ile Ala Ile Ile Arg Glu Val Gly 270                 275                 280 gtg gac acc ggt gga tgt aac atc cag ttc gct atc aac cca gtt gat     2360 Val Asp Thr Gly Gly Cys Asn Ile Gln Phe Ala Ile Asn Pro Val Asp 285                 290                 295 ggc cgc atc atc acc att gag atg aac cca cgt gtg tct cgt tcc tcc     2408 Gly Arg Ile Ile Thr Ile Glu Met Asn Pro Arg Val Ser Arg Ser Ser 300                 305                 310 gcg ctg gca tcc aag gca acg ggc ttc cca att gcc aag atg gct gcc     2456 Ala Leu Ala Ser Lys Ala Thr Gly Phe Pro Ile Ala Lys Met Ala Ala 315                 320                 325 aag ctg gct atc gga tac acc ctg gat gag atc acc aac gac atc act     2504 Lys Leu Ala Ile Gly Tyr Thr Leu Asp Glu Ile Thr Asn Asp Ile Thr 330                 335                 340                 345 ggt gaa acc cca gct gcg ttt gag ccc acc atc gac tac gtc gtg gtc     2552 Gly Glu Thr Pro Ala Ala Phe Glu Pro Thr Ile Asp Tyr Val Val Val 350                 355                 360 aag gcc cca cgc ttt gct ttc gag aag ttt gtc ggc gct gat gac act     2600 Lys Ala Pro Arg Phe Ala Phe Glu Lys Phe Val Gly Ala Asp Asp Thr 365                 370                 375 ttg acc acc acc atg aag tcc gtc ggt gag gtc atg tcc ctg ggc cgt     2648 Leu Thr Thr Thr Met Lys Ser Val Gly Glu Val Met Ser Leu Gly Arg 380                 385                 390 aac tac att gca gca ctg aac aag gca ctg cgt tcc ctg gaa acc aag     2696 Asn Tyr Ile Ala Ala Leu Asn Lys Ala Leu Arg Ser Leu Glu Thr Lys 395                 400                 405 cag cag ggt ttc tgg acc aag cct gat gag ttc ttc gca ggg gag cgc     2744 Gln Gln Gly Phe Trp Thr Lys Pro Asp Glu Phe Phe Ala Gly Glu Arg 410                 415                 420                 425 gct acc gat aag gca gct gtt ctg gaa gat ctc aag cgc cca acc gaa     2792 Ala Thr Asp Lys Ala Ala Val Leu Glu Asp Leu Lys Arg Pro Thr Glu 430                 435                 440 ggc cgc ctc tac gac gtt gag ctg gca atg cgc ctt ggc gca agc gtg     2840 Gly Arg Leu Tyr Asp Val Glu Leu Ala Met Arg Leu Gly Ala Ser Val 445                 450                 455 gaa gaa ctc tac gaa gca tct tct att gat cct tgg ttc ctc gcc gag     2888 Glu Glu Leu Tyr Glu Ala Ser Ser Ile Asp Pro Trp Phe Leu Ala Glu 460                 465                 470 ctt gaa gct ctc gtg cag ttc cgc cag aag ctc gtt gac gca cca ttc     2936 Leu Glu Ala Leu Val Gln Phe Arg Gln Lys Leu Val Asp Ala Pro Phe 475                 480                 485 ctc aac gaa gat ctc ctg cgc gaa gca aag ttc atg ggt ctg tcc gac     2984 Leu Asn Glu Asp Leu Leu Arg Glu Ala Lys Phe Met Gly Leu Ser Asp 490                 495                 500                 505 ctg cag atc gca gcc ctt cgc cca gag ttc gct ggc gaa gac ggc gta     3032 Leu Gln Ile Ala Ala Leu Arg Pro Glu Phe Ala Gly Glu Asp Gly Val 510                 515                 520 cgc acc ttg cgt ctg tcc cta ggc atc cgc cca gta ttc aag act gtg     3080 Arg Thr Leu Arg Leu Ser Leu Gly Ile Arg Pro Val Phe Lys Thr Val 525                 530                 535 gat acc tgt gca gca gag ttt gaa gct aag act ccg tac cac tac tcc     3128 Asp Thr Cys Ala Ala Glu Phe Glu Ala Lys Thr Pro Tyr His Tyr Ser 540                 545                 550 gca tac gag ctg gat cca gca gct gag tct gag gtc gca cca cag act     3176 Ala Tyr Glu Leu Asp Pro Ala Ala Glu Ser Glu Val Ala Pro Gln Thr 555                 560                 565 gag cgt gaa aag gtc ctg atc ttg ggc tcc ggt cca aac cgc atc ggc     3224 Glu Arg Glu Lys Val Leu Ile Leu Gly Ser Gly Pro Asn Arg Ile Gly 570                 575                 580                 585 cag ggc atc gag ttc gac tat tcc tgt gtt cac gca gct ctt gag ctc     3272 Gln Gly Ile Glu Phe Asp Tyr Ser Cys Val His Ala Ala Leu Glu Leu 590                 595                 600 tcc cgc gtc ggc tac gaa act gtc atg gtc aac tgc aac cca gag acc     3320 Ser Arg Val Gly Tyr Glu Thr Val Met Val Asn Cys Asn Pro Glu Thr 605                 610                 615 gtg tcc acc gac tac gac acc gct gac cgc ctg tac ttc gag cca ctg     3368 Val Ser Thr Asp Tyr Asp Thr Ala Asp Arg Leu Tyr Phe Glu Pro Leu 620                 625                 630 acc ttc gaa gac gtc atg gag gtc tac cac gct gag gcg cag tcc ggc     3416 Thr Phe Glu Asp Val Met Glu Val Tyr His Ala Glu Ala Gln Ser Gly 635                 640                 645 acc gtc gca ggt gtt atc gtc cag ctt ggt ggc cag act cct ctg ggc     3464 Thr Val Ala Gly Val Ile Val Gln Leu Gly Gly Gln Thr Pro Leu Gly 650                 655                 660                 665 ttg gca gat cgt ttg aag aag gct ggc gtc cct gtc att ggt acc tcc     3512 Leu Ala Asp Arg Leu Lys Lys Ala Gly Val Pro Val Ile Gly Thr Ser 670                 675                 680 cca gag gca atc gac atg gct gag gac cgt ggc gag ttc ggt gca ctg     3560 Pro Glu Ala Ile Asp Met Ala Glu Asp Arg Gly Glu Phe Gly Ala Leu 685                 690                 695 ctg aac cgc gag cag ctt cct gct cca gca ttc ggc acc gca acc tct     3608 Leu Asn Arg Glu Gln Leu Pro Ala Pro Ala Phe Gly Thr Ala Thr Ser 700                 705                 710 ttc gaa gag gct cgc aca gta gcc gat gag atc agc tac cca gtg ctg     3656 Phe Glu Glu Ala Arg Thr Val Ala Asp Glu Ile Ser Tyr Pro Val Leu 715                 720                 725 gtt cgc cct tcc tac gtc ttg ggt ggc cgt ggc atg gag att gtc tac     3704 Val Arg Pro Ser Tyr Val Leu Gly Gly Arg Gly Met Glu Ile Val Tyr 730                 735                 740                 745 gat gag gct tcc ctc gag gat tac atc aac cgc gca act gag ttg tct     3752 Asp Glu Ala Ser Leu Glu Asp Tyr Ile Asn Arg Ala Thr Glu Leu Ser 750                 755                 760 tct gac cac cca gtg ctg gtt gac cgc ttc ctg gac aac gct att gag     3800 Ser Asp His Pro Val Leu Val Asp Arg Phe Leu Asp Asn Ala Ile Glu 765                 770                 775 atc gac gtc gac gca ctg tgc gac ggc gac gaa gtc tac ctg gcg ggc     3848 Ile Asp Val Asp Ala Leu Cys Asp Gly Asp Glu Val Tyr Leu Ala Gly 780                 785                 790 gtc atg gaa cac atc gag gaa gcc ggc att cac tcc ggt gac tcc gca     3896 Val Met Glu His Ile Glu Glu Ala Gly Ile His Ser Gly Asp Ser Ala 795                 800                 805 tgt gca ctt cct cca atg act ttg ggc gca cag gac atc gag aag gtc     3944 Cys Ala Leu Pro Pro Met Thr Leu Gly Ala Gln Asp Ile Glu Lys Val 810                 815                 820                 825 cgc gaa gca acc aag aag ctg gct ctg ggc atc ggc gta cag ggc ctg     3992 Arg Glu Ala Thr Lys Lys Leu Ala Leu Gly Ile Gly Val Gln Gly Leu 830                 835                 840 atg aac gtc cag tac gca ctc aag gac gac atc ctc tac gtc atc gag     4040 Met Asn Val Gln Tyr Ala Leu Lys Asp Asp Ile Leu Tyr Val Ile Glu 845                 850                 855 gca aac cca cgt gca tcc cgc acc gtg ccg ttc gtc tcc aag gca acg     4088 Ala Asn Pro Arg Ala Ser Arg Thr Val Pro Phe Val Ser Lys Ala Thr 860                 865                 870 ggc gtc aac ctg gcc aag gca gca tcc cgt atc gca gtg ggc gcc acc     4136 Gly Val Asn Leu Ala Lys Ala Ala Ser Arg Ile Ala Val Gly Ala Thr 875                 880                 885 atc aag gat ctc caa gat gag ggc atg att cct acc gag tac gac ggc     4184 Ile Lys Asp Leu Gln Asp Glu Gly Met Ile Pro Thr Glu Tyr Asp Gly 890                 895                 900                 905 ggc tcc ttg cca ctg gac gct cca atc gct gtg aag gaa gca gtg ttg     4232 Gly Ser Leu Pro Leu Asp Ala Pro Ile Ala Val Lys Glu Ala Val Leu 910                 915                 920 ccg ttc aac cgc ttc cgt cgc cca gat gga aag acc ctg gac acc ctg     4280 Pro Phe Asn Arg Phe Arg Arg Pro Asp Gly Lys Thr Leu Asp Thr Leu 925                 930                 935 ctt tcc cca gag atg aag tcc act ggc gag gtc atg ggc ttg gcc aac     4328 Leu Ser Pro Glu Met Lys Ser Thr Gly Glu Val Met Gly Leu Ala Asn 940                 945                 950 aac ttc ggc gct gca tat gca aag gct gaa gct ggc gcg ttt ggt gca     4376 Asn Phe Gly Ala Ala Tyr Ala Lys Ala Glu Ala Gly Ala Phe Gly Ala 955                 960                 965 ttg cca acc gaa ggc acc gtc ttc gtg acc gtg gct aac cgc gac aag     4424 Leu Pro Thr Glu Gly Thr Val Phe Val Thr Val Ala Asn Arg Asp Lys 970                 975                 980                 985 cgc acc ctg atc ctg cca atc cag cgc ctg gcg tcg atg ggc tac aag     4472 Arg Thr Leu Ile Leu Pro Ile Gln Arg Leu Ala Ser Met Gly Tyr Lys 990                 995                1000 atc ctc gcc acc gaa ggc acc gca ggc atg ctg cgc cgc aac ggc att     4520 Ile Leu Ala Thr Glu Gly Thr Ala Gly Met Leu Arg Arg Asn Gly Ile 1005                1010                1015 gat tgt gaa gtt gtg ctc aag gct tcc gac atc cgc gaa ggt gta gag     4568 Asp Cys Glu Val Val Leu Lys Ala Ser Asp Ile Arg Glu Gly Val Glu 1020                1025                1030 ggc aag tcc atc gtg gat cgt atc cgc gaa ggc gaa gtt gac ctc atc     4616 Gly Lys Ser Ile Val Asp Arg Ile Arg Glu Gly Glu Val Asp Leu Ile 1035                1040                1045 ctc aac acc cca gct ggt tct gct ggc gct cgc cac gat ggc tac gat     4664 Leu Asn Thr Pro Ala Gly Ser Ala Gly Ala Arg His Asp Gly Tyr Asp 1050               1055                1060                1065 atc cgc gca gca gca gtg acc gtg ggt gtt cca ctg atc acc act gtc     4712 Ile Arg Ala Ala Ala Val Thr Val Gly Val Pro Leu Ile Thr Thr Val 1070                1075                1080 cag ggt gtc acc gca gct gtc cag ggc att gag gcc ctg cgt gag ggc     4760 Gln Gly Val Thr Ala Ala Val Gln Gly Ile Glu Ala Leu Arg Glu Gly 1085                1090                1095 gtt gtc agc gtc cgc gcg ctg cag gaa ctc gac cac gca gtc aag gct     4808 Val Val Ser Val Arg Ala Leu Gln Glu Leu Asp His Ala Val Lys Ala 1100                1105                1110 taagccctat gacattcggc gagaagctt                                     4837 <210> SEQ ID NO 2 <211> LENGTH: 393 <212> TYPE: PRT <213> ORGANISM: Brevibacterium lactofermentum <400> SEQUENCE: 2 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> SEQ ID NO 3 <211> LENGTH: 1113 <212> TYPE: PRT <213> ORGANISM: Brevibacterium lactofermentum <400> SEQUENCE: 3 Met Pro Lys Arg Ser Asp Ile Asn His Val Leu Val Ile Gly Ser Gly 1               5                  10                  15 Pro Ile Val Ile Gly Gln Ala Cys Glu Phe Asp Tyr Ser Gly Thr Gln 20                  25                  30 Ala Cys Arg Val Leu Lys Glu Glu Gly Leu Arg Val Thr Leu Ile Asn 35                  40                  45 Ser Asn Pro Ala Thr Ile Met Thr Asp Pro Glu Met Ala Asp His Thr 50                  55                  60 Tyr Val Glu Pro Ile Glu Pro Glu Tyr Ile Asp Lys Ile Phe Ala Lys 65                  70                  75                  80 Glu Ile Glu Gln Gly His Pro Ile Asp Ala Val Leu Ala Thr Leu Gly 85                  90                  95 Gly Gln Thr Ala Leu Asn Ala Ala Ile Gln Leu Asp Arg Leu Gly Ile 100                 105                 110 Leu Glu Lys Tyr Gly Val Glu Leu Ile Gly Ala Asp Ile Asp Ala Ile 115                 120                 125 Glu Arg Gly Glu Asp Arg Gln Lys Phe Lys Asp Ile Val Thr Thr Ile 130                 135                 140 Gly Gly Glu Ser Ala Arg Ser Arg Val Cys His Asn Met Asp Glu Val 145                 150                 155                 160 His Glu Thr Val Ala Glu Leu Gly Leu Pro Val Val Val Arg Pro Ser 165                 170                 175 Phe Thr Met Gly Gly Leu Gly Ser Gly Leu Ala Tyr Asn Thr Glu Asp 180                 185                 190 Leu Glu Arg Ile Ala Gly Gly Gly Leu Ala Ala Ser Pro Glu Ala Asn 195                 200                 205 Val Leu Ile Glu Glu Ser Ile Leu Gly Trp Lys Glu Phe Glu Leu Glu 210                 215                 220 Leu Met Arg Asp Thr Ala Asp Asn Val Val Val Ile Cys Ser Ile Glu 225                 230                 235                 240 Asn Val Asp Ala Leu Gly Val His Thr Gly Asp Ser Val Thr Val Ala 245                 250                 255 Pro Ala Leu Thr Leu Thr Asp Arg Glu Phe Gln Lys Met Arg Asp Gln 260                 265                 270 Gly Ile Ala Ile Ile Arg Glu Val Gly Val Asp Thr Gly Gly Cys Asn 275                 280                 285 Ile Gln Phe Ala Ile Asn Pro Val Asp Gly Arg Ile Ile Thr Ile Glu 290                 295                 300 Met Asn Pro Arg Val Ser Arg Ser Ser Ala Leu Ala Ser Lys Ala Thr 305                 310                 315                 320 Gly Phe Pro Ile Ala Lys Met Ala Ala Lys Leu Ala Ile Gly Tyr Thr 325                 330                 335 Leu Asp Glu Ile Thr Asn Asp Ile Thr Gly Glu Thr Pro Ala Ala Phe 340                 345                 350 Glu Pro Thr Ile Asp Tyr Val Val Val Lys Ala Pro Arg Phe Ala Phe 355                 360                 365 Glu Lys Phe Val Gly Ala Asp Asp Thr Leu Thr Thr Thr Met Lys Ser 370                 375                 380 Val Gly Glu Val Met Ser Leu Gly Arg Asn Tyr Ile Ala Ala Leu Asn 385                 390                 395                 400 Lys Ala Leu Arg Ser Leu Glu Thr Lys Gln Gln Gly Phe Trp Thr Lys 405                 410                 415 Pro Asp Glu Phe Phe Ala Gly Glu Arg Ala Thr Asp Lys Ala Ala Val 420                 425                 430 Leu Glu Asp Leu Lys Arg Pro Thr Glu Gly Arg Leu Tyr Asp Val Glu 435                 440                 445 Leu Ala Met Arg Leu Gly Ala Ser Val Glu Glu Leu Tyr Glu Ala Ser 450                 455                 460 Ser Ile Asp Pro Trp Phe Leu Ala Glu Leu Glu Ala Leu Val Gln Phe 465                 470                 475                 480 Arg Gln Lys Leu Val Asp Ala Pro Phe Leu Asn Glu Asp Leu Leu Arg 485                 490                 495 Glu Ala Lys Phe Met Gly Leu Ser Asp Leu Gln Ile Ala Ala Leu Arg 500                 505                 510 Pro Glu Phe Ala Gly Glu Asp Gly Val Arg Thr Leu Arg Leu Ser Leu 515                 520                 525 Gly Ile Arg Pro Val Phe Lys Thr Val Asp Thr Cys Ala Ala Glu Phe 530                 535                 540 Glu Ala Lys Thr Pro Tyr His Tyr Ser Ala Tyr Glu Leu Asp Pro Ala 545                 550                 555                 560 Ala Glu Ser Glu Val Ala Pro Gln Thr Glu Arg Glu Lys Val Leu Ile 565                 570                 575 Leu Gly Ser Gly Pro Asn Arg Ile Gly Gln Gly Ile Glu Phe Asp Tyr 580                 585                 590 Ser Cys Val His Ala Ala Leu Glu Leu Ser Arg Val Gly Tyr Glu Thr 595                 600                 605 Val Met Val Asn Cys Asn Pro Glu Thr Val Ser Thr Asp Tyr Asp Thr 610                 615                 620 Ala Asp Arg Leu Tyr Phe Glu Pro Leu Thr Phe Glu Asp Val Met Glu 625                 630                 635                 640 Val Tyr His Ala Glu Ala Gln Ser Gly Thr Val Ala Gly Val Ile Val 645                 650                 655 Gln Leu Gly Gly Gln Thr Pro Leu Gly Leu Ala Asp Arg Leu Lys Lys 660                 665                 670 Ala Gly Val Pro Val Ile Gly Thr Ser Pro Glu Ala Ile Asp Met Ala 675                 680                 685 Glu Asp Arg Gly Glu Phe Gly Ala Leu Leu Asn Arg Glu Gln Leu Pro 690                 695                 700 Ala Pro Ala Phe Gly Thr Ala Thr Ser Phe Glu Glu Ala Arg Thr Val 705                 710                 715                 720 Ala Asp Glu Ile Ser Tyr Pro Val Leu Val Arg Pro Ser Tyr Val Leu 725                 730                 735 Gly Gly Arg Gly Met Glu Ile Val Tyr Asp Glu Ala Ser Leu Glu Asp 740                 745                 750 Tyr Ile Asn Arg Ala Thr Glu Leu Ser Ser Asp His Pro Val Leu Val 755                 760                 765 Asp Arg Phe Leu Asp Asn Ala Ile Glu Ile Asp Val Asp Ala Leu Cys 770                 775                 780 Asp Gly Asp Glu Val Tyr Leu Ala Gly Val Met Glu His Ile Glu Glu 785                 790                 795                 800 Ala Gly Ile His Ser Gly Asp Ser Ala Cys Ala Leu Pro Pro Met Thr 805                 810                 815 Leu Gly Ala Gln Asp Ile Glu Lys Val Arg Glu Ala Thr Lys Lys Leu 820                 825                 830 Ala Leu Gly Ile Gly Val Gln Gly Leu Met Asn Val Gln Tyr Ala Leu 835                 840                 845 Lys Asp Asp Ile Leu Tyr Val Ile Glu Ala Asn Pro Arg Ala Ser Arg 850                 855                 860 Thr Val Pro Phe Val Ser Lys Ala Thr Gly Val Asn Leu Ala Lys Ala 865                 870                 875                 880 Ala Ser Arg Ile Ala Val Gly Ala Thr Ile Lys Asp Leu Gln Asp Glu 885                 890                 895 Gly Met Ile Pro Thr Glu Tyr Asp Gly Gly Ser Leu Pro Leu Asp Ala 900                 905                 910 Pro Ile Ala Val Lys Glu Ala Val Leu Pro Phe Asn Arg Phe Arg Arg 915                 920                 925 Pro Asp Gly Lys Thr Leu Asp Thr Leu Leu Ser Pro Glu Met Lys Ser 930                 935                 940 Thr Gly Glu Val Met Gly Leu Ala Asn Asn Phe Gly Ala Ala Tyr Ala 945                 950                 955                 960 Lys Ala Glu Ala Gly Ala Phe Gly Ala Leu Pro Thr Glu Gly Thr Val 965                 970                 975 Phe Val Thr Val Ala Asn Arg Asp Lys Arg Thr Leu Ile Leu Pro Ile 980                 985                 990 Gln Arg Leu Ala Ser Met Gly Tyr Lys Ile Leu Ala Thr Glu Gly Thr 995                1000                1005 Ala Gly Met Leu Arg Arg Asn Gly Ile Asp Cys Glu Val Val Leu Lys 1010                1015                1020 Ala Ser Asp Ile Arg Glu Gly Val Glu Gly Lys Ser Ile Val Asp Arg 1025               1030                1035                1040 Ile Arg Glu Gly Glu Val Asp Leu Ile Leu Asn Thr Pro Ala Gly Ser 1045                1050                1055 Ala Gly Ala Arg His Asp Gly Tyr Asp Ile Arg Ala Ala Ala Val Thr 1060                1065                1070 Val Gly Val Pro Leu Ile Thr Thr Val Gln Gly Val Thr Ala Ala Val 1075                1080                1085 Gln Gly Ile Glu Ala Leu Arg Glu Gly Val Val Ser Val Arg Ala Leu 1090                1095                1100 Gln Glu Leu Asp His Ala Val Lys Ala 1105               1110 <210> SEQ ID NO 4 <211> LENGTH: 32 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence:primer for amplifying kanamycin resistant gene of Streptococcus faecalis <400> SEQUENCE: 4 cccgttaact gcttgaaacc caggacaata ac                                   32 <210> SEQ ID NO 5 <211> LENGTH: 30 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence:primer for amplifying kanamycin resistant gene of Streptococcus faecalis <400> SEQUENCE: 5 cccgttaaca tgtacttcag aaaagattag                                      30 <210> SEQ ID NO 6 <211> LENGTH: 26 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence:primer for amplifying Escherichia coli cloning vector pHSG399 <400> SEQUENCE: 6 gatatctacg tgccgatcaa cgtctc                                          26 <210> SEQ ID NO 7 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence:primer for amplifying Escherichia coli cloning vector pHSG399 <400> SEQUENCE: 7 aggccttttt ttaaggcagt tattg                                           25

## Claims

1. A method for producing L-arginine, comprising: or a fragment thereof that encodes a polypeptide with carbamoyl-phosphate synthetase activity in a medium and under conditions suitable for production of L-arginine, and

2. A method for producing L-arginine, comprising: L-arginine, a host cell comprising a variant or mutant of the nucleic acid sequence of SEQ ID NO: 1 that encodes a polypeptide with carbamoyl-phosphate synthetase activity, and that hybridizes under stringent conditions of 60.degree. C., 1.times.SSC, and 0.1% SDS with the nucleic acid sequence of SEQ ID NO: 1; and

3. The method of claim 1, wherein said host cell is a coryneform bacterium.

4. The method of claim 2, wherein said host cell is a coryneform bacterium.