US20020137150A1

US20020137150A1 - Bacterium producing L-glutamic acid and method for producing L-glutamic acid

Info

Publication number: US20020137150A1
Application number: US09/895,382
Authority: US
Inventors: Hiromi Ohtaki; Jun Nakamura; Hiroshi Izui; Tsuyoshi Nakamatsu
Original assignee: Ajinomoto Co Inc
Current assignee: Ajinomoto Co Inc
Priority date: 2000-07-05
Filing date: 2001-07-02
Publication date: 2002-09-26
Also published as: PL348447A1; CN1763200A; US7307160B1; EP1174508A3; PE20020212A1; CN1935983A; JP2002017364A; DE60132277D1; ATE383427T1; US20100124777A1; JP4304837B2; KR20020003519A; MY134051A; BR0102669A; AU784207B2; EP1174508A2; DE60132277T2; CN1335394A; CN1664106A; KR100809856B1

Abstract

L-Glutamic acid is produced by culturing a coryneform bacterium having L-glutamic acid producing ability, in which trehalose synthesis ability is decreased or deleted by, for example, disrupting a gene coding for trehalose-6-phosphate synthase, a gene coding for maltooligosyltrehalose synthase, or both of these genes to produce and accumulate L-glutamic acid in the medium, and collecting the L-glutamic acid from the medium.

Description

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a novel L-glutamic acid producing bacterium and a method for producing L-glutamic acid by fermentation utilizing it. L-glutamic acid is an important amino acid as foodstuffs, drugs and so forth.

2. Description of the Related Art

Conventionally, L-glutamic acid is mainly produced by fermentative methods using so-called L-glutamic acid producing coryneform bacteria belonging to the genus Brevibacterium, Corynebacterium or Microbacterium, or mutant strains thereof (Amino Acid Fermentation, pp.195-215, Gakkai Shuppan Center, 1986).

It is known that, in the production of L-glutamic acid by fermentation, trehalose is also produced as a secondary product. Therefore, techniques have been developed for decomposing or metabolizing the produced trehalose. Such techniques include the method of producing an amino acid by fermentation using a coryneform bacterium in which proliferation ability on trehalose is induced (Japanese Patent Laid-open (Kokai) No. 5-276935) and the method of producing amino acid by fermentation using a coryneform bacterium in which a gene coding for trehalose catabolic enzyme is amplified (Korean Patent Publication (B1) No. 165836). However, it is not known how to suppress the formation of trehalose itself in an L-glutamic acid producing bacterium.

In Escherichia coli, the synthesis of trehalose is catalyzed by trehalose-6-phosphate synthase. This enzyme is known to be encoded by otsA gene. Further, it has been also reported that an otsA gene-disrupted strain of Escherichia coli can scarcely grow in a hyperosmotic medium (H. M. Glaever, et al., J. Bacteriol., 170(6), 2841-2849 (1998)). However, the relationship between disruption of otsA gene and production of substances has not been known.

On the other hand, although the treY gene is known for Brevibacterium helvolum among bacteria belonging to the genus Brevibacterium bacteria, any otsA gene is not known for them. As for bacteria belonging to the genus Mycobacterium bacteria, there is known a pathway via a reaction catalyzed by a product encoded by treS gene (trehalose synthase (TreS)), which gene is different from the otsA gene and treY gene, as a gene coding for a enzyme in trehalose biosynthesis pathway (De Smet K. A., et al., Microbiology, 146 (1), 199-208 (2000)). However, this pathway utilizes maltose as a substrate and does not relate to usual L-glutamic acid fermentation that utilizes glucose, fructose or sucrose as a starting material.

SUMMARY OF THE INVENTION

An object of the present invention is to improve production efficiency of L-glutamic acid in L-glutamic acid production by fermentation using coryneform bacteria through suppression of the production of trehalose as a secondary product.

The inventors of the present invention assiduously studied in order to achieve the aforementioned object. As a result, they found that bacterium belonging to the genus Brevibacterium contained otsA gene and treY gene like Mycobacterium tuberculosis, and the production efficiency of L-glutamic acid was improved by disrupting at least one of these genes. Thus, they accomplished the present invention.

That is, the present invention provides the followings.

(1) A coryneform bacterium having L-glutamic acid producing ability, wherein trehalose synthesis ability is decreased or deleted in the bacterium.

(2) The coryneform bacteria according to (1), wherein the trehalose synthesis ability is decreased or deleted by introducing a mutation into a chromosomal gene coding for an enzyme in a trehalose synthesis pathway or disrupting the gene.

(3) The coryneform bacteria according to (2), wherein the gene coding for the enzyme in trehalose synthesis pathway consists of a gene coding for trehalose-6-phosphate synthase, a gene coding for maltooligosyltrehalose synthase, or both of these genes.

(4) The coryneform bacteria according to (3), wherein the gene coding for trehalose-6-phosphate synthase codes for the amino acid sequence of SEQ ID NO: 30, and the gene coding for maltooligosyltrehalose synthase codes for the amino acid sequence of SEQ ID NO: 32.

(5) A method for producing L-glutamic acid comprising culturing a coryneform bacterium according to any one of (1) to (4) in a medium to produce and accumulate L-glutamic acid in the medium, and collecting the L-glutamic acid from the medium.

(6) A DNA coding for a protein defined in the following (A) or (B):

(A) a protein having the amino acid sequence of SEQ ID NO: 30,

(B) a protein having an amino acid sequence of SEQ ID NO: 30 including substitution, deletion, insertion or addition of one or several amino acid residues and having trehalose-6-phosphate synthase activity.

(7) A DNA according to (6), which is a DNA defined in the following (a) or (b):

(a) a DNA containing a nucleotide sequence comprising at least the residues of nucleotide numbers 484-1938 in the nucleotide sequence of SEQ ID NO: 29,

(b) a DNA hybridizable with a nucleotide sequence comprising at least the residues of nucleotide numbers 484-1938 in the nucleotide sequence of SEQ ID NO: 29 under a stringent condition, showing homology of 55% or more to the foregoing nucleotide sequence, and coding for a protein having trehalose-6-phosphate synthase activity.

(8) A DNA coding for a protein defined in the following (A) or (B):

(A) a protein having the amino acid sequence of SEQ ID NO: 32,

(B) a protein having an amino acid sequence of SEQ ID NO: 32 including substitution, deletion, insertion or addition of one or several amino acid residues and having maltooligosyltrehalose synthase activity.

(9) A DNA according to (8), which is a DNA defined in the following (a) or (b):

(a) a DNA containing a nucleotide sequence comprising at least the residues of nucleotide numbers 82-2514 in the nucleotide sequence of SEQ ID NO: 31,

(b) a DNA hybridizable with a nucleotide sequence comprising at least the residues of nucleotide numbers 82-2514 in the nucleotide sequence of SEQ ID NO: 31 under a stringent condition, showing homology of 60% or more to the foregoing nucleotide sequence, and coding for a protein having maltooligosyltrehalose synthase activity.

The trehalose-6-phosphate synthase activity means an activity to catalyze a reaction in which α,α-trehalose-6-phosphate and UDP are produced from UDP-glucose and glucose-6-phosphate, and the maltooligosyltrehalose synthase activity means an activity to catalyze a reaction in which maltotriosyltrehalose is produced from maltopentose.

According to the present invention, production efficiency of L-glutamic acid in L-glutamic acid production by fermentation using coryneform bacteria can be improved through inhibition of the production of trehalose as a secondary product.

PREFERREED EMBODIMENTS OF THE INVENTION

Hereafter, the present invention will be explained in detail.

The coryneform bacterium of the present invention is a coryneform bacterium having L-glutamic acid producing ability, in which trehalose synthesis ability is decreased or deleted.

The coryneform bacteria referred to in the present invention include the group of microorganisms defined in Bergey's Manual of Determinative Bacteriology, 8th edition, p.599 (1974), which are aerobic Gram-positive rods having no acid resistance and no spore-forming ability aerobic. They have hitherto been classified into the genus Brevibacterium, but united into the genus Corynebacterium at present ( Int. J. Syst. Bacteriol., 41, 255 (1981)), and include bacteria belonging to the genus Brevibacterium or Microbacterium closely relative to the genus Corynebacterium. Examples of such coryneform bacteria are mentioned below.

Corynebacterium acetoacidophilum

Corynebacterium acetoglutamicum

Corynebacterium alkanolyticum

Corynebacterium callunae

Corynebacterium glutamicum

Corynebacterium lilium (Corynebacterium glutamicum)

Corynebacterium melassecola

Corynebacterium thermoaminogenes

Corynebacterium herculis

Brevibacterium divaricatum (Corynebacterium glutamicum)

Brevibacterium flavum (Corynebacterium glutamicum)

Brevibacterium immariophilum

Brevibacterium lactofermentum (Corynebacterium glutamicum)

Brevibacterium roseum

Brevibacterium saccharolyticum

Brevibacterium thiogenitalis

Brevibacterium ammoniagenes (Corynebacterium ammoniagenes)

Brevibacterium album

Brevibacterium cerium

Microbacterium ammoniaphilum

Specifically, the following strains can be exemplified.

Corynebacterium acetoacidophilum ATCC 13870

Corynebacterium acetoglutamicum ATCC 15806

Corynebacterium alkanolyticum ATCC21511

Corynebacterium callunae ATCC 15991

Corynebacterium glutamicum ATCC 13020, 13032, 13060

Corynebacterium lilium (Corynebacterium glutamicum) ATCC 15990

Corynebacterium melassecola ATCC 17965

Corynebacterium thermoaminogenes AJ12340 (FERM BP-1539)

Corynebacterium herculis ATCC13868

Brevibacterium divaricatum (Corynebacterium glutamicum) ATCC 14020

Brevibacterium flavum (Corynebacterium glutamicum) ATCC 13826, ATCC 14067

Brevibacterium immariophilum ATCC 14068

Brevibacterium lactofermentum (Corynebacterium glutamicum) ATCC 13665, ATCC 13869

Brevibacterium roseum ATCC 13825

Brevibacterium saccharolyticum ATCC 14066

Brevibacterium thiogenitalis ATCC 19240

Brevibacterium ammoniagenes (Corynebacterium ammoniagenes) ATCC 6871

Brevibacterium album ATCC 15111

Brevibacterium cerium ATCC 15112

Microbacterium ammoniaphilum ATCC 15354

The trehalose synthesis ability of such coryneform bacteria as mentioned above can be decreased or deleted by mutagenizing or disrupting a gene coding for an enzyme in trehalose synthesis pathway using mutagenesis treatment or genetic recombination technique. Such a mutation may be a mutation that suppresses transcription or translation of the gene coding for the enzyme in trehalose synthesis pathway, or a mutation that causes elimination or decrease of an enzyme in trehalose systhesis pathway. The enzyme in trehalose systhesis pathway may be exemplified by, for example, trehalose-6-phosphate synthase, maltooligosyltrehalose synthases, or both of these.

The disruption of a gene coding for an enzyme in trehalose systhesis pathway can be performed by gene substitution utilizing homologous recombination. A gene on a chromosome of a coryneform bacterium can be disrupted by transforming the coryneform bacterium with DNA containing a gene coding for an enzyme in trehalose systhesis pathway modified so that a part thereof should be deleted and hence the enzyme in trehalose systhesis pathway should not normally function (deletion type gene), and allowing recombination between the deletion type gene and a normal gene on the chromosome. Such gene disruption by homologous recombination has already been established. To this end, there can be mentioned a method utilizing a linear DNA or a cyclic DNA that does not replicate in coryneform bacteria and a method utilizing a plasmid containing a temperature sensitive replication origin. However, a method utilizing a cyclic DNA that does not replicate in coryneform bacteria or a plasmid containing a temperature sensitive replication origin is preferred.

The gene coding for an enzyme in trehalose systhesis pathway may be exemplified by, for example, the otsA gene or treY gene, or it may consist of both of these. Since the nucleotide sequences of the otsA gene and treY gene of Brevibacterium lactofermentum and flanking regions thereof have been elucidated by the present invention, those genes can be easily obtained by preparing primers based on the sequences and performing PCR (polymerase chain reaction, see White, T. J. et al., Trends Genet., 5, 185 (1989)) using the primers and chromosomal DNA of Brevibacterium lactofermentum as a template.

The nucleotide sequence comprising the otsA gene and the nucleotide sequence comprising the treY gene of Brevibacterium lactofermentum obtained in the examples described later are shown in SEQ ID NOS: 29 and 31, respectively. Further, the amino acid sequences encoded by these nucleotide sequences are shown in SEQ ID NOS: 30 and 32, respectively.

The otsA gene and treY gene each may be one coding for a protein including substitution, deletion, insertion or addition of one or several amino acids at one or a plurality of positions, provided that the activity of trehalose-6-phosphate synthase or maltooligosyltrehalose synthase encoded thereby is not deteriorated. While the number of “several” amino acids differs depending on positions or types of amino acid residues in the three-dimensional structure of the protein, it is preferably 1-40, more preferably 1-20, further preferably 1-10.

A DNA coding for the substantially same protein as trehalose-6-phosphate synthase or maltooligosyltrehalose synthase described above can be obtained by, for example, modifying each of the nucleotide sequences by, for example, the site-directed mutagenesis method so that one or more amino acid residues at a specified site should involve substitution, deletion, insertion, addition or inversion. Such a DNA modified as described above may also be obtained by a conventionally known mutation treatment. The mutation treatment includes a method of treating DNA coding for trehalose-6-phosphate synthase or maltooligosyltrehalose in vitro, for example, with hydroxylamine, and a method for treating a microorganism, for example, a bacterium belonging to the genus Escherichia harboring a DNA coding for trehalose-6-phosphate synthase or maltooligosyltrehalose with ultraviolet irradiation or a mutating agent usually used for mutation treatment such as N-methyl-N′-nitro-N-nitrosoguanidine (NTG) and nitrous acid.

The substitution, deletion, insertion, addition, or inversion of nucleotide as described above also includes a naturally occurring mutant or variant on the basis of, for example, individual difference or difference in species or genus of microorganisms that harbor trehalose-6-phosphate synthase or maltooligosyltrehalose.

A DNA coding for the substantially same protein as trehalose-6-phosphate synthase or maltooligosyltrehalose synthase described above can be obtained by expressing such a DNA having a mutation as described above in a suitable cell, and examining the trehalose-6-phosphate synthase activity or maltooligosyltrehalose synthase activity of the expression product.

A DNA coding for substantially the same protein as trehalose-6-phosphate synthase can also be obtained by isolating a DNA hybridizable with a DNA having, for example, a nucleotide sequence corresponding to nucleotide numbers of 484-1938 of the nucleotide sequence shown in SEQ ID NO: 29 or a probe that can be prepared from the nucleotide sequence under a stringent condition, showing homology of 55% or more, preferably 65% or more, more preferably 75% or more, to the foregoing nucleotide sequence, and having trehalose-6-phosphate synthase activity from a DNA coding for trehalose-6-phosphate synthase having a mutation or from a cell harboring it. Similarly, a DNA coding for substantially the same protein as maltooligosyltrehalose synthase can also be obtained by isolating a DNA hybridizable with a DNA having, for example, a nucleotide sequence corresponding to nucleotide numbers of 82-2514 of the nucleotide sequence shown in SEQ ID NO: 31 or a probe that can be prepared from the nucleotide sequence under a stringent condition, showing homology of 60% or more, preferably 70% or more, more preferably 80% or more, to the foregoing nucleotide sequence, and having maltooligosyltrehalose synthase activity from a DNA coding for maltooligosyltrehalose synthase having a 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 55%, preferably not less than 60%, are hybridized with each other, and DNA's having homology lower than the above level 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., 1× SSC, 0.1% SDS, preferably 0.1× SSC, 0.1% SDS, at 60° C.

As the probe, a partial sequence of each gene can also be used. Such a probe can be produced by PCR using oligonucleotides produced based on the nucleotide sequence of each gene as primers and a DNA fragment containing each gene as a template. When a DNA fragment in a length of about 300 bp is used as the probe, the washing conditions for the hybridization may consists of 50° C., 2× SSC and 0.1% SDS.

Genes hybridizable under such conditions as described above include those having a stop codon generated in a coding region of the genes, and those having no activity due to mutation of active center. However, such mutants can be easily removed by ligating each of the genes with a commercially available expression vector, and measuring trehalose-6-phosphate synthase activity or maltooligosyltrehalose synthase activity.

When an otsA gene or treY gene is used for the disruption of these genes on chromosomes of coryneform bacteria, the encoded trehalose-6-phosphate synthase or maltooligosyltrehalose synthase are not required to have their activities. Further, the otsA gene or treY gene used for the gene disruption may be a gene derived from another microorganism, so long as they can undergo homologous recombination with these genes of coryneform bacteria. For example, an otsA gene of bacterium belonging to the genus Escherichia or Mycobacterium, treY gene of bacterium belonging to the genus Arthrobacter, Brevibacterium helvolum, or bacterium belonging to the genus Rhizobium can be mentioned.

A deletion type gene of the otsA gene or treY gene can be prepared by excising a certain region with restriction enzyme(s) from a DNA fragment containing one of these genes or a part of them to delete at least a part of coding region or an expression regulatory sequence such as promoter.

Further, a deletion type gene can also be obtained by performing PCR using primers designed so that a part of gene should be deleted. Furthermore, a deletion type gene may be one obtained by single nucleotide mutation, for example, a frame shift mutation.

Gene disruption of the otsA gene will be explained hereafter. Gene disruption of the treY gene can be performed similarly.

An otsA gene on a host chromosome can be replaced with a deletion type otsA gene as follows. That is, a deletion type otsA gene and a marker gene for resistance to a drug, such as kanamycin, chloramphenicol, tetracycline and streptomycin, are inserted into a plasmid that cannot autonomously replicate in coryneform bacteria to prepare a recombinant DNA. A coryneform bacterium can be transformed with the recombinant DNA, and the transformant strain can be cultured in a medium containing the drug to obtain a transformant strain in which the recombinant DNA was introduced into chromosomal DNA. Alternatively, such a transformant strain can be obtained by using a temperature sensitive plasmid as the plasmid, and culturing the transformants at a temperature at which the temperature sensitive plasmid cannot replicate.

In a strain in which the recombinant DNA is incorporated into a chromosome as described above, the recombinant DNA causes recombination with an otsA gene sequence that originally exists on the chromosome, and two of fused genes comprising the chromosomal otsA gene and the deletion type otsA gene are inserted into the chromosome so that other portions of the recombinant DNA (vector portion and drug resistance marker gene) should be interposed between them.

Then, in order to leave only the deletion type otsA gene on the chromosomal DNA, one copy of the otsA gene is eliminated from the chromosomal DNA together with the vector portion (including the drug resistance marker gene) by recombination of two of the otsA genes. In that case, the normal otsA gene is left on the chromosomal DNA and the deletion type otsA gene is excised, or conversely, the deletion type otsA gene is left on the chromosomal DNA and the normal otsA gene is excised. It can be confirmed which type of the gene is left on the chromosomal DNA by investigating structure of the otsA gene on the chromosome by PCR, hybridization or the like.

The coryneform bacterium used for the present invention may have enhanced activity of an enzyme that catalyzes the biosynthesis of L-glutamic acid in addition to the deletion or decrease of trehalose synthesis ability. Examples of the enzyme that catalyzes the biosynthesis of L-glutamic acid include glutamate dehydrogenase, glutamine synthetase, glutamate synthase, isocitrate dehydrogenase, aconitate hydratase, citrate synthase, pyruvate carboxylase, phosphoenolpyruvate carboxylase, phosphoenolpyruvate synthase, enolase, phosphoglyceromutase, phosphoglycerate kinase, glyceraldehyde-3-phosphate dehydrogenase, triosephosphate isomerase, fructose bisphosphate aldolase, phosphofructokinase, glucose phosphate isomerase and so forth.

Further, in the coryneform bacterium used for the present invention, an enzyme that catalyzes a reaction for generating a compound other than L-glutamic acid by branching off from the biosynthetic pathway of L-glutamic acid may be declined or made deficient. Examples of such an enzyme include α-ketoglutarate dehydrogenase, isocitrate lyase, phosphate acetyltransferase, acetate kinase, acetohydroximate synthase, acetolactate synthase, formate acetyltransferase, lactate dehydrogenase, L-glutamate decarboxylase, 1-pyrroline dehydrogenase and so forth.

Furthermore, by introducing a temperature sensitive mutation for a biotin activity inhibiting substance such as surface active agents into a coryneform bacterium having L-glutamic acid producing ability, the bacterium becomes to be able to produce L-glutamic acid in a medium containing an excessive amount of biotin in the absence of a biotin activity inhibiting substance (see WO96/06180). As such a coryneform bacterium, the Brevibacterium lactofermentum AJ13029 strain disclosed in WO96/06180 can be mentioned. The AJ13029 strain was deposited at the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology (currently, the independent administrative corporation, National Institute of Advanced Industrial Science and Technology, International Patent Organism Depositary (Chuo Dai-6, 1-1 Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, Japan, postal code: 305-5466) on Sep. 2, 1994, and received an accession number of FERM P-14501. Then, it was transferred to an international deposit under the provisions of the Budapest Treaty on Aug. 1, 1995, and received an accession number of FERM BP-5189.

When a coryneform bacterium having L-glutamic acid producing ability, in which trehalose synthesis ability is decreased or deleted, is cultured in a suitable medium, L-glutamic acid is accumulated in the medium.

The medium used for producing L-glutamic acid is a usual medium that contains a carbon source, a nitrogen source, inorganic ions and other organic trace nutrients as required. As the carbon source, it is possible to use sugars such as glucose, lactose, galactose, fructose, sucrose, maltose, blackstrap molasses and starch hydrolysate; alcohols such as ethanol and inositol; or organic acids such as acetic acid, fumaric acid, citric acid and succinic acid.

As the nitrogen source, there can be used inorganic ammonium salts such as ammonium sulfate, ammonium nitrate, ammonium chloride, ammonium phosphate and ammonium acetate, ammonia, organic nitrogen such as peptone, meat extract, yeast extract, corn steep liquor and soybean hydrolysate, ammonia gas, aqueous ammonia and so forth.

As the inorganic ions (or sources thereof), added is a small amount of potassium phosphate, magnesium sulfate, iron ions, manganese ions and so forth. As for the organic trace nutrients, it is desirable to add required substances such as vitamin B ₁, yeast extract and so forth in a suitable amount as required.

The culture is preferably performed under an aerobic condition performed by shaking, stirring for aeration or the like for 16 to 72 hours. The culture temperature is controlled to be at 30° C. to 45° C., and pH is controlled to be 5 to 9 during the culture. For such adjustment of pH, inorganic or organic acidic or alkaline substances, ammonia gas and so forth can be used.

Collection of L-glutamic acid from fermentation broth can be performed by, for example, methods utilizing ion exchange resins, crystallization and so forth. Specifically, L-glutamic acid can be adsorbed on an anion exchange resin and isolated from it, or crystallized by neutralization.

EXAMPLES

Hereafter, the present invention will be explained more specifically with reference to the following examples. [0102]

Example 1

Construction of otsA Gene-disrupted Strain of Brevibacterium lactofermentum

<1> Cloning of otsA Gene [0103]
Since otsA gene of [0104] Brevibacterium lactofermentum was not known, it was obtained by utilizing a nucleotide sequence of otsA gene of another microorganism for reference. The otsA genes of Escherichia and Mycobacterium had been hitherto elucidated for their entire nucleotide sequences (Kaasen I., et al., Gene, 145 (1), 9-15 (1994); De Smet K. A., et al., Microbiology, 146 (1), 199-208 (2000)). Therefore, referring to an amino acid sequence deduced from these nucleotide sequences, DNA primers P1 (SEQ ID NO: 1) and P2 (SEQ ID NO: 2) for PCR were synthesized first. The DNA primers P1 and P2 corresponded to the regions of the nucleotide numbers of 1894-1913 and 2531-2549 of the nucleotide sequence of the otsA gene of Escherichia coli (GenBank accession X69160), respectively. They also corresponded to the regions of the nucleotide numbers 40499-40518 and 41166-41184 of the otsA gene of Mycobacterium tuberculosis (GenBank accession Z95390), respectively.
Then, PCR was performed by using the primers P1 and P2 and chromosomal DNA of [0105] Brevibacterium lactofermentum ATCC 13869 as a template with a cycle consisting of reactions at 94° C. for 0.5 minute, 50° C. for 0.5 minute and 72° C. for 4 minutes, which was repeated for 30 cycles. As a result, a substantially single kind of amplified fragment of about 0.6 kbp was obtained. This amplified fragment was cloned into a plasmid vector pCR2.1 by using “Original TA Cloning Kit” produced by Invitrogen to obtain pCotsA. Then, the nucleotide sequence of the cloned fragment was determined.
Based on the nucleotide sequence of the partial fragment of otsA gene obtained as described above, DNA primers P10 (SEQ ID NO: 8) and P12 (SEQ ID NO: 10) were newly synthesized, and unknown regions flanking to the partial fragment was amplified by “inverse PCR” (Triglia, T. et al., [0106] Nucleic Acids Res., 16, 81-86 (1988); Ochman H., et al., Genetics, 120, 621-623 (1988)). The chromosomal DNA of Brevibacterium lactofermentum ATCC 13869 was digested with a restriction enzyme BamHI, BglII, ClaI, HindIII, KpnI, MluI, MunL, SalI or XhoI, and self-ligated by using T4 DNA ligase (Takara Shuzo). By using resultant DNA as a template and the DNA primers P10 and P12, PCR was performed with a cycle consisting of reactions at 94° C. for 0.5 minute, 55° C. for 1 minute and 72° C. for 4 minutes, which was repeated for 30 cycles. As a result, when ClaI or BglII was used as the restriction enzyme, an amplified fragment of 4 kbp was obtained for each case. The nucleotide sequences of these amplified fragments were directly determined by using the DNA primers P5 to P9 (SEQ ID NOS: 3-7) and P11 to P15 (SEQ ID NOS: 9-13). Thus, the entire nucleotide sequence of otsA gene of Brevibacterium lactofermentum ATCC 13869 was determined as shown in SEQ ID NO: 29. The amino acid sequence encoded by this nucleotide sequence is shown in SEQ ID NOS: 29 and 30.
When homology of the sequence of the aforementioned otsA gene was determined with respect to the otsA gene of [0107] Escherichia coli (GenBank accession X69160) and the otsA gene of Mycobacterium tuberculosis (GenBank accession Z95390), the nucleotide sequence showed homologies of 46.3% and 55.9%, respectively, and the amino acid sequence showed homologies of 30.9% and 51.7%, respectively. The homologies were calculated by using software, “GENETIX-WIN” (Software Development), based on the Lipman-Person method (Science, 227, 1435-1441 (1985)).
<2> Preparation of Plasmid for otsA Gene Disruption [0108]
In order to examine presence or absence of improvement effect in L-glutamic acid productivity by disruption of a gene coding for an enzyme in trehalose biosysthesis pathway in coryneform bacteria, a plasmid for otsA gene disruption was produced. A plasmid for otsA gene disruption was produced as follows. PCR was performed by using the plasmid pCotsA previously constructed in the cloning of the otsA gene as a template and the primers P29 (SEQ ID NO: 33) and P30 (SEQ ID NO: 34) comprising ClaI site with a cycle consisting of reactions at 94° C. for 0.5 minute, 55° C. for 0.5 minute and 72° C. for 8 minutes, which was repeated for 30 cycles. The amplified fragment was digested with ClaI, blunt-ended by using T4 DNA polymerase (Takara Shuzo), and self-ligated by using T4 ligase (Takara Shuzo) to construct a plasmid pCotsAC containing the otsA gene having a frame shift mutation (1258-1300th nucleotides of SEQ ID NO: 29 were deleted) at an approximately central part thereof. [0109]
<3> Preparation of otsA Gene-disrupted Strain [0110]
By using the plasmid pCotsAC for gene disruption, a L-glutamic acid producing bacterium, [0111] Brevibacterium lactofermentum ATCC 13869, was transformed by the electric pulse method, and transformants were selected as to the ability to grow in CM2B medium containing 20 mg/L of kanamycin. Because the plasmid pCotsAC for otsA gene disruption did not have a replication origin that could function in Brevibacterium lactofermentum, resultant transformants obtained by using the plasmid suffered homologous recombination occurred between the otsA genes on the chromosome of Brevibacterium lactofermentum and the plasmid pCotsAC for gene disruption. From the homologous recombinant strains obtained as described above, strains in which the vector portion of the plasmid pCotsAC for gene disruption was eliminated due to re-occurrence of homologous recombination were selected based on acquired kanamycin sensitivity as a marker.
From the strains obtained as described above, a strain introduced with the desired frame shift mutation was selected. Selection of such a strain was performed by PCR using chromosomal DNA extracted from a strain that became kanamycin sensitive as a template and the DNA primers P8 (SEQ ID NO: 14) and P13 (SEQ ID NO: 11) with a cycle consisting of reactions at 94° C. for 0.5 minute, 55° C. for 0.5 minute and 72° C. for 1 minutes, which was repeated for 30 cycles, and sequencing of the obtained amplified fragment using the DNA primer P8 to confirm disfunction of the otsA gene due to introduction of frame shift mutation. The strain obtained as described above was designated as AOA strain. [0112]

Example 2

Construction of treY Gene-disrupted Strain

<1> Cloning of treY Gene [0113]
Since treY gene of [0114] Brevibacterium lactofermentum was not known, it was obtained by using nucleotide sequences of treY genes of the other microorganisms for reference. The nucleotide sequences of treY genes were hitherto elucidated for the genera Arthrobacter, Brevibacterium and Rhizobium (Maruta K., et al., Biochim. Biophys. Acta, 1289 (1), 10-13 (1996); Genbank accession AF039919; Maruta K., et al., Biosci. Biotechnol. Biochem., 60 (4), 717-720 (1996)). Therefore, referring to an amino acid sequence deduced from these nucleotide sequences, the PCR DNA primers P3 (SEQ ID NO: 14) and P4 (SEQ ID NO: 15) were synthesized first. The DNA primers P3 and P4 correspond to the regions of the nucleotide numbers of 975-992 and 2565-2584 of the nucleotide sequence of the treY gene of Arthrobacter species (GenBank accession D63343), respectively. Further, they correspond to the regions of the nucleotide numbers 893-910 and 2486-2505 of the treY gene of Brevibacterium helvolum (GenBank accession AF039919), respectively. Furthermore, they correspond to the regions of the nucleotide numbers of 862-879 and 2452-2471 of treY gene of Rhizobium species (GenBank accession D78001).
Then, PCR was performed by using the primers P3 and P4 and chromosomal DNA of [0115] Brevibacterium lactofermentum ATCC13869 as a template with a cycle consisting of reactions at 94° C. for 0.5 minute, 55° C. for 0.5 minute and 72° C. for 2 minutes, which was repeated for 30 cycles. As a result, a substantially single kind of an amplified fragment of about 1.6 kbp was obtained. This amplified fragment was cloned into a plasmid vector pCR2.1 by using “Original TA Cloning Kit” produced by Invitrogen. Then, the nucleotide sequence was determined for about 0.6 kb.
Based on the nucleotide sequence of the partial fragment of treY gene obtained as described above, the DNA primers P16 (SEQ ID NO: 16) and P26 (SEQ ID NO: 26) were newly synthesized, and unknown regions flanking to the partial fragment was amplified by “inverse PCR” (Triglia, T. et al., [0116] Nucleic Acids Res., 16, 81-86 (1988); Ochman H., et al., Genetics, 120, 621-623 (1988)). The chromosomal DNA of Brevibacterium lactofermentum ATCC 13869 was digested with a restriction enzyme BamHI, HindIII, SalI or XhoI, and self-ligated by using T4 DNA ligase (Takara Shuzo). By using this as a template and the DNA primers P16 and P26, PCR was performed with a cycle consisting of reactions at 94° C. for 0.5 minute, 55° C. for 1 minute and 72° C. for 4 minutes, which was repeated for 30 cycles. As a result, when HindIII or SalI was used as the restriction enzyme, an amplified fragment of 0.6 kbp or 1.5 kbp was obtained, respectively. The nucleotide sequences of these amplified fragments were directly determined by using the DNA primers P16 to P28 (SEQ ID NOS: 16-28). Thus, the entire nucleotide sequence of treY gene of Brevibacterium lactofermentum ATCC 13869 was determined as shown in SEQ ID NO: 31. The amino acid sequence encoded by this nucleotide sequence is shown in SEQ ID NOS: 31 and 32.
When homology of the sequence of the aforementioned treY gene was determined with respect to the treY gene of Arthrobacter sp. (GenBank accession D63343), treY gene of [0117] Brevibacterium helvolum (GenBank accession AF039919) and treY gene of Rhizobium sp. (GenBank accession D78001), the nucleotide sequence showed homologies of 52.0%, 52.3% and 51.9%, respectively, and the amino acid sequence showed homologies of 40.9%, 38.5% and 39.8%, respectively. The homologies were calculated by using software, “GENETIX-WIN” (Software Development), based on the Lipman-Person method (Science, 227, 1435-1441 (1985)).
<2> Preparation of Plasmid for Trey Gene Disruption [0118]
In order to examine presence or absence of improvement effect in L-glutamic acid productivity by disruption of the gene coding for the enzyme in trehalose biosysthesis pathway in coryneform bacteria, a plasmid for treY gene disruption was produced. First, PCR was performed by using the primers P17 (SEQ ID NO: 17) and P25 (SEQ ID NO: 25) and the chromosomal DNA of ATCC 13869 as a template with a cycle consisting of reactions at 94° C. for 0.5 minute, 60° C. for 0.5 minute and 72° C. for 2 minutes, which was repeated for 30 cycles. The amplified fragment was digested with EcoRI and ligated to pHSG299 (Takara Shuzo) digested with EcoRI by using T4 DNA ligase (Takara Shuzo) to obtain a plasmid pHtreY. Further, this pHtreY was digested with AflII (Takara Shuzo), blunt-ended by using T4 DNA polymerase (Takara Shuzo), and self-ligated by using T4 ligase (Takara Shuzo) to construct a plasmid pHtreYA containing the treY gene having a frame shift mutation (four nucleotides were inserted after the 1145th nucleotide in the sequence of SEQ ID NO: 31) at an approximately central part thereof. [0119]
<3> Preparation of treY Gene-disrupted Strain [0120]
By using the plasmid pCtreYA for gene disruption, a L-glutamic acid producing bacterium, [0121] Brevibacterium lactofermentum ATCC 13869, was transformed by the electric pulse method, and transformants were selected as to the ability to grow in CM2B medium containing 20 mg/L of kanamycin. Because the plasmid pCtreYA for treY gene disruption does not have a replication origin that could function in Brevibacterium lactofermentum, the transformants obtained by using the plasmid suffered recombination occurred between the treY genes on the Brevibacterium lactofermentum chromosome and the plasmid pCtreYA for gene disruption. From the homologous recombinant strains obtained as described above, strains in which the vector portion of the plasmid pCtreYA for gene disruption was eliminated due to re-occurrence of homologous recombination were selected based on acquired kanamycin sensitivity as a marker.
From the strains obtained as described above, a strain introduced with the desired frame shift mutation was selected. Selection of such a strain was performed by PCR using the DNA primers P19 (SEQ ID NO: 19) and P25 (SEQ ID NO: 25) with a cycle consisting of reactions at 94° C. for 0.5 minute, 55° C. for 0.5 minute and 72° C. for 1.5 minutes, which was repeated for 30 cycles, and sequencing the obtained fragment using the DNA primer P21 or P23 to confirm dysfunction of the trey gene due to introduction of frame shift mutation. The strain obtained as described above was designated as ΔTA strain. [0122]

Example 3

Evaluation of L-glutamic Acid Producing Ability of ΔOA Strain and ΔTA Strain

The ATCC 13869 strain, AOA strain and ATA strain were each cultured for producing L-glutamic acid as follows. Each of these strains was refreshed by culturing it on a CM2B plate medium, and each refreshed strain was cultured in a medium containing 80 g of glucose, 1 g of KH[0123] ₂PO₄, 0.4 g of MgSO₄, 30 g of (NH₄)₂SO₄, 0.01 g of FeSO₄.7H₂O, 0.01 g MnSO₄.7H₂O, 15 ml of soybean hydrolysate solution, 200 μg of thiamin hydrochloride, 3 μg of biotin and 50 g of CaCO₃in 1 L of pure water (adjusted to pH 8.0 with KOH) at 31.5° C. After the culture, amount of L-glutamic acid accumulated in the medium and absorbance at 620 nm of the culture broth diluted 51 times were measured. The results are shown in Table 1.
The [0124] Brevibacterium lactofermentum strains of which otsA gene or treY gene was disrupted showed growth in a degree similar to that of the parent strain, and in addition, increased L-glutamic acid production compared with the parent strain.

Table 1

[0125]

TABLE 1

Strain OD₆₂₀(x51) L-Glutamic acid (g/L) Yield (%)

ATCC 13869 0.930 40.2 48.4

ΔOA 1.063 43.8 52.8

ΔTA 0.850 45.6 54.9
(Explanation of Sequence Listing) [0126]
SEQ ID NO: 1: Primer P1 for amplification of otsA [0127]
SEQ ID NO: 2: Primer P2 for amplification of otsA [0128]
SEQ ID NO: 3: Primer P5 [0129]
SEQ ID NO: 4: Primer P6 [0130]
SEQ ID NO: 5: Primer P7 [0131]
SEQ ID NO: 6: Primer P8 [0132]
SEQ ID NO: 7: Primer P9 [0133]
SEQ ID NO: 8: Primer P10 [0134]
SEQ ID NO: 9: Primer P11 [0135]
SEQ ID NO: 10: Primer P12 [0136]
SEQ ID NO: 11: Primer P13 [0137]
SEQ ID NO: 12: Primer P14 [0138]
SEQ ID NO: 13: Primer P15 [0139]
SEQ ID NO: 14: Primer P3 for amplification of treY [0140]
SEQ ID NO: 15: Primer P4 for amplification of treY [0141]
SEQ ID NO: 16: Primer P16 [0142]
SEQ ID NO: 17: Primer P17 [0143]
SEQ ID NO: 18: Primer P18 [0144]
SEQ ID NO: 19: Primer P19 [0145]
SEQ ID NO: 20: Primer P20 [0146]
SEQ ID NO: 21: Primer P21 [0147]
SEQ ID NO: 22: Primer P22 [0148]
SEQ ID NO: 23: Primer P23 [0149]
SEQ ID NO: 24: Primer P24 [0150]
SEQ ID NO: 25: Primer P25 [0151]
SEQ ID NO: 26: Primer P26 [0152]
SEQ ID NO: 27: Primer P27 [0153]
SEQ ID NO: 28: Primer P28 [0154]
SEQ ID NO: 29: Nucleotide sequence of otsA gene [0155]
SEQ ID NO: 30: Amino acid sequence of OtsA [0156]
SEQ ID NO: 31: Nucleotide sequence of treY gene [0157]
SEQ ID NO: 32: Amino acid sequence of TreY [0158]
SEQ ID NO: 33: Primer P29 [0159]
SEQ ID NO: 34: Primer P30 [0160]
1 34 1 20 DNA Artificial Sequence Synthetic DNA 1 canathggnt tyttyytnca 20 2 19 DNA Artificial Sequence Synthetic DNA 2 canarrttca tnccrtcnc 19 3 23 DNA Artificial Sequence Synthetic DNA 3 gaatcatcca tataagatcc ggc 23 4 24 DNA Artificial Sequence Synthetic DNA 4 tagctttgta gttgttgcta accg 24 5 24 DNA Artificial Sequence Synthetic DNA 5 agcgaacttg aggtttactt cccg 24 6 24 DNA Artificial Sequence Synthetic DNA 6 tgctggttcc tggcattttg cgcc 24 7 20 DNA Artificial Sequence Synthetic DNA 7 tcgaacaatc tcttcacgcc 20 8 21 DNA Artificial Sequence Synthetic DNA 8 gaatcccacc aaatctgcgc c 21 9 20 DNA Artificial Sequence Synthetic DNA 9 tgatgttgaa atgtttgggg 20 10 20 DNA Artificial Sequence Synthetic DNA 10 gatgtcatgc tggttacgcc 20 11 22 DNA Artificial Sequence Synthetic DNA 11 caaagcacca gtgccgtcgc gg 22 12 24 DNA Artificial Sequence Synthetic DNA 12 tgttcgtttt cattcgcgtt gccg 24 13 24 DNA Artificial Sequence Synthetic DNA 13 atagtttcct ggattgtttg gcgc 24 14 18 DNA Artificial Sequence Synthetic DNA 14 caraayccnt ggtggtgg 18 15 20 DNA Artificial Sequence Synthetic DNA 15 ggncgncgrt trtcnggrtc 20 16 20 DNA Artificial Sequence Synthetic DNA 16 cgagctcttc attgatggcg 20 17 20 DNA Artificial Sequence Synthetic DNA 17 gcagctacac acgagttggg 20 18 20 DNA Artificial Sequence Synthetic DNA 18 gcaacaccta aatggttggg 20 19 20 DNA Artificial Sequence Synthetic DNA 19 gcaagaagtc tacaagcgcc 20 20 16 DNA Artificial Sequence Synthetic DNA 20 gccaacgtat tcacgg 16 21 20 DNA Artificial Sequence Synthetic DNA 21 tgatgaacca ctcgatcccc 20 22 20 DNA Artificial Sequence Synthetic DNA 22 aagacaccac cttctaccgc 20 23 20 DNA Artificial Sequence Synthetic DNA 23 caagtggaat tctgcagcgg 20 24 21 DNA Artificial Sequence Synthetic DNA 24 cctcctacaa aacctgctgg g 21 25 20 DNA Artificial Sequence Synthetic DNA 25 tcgcggatag cttttagggc 20 26 20 DNA Artificial Sequence Synthetic DNA 26 tgagttttta gaagactccc 20 27 20 DNA Artificial Sequence Synthetic DNA 27 cgcttcagtg gtgttgtccc 20 28 24 DNA Artificial Sequence Synthetic DNA 28 cgtaccactc cacggaaatt cccg 24 29 2369 DNA Brevibacterium lactofermentum CDS (484)..(1938) 29 acagaatcag cgccggcaga gaaacgtcca aagactaatc agagattcgg tataaaggta 60 aaaatcaacc tgcttaggcg tctttcgctt aaatagcgta gaatatcggg tcgatcgctt 120 ttaaacactc aggaggatcc ttgccggcca aaatcacgga cactcgtccc accccagaat 180 cccttcacgc tgttgaagag gaaaccgcag ccggtgcccg caggattgtt gccacctatt 240 ctaaggactt cttcgacggc gtcactttga tgtgcatgct cggcgttgaa cctcagggcc 300 tgcgttacac caaggtcgct tctgaacacg aggaagctca gccaaagaag gctacaaagc 360 ggactcgtaa ggctaccagc taagaaggct gctgctaaga aaacgaccaa gaagaccact 420 aagaaaacta ctaaaaagac caccgcaaag aagaccacaa agaagtctta agccggatct 480 tat atg gat gat tcc aat agc ttt gta gtt gtt gct aac cgt ctg cca 528 Met Asp Asp Ser Asn Ser Phe Val Val Val Ala Asn Arg Leu Pro 1 5 10 15 gtg gat atg act gtc cac cca gat ggt agc tat agc atc tcc ccc agc 576 Val Asp Met Thr Val His Pro Asp Gly Ser Tyr Ser Ile Ser Pro Ser 20 25 30 ccc ggt ggc ctt gtc acg ggg ctt tcc ccc gtt ctg gaa caa cat cgt 624 Pro Gly Gly Leu Val Thr Gly Leu Ser Pro Val Leu Glu Gln His Arg 35 40 45 gga tgt tgg gtc gga tgg cct gga act gta gat gtt gca ccc gaa cca 672 Gly Cys Trp Val Gly Trp Pro Gly Thr Val Asp Val Ala Pro Glu Pro 50 55 60 ttt cga aca gat acg ggt gtt ttg ctg cac cct gtt gtc ctc act gca 720 Phe Arg Thr Asp Thr Gly Val Leu Leu His Pro Val Val Leu Thr Ala 65 70 75 agt gac tat gaa ggc ttc tac gag ggc ttt tca aac gca acg ctg tgg 768 Ser Asp Tyr Glu Gly Phe Tyr Glu Gly Phe Ser Asn Ala Thr Leu Trp 80 85 90 95 cct ctt ttc cac gat ctg att gtt act ccg gtg tac aac acc gat tgg 816 Pro Leu Phe His Asp Leu Ile Val Thr Pro Val Tyr Asn Thr Asp Trp 100 105 110 tgg cat gcg ttt cgg gaa gta aac ctc aag ttc gct gaa gcc gtg agc 864 Trp His Ala Phe Arg Glu Val Asn Leu Lys Phe Ala Glu Ala Val Ser 115 120 125 caa gtg gcg gca cac ggt gcc act gtg tgg gtg cag gac tat cag ctg 912 Gln Val Ala Ala His Gly Ala Thr Val Trp Val Gln Asp Tyr Gln Leu 130 135 140 ttg ctg gtt cct ggc att ttg cgc cag atg cgc ctt gat ttg aag atc 960 Leu Leu Val Pro Gly Ile Leu Arg Gln Met Arg Leu Asp Leu Lys Ile 145 150 155 ggt ttc ttc ctc cac att ccc ttc cct tcc cct gat ctg ttc cgt cag 1008 Gly Phe Phe Leu His Ile Pro Phe Pro Ser Pro Asp Leu Phe Arg Gln 160 165 170 175 ctg ccg tgg cgt gaa gag att gtt cga ggc atg ctg ggc gca gat ttg 1056 Leu Pro Trp Arg Glu Glu Ile Val Arg Gly Met Leu Gly Ala Asp Leu 180 185 190 gtg gga ttc cat ttg gtt caa aac gca gaa aac ttc ctt gcg tta acc 1104 Val Gly Phe His Leu Val Gln Asn Ala Glu Asn Phe Leu Ala Leu Thr 195 200 205 cag cag gtt gcc ggc act gcc ggg tct cat gtg ggt cag ccg gac acc 1152 Gln Gln Val Ala Gly Thr Ala Gly Ser His Val Gly Gln Pro Asp Thr 210 215 220 ttg cag gtc agt ggt gaa gca ttg gtg cgt gag att ggc gct cat gtt 1200 Leu Gln Val Ser Gly Glu Ala Leu Val Arg Glu Ile Gly Ala His Val 225 230 235 gaa acc gct gac gga agg cga gtt agc gtc ggg gcg ttc ccg atc tcg 1248 Glu Thr Ala Asp Gly Arg Arg Val Ser Val Gly Ala Phe Pro Ile Ser 240 245 250 255 att gat gtt gaa atg ttt ggg gag gcg tcg aaa agc gcc gtt ctt gat 1296 Ile Asp Val Glu Met Phe Gly Glu Ala Ser Lys Ser Ala Val Leu Asp 260 265 270 ctt tta aaa acg ctc gac gag ccg gaa acc gta ttc ctg ggc gtt gac 1344 Leu Leu Lys Thr Leu Asp Glu Pro Glu Thr Val Phe Leu Gly Val Asp 275 280 285 cga ctg gac tac acc aag ggc att ttg cag cgc ctg ctt gcg ttt gag 1392 Arg Leu Asp Tyr Thr Lys Gly Ile Leu Gln Arg Leu Leu Ala Phe Glu 290 295 300 gaa ctg ctg gaa tcc ggc gcg ttg gag gcc gac aaa gct gtg ttg ctg 1440 Glu Leu Leu Glu Ser Gly Ala Leu Glu Ala Asp Lys Ala Val Leu Leu 305 310 315 cag gtc gcg acg cct tcg cgt gag cgc att gat cac tat cgt gtg tcg 1488 Gln Val Ala Thr Pro Ser Arg Glu Arg Ile Asp His Tyr Arg Val Ser 320 325 330 335 cgt tcg cag gtc gag gaa gcc gtc ggc cgt atc aat ggt cgt ttc ggt 1536 Arg Ser Gln Val Glu Glu Ala Val Gly Arg Ile Asn Gly Arg Phe Gly 340 345 350 cgc atg ggg cgt ccc gtg gtg cat tat cta cac agg tca ttg agc aaa 1584 Arg Met Gly Arg Pro Val Val His Tyr Leu His Arg Ser Leu Ser Lys 355 360 365 aat gat ctc cag gtg ctg tat acc gca gcc gat gtc atg ctg gtt acg 1632 Asn Asp Leu Gln Val Leu Tyr Thr Ala Ala Asp Val Met Leu Val Thr 370 375 380 cct ttt aaa gac ggt atg aac ttg gtg gct aaa gaa ttc gtg gcc aac 1680 Pro Phe Lys Asp Gly Met Asn Leu Val Ala Lys Glu Phe Val Ala Asn 385 390 395 cac cgc gac ggc act ggt gct ttg gtg ctg tcc gaa ttt gcc ggc gcg 1728 His Arg Asp Gly Thr Gly Ala Leu Val Leu Ser Glu Phe Ala Gly Ala 400 405 410 415 gcc act gag ctg acc ggt gcg tat tta tgc aac cca ttt gat gtg gaa 1776 Ala Thr Glu Leu Thr Gly Ala Tyr Leu Cys Asn Pro Phe Asp Val Glu 420 425 430 tcc atc aaa cgg caa atg gtg gca gct gtc cat gat ttg aag cac aat 1824 Ser Ile Lys Arg Gln Met Val Ala Ala Val His Asp Leu Lys His Asn 435 440 445 ccg gaa tct gcg gca acg cga atg aaa acg aac agc gag cag gtc tat 1872 Pro Glu Ser Ala Ala Thr Arg Met Lys Thr Asn Ser Glu Gln Val Tyr 450 455 460 acc cac gac gtc aac gtg tgg gct aat agt ttc ctg gat tgt ttg gcg 1920 Thr His Asp Val Asn Val Trp Ala Asn Ser Phe Leu Asp Cys Leu Ala 465 470 475 cag tcg gga gaa aac tca tgaaccgcgc acgaatcgcg accataggcg 1968 Gln Ser Gly Glu Asn Ser 480 485 ttcttccgct tgctttactg ctggcgtcct gtggttcaga caccgtggaa atgacagatt 2028 ccacctggtt ggtgaccaat atttacaccg atccagatga gtcgaattcg atcagtaatc 2088 ttgtcatttc ccagcccagc ttagattttg gcaattcttc cctgtctggt ttcactggct 2148 gtgtgccttt tacggggcgt gcggaattct tccaaaatgg tgagcaaagc tctgttctgg 2208 atgccgatta tgtgaccttg tcttccctgg atttcgataa acttcccgat gattgccaag 2268 gacaagaact caaagttcat aacgagctgg ttgatcttct gcctggttct tttgaaatct 2328 ccaggacttc tggttcagaa atcttgctga ctagcgatgt c 2369 30 485 PRT Brevibacterium lactofermentum 30 Met Asp Asp Ser Asn Ser Phe Val Val Val Ala Asn Arg Leu Pro Val 1 5 10 15 Asp Met Thr Val His Pro Asp Gly Ser Tyr Ser Ile Ser Pro Ser Pro 20 25 30 Gly Gly Leu Val Thr Gly Leu Ser Pro Val Leu Glu Gln His Arg Gly 35 40 45 Cys Trp Val Gly Trp Pro Gly Thr Val Asp Val Ala Pro Glu Pro Phe 50 55 60 Arg Thr Asp Thr Gly Val Leu Leu His Pro Val Val Leu Thr Ala Ser 65 70 75 80 Asp Tyr Glu Gly Phe Tyr Glu Gly Phe Ser Asn Ala Thr Leu Trp Pro 85 90 95 Leu Phe His Asp Leu Ile Val Thr Pro Val Tyr Asn Thr Asp Trp Trp 100 105 110 His Ala Phe Arg Glu Val Asn Leu Lys Phe Ala Glu Ala Val Ser Gln 115 120 125 Val Ala Ala His Gly Ala Thr Val Trp Val Gln Asp Tyr Gln Leu Leu 130 135 140 Leu Val Pro Gly Ile Leu Arg Gln Met Arg Leu Asp Leu Lys Ile Gly 145 150 155 160 Phe Phe Leu His Ile Pro Phe Pro Ser Pro Asp Leu Phe Arg Gln Leu 165 170 175 Pro Trp Arg Glu Glu Ile Val Arg Gly Met Leu Gly Ala Asp Leu Val 180 185 190 Gly Phe His Leu Val Gln Asn Ala Glu Asn Phe Leu Ala Leu Thr Gln 195 200 205 Gln Val Ala Gly Thr Ala Gly Ser His Val Gly Gln Pro Asp Thr Leu 210 215 220 Gln Val Ser Gly Glu Ala Leu Val Arg Glu Ile Gly Ala His Val Glu 225 230 235 240 Thr Ala Asp Gly Arg Arg Val Ser Val Gly Ala Phe Pro Ile Ser Ile 245 250 255 Asp Val Glu Met Phe Gly Glu Ala Ser Lys Ser Ala Val Leu Asp Leu 260 265 270 Leu Lys Thr Leu Asp Glu Pro Glu Thr Val Phe Leu Gly Val Asp Arg 275 280 285 Leu Asp Tyr Thr Lys Gly Ile Leu Gln Arg Leu Leu Ala Phe Glu Glu 290 295 300 Leu Leu Glu Ser Gly Ala Leu Glu Ala Asp Lys Ala Val Leu Leu Gln 305 310 315 320 Val Ala Thr Pro Ser Arg Glu Arg Ile Asp His Tyr Arg Val Ser Arg 325 330 335 Ser Gln Val Glu Glu Ala Val Gly Arg Ile Asn Gly Arg Phe Gly Arg 340 345 350 Met Gly Arg Pro Val Val His Tyr Leu His Arg Ser Leu Ser Lys Asn 355 360 365 Asp Leu Gln Val Leu Tyr Thr Ala Ala Asp Val Met Leu Val Thr Pro 370 375 380 Phe Lys Asp Gly Met Asn Leu Val Ala Lys Glu Phe Val Ala Asn His 385 390 395 400 Arg Asp Gly Thr Gly Ala Leu Val Leu Ser Glu Phe Ala Gly Ala Ala 405 410 415 Thr Glu Leu Thr Gly Ala Tyr Leu Cys Asn Pro Phe Asp Val Glu Ser 420 425 430 Ile Lys Arg Gln Met Val Ala Ala Val His Asp Leu Lys His Asn Pro 435 440 445 Glu Ser Ala Ala Thr Arg Met Lys Thr Asn Ser Glu Gln Val Tyr Thr 450 455 460 His Asp Val Asn Val Trp Ala Asn Ser Phe Leu Asp Cys Leu Ala Gln 465 470 475 480 Ser Gly Glu Asn Ser 485 31 2956 DNA Brevibacterium lactofermentum CDS (82)..(2514) 31 ttttcccacg cagggaaggc gtgaacacta agatcgagga cgtaccgcac gattttgcct 60 aacttttaag ggtgtttcat c atg gca cgt cca att tcc gca acg tac agg 111 Met Ala Arg Pro Ile Ser Ala Thr Tyr Arg 1 5 10 ctt caa atg cga gga cct caa gca gat agc gcc ggg cgt ttc ttt ggt 159 Leu Gln Met Arg Gly Pro Gln Ala Asp Ser Ala Gly Arg Phe Phe Gly 15 20 25 ttt gcg cag gcc aaa gcc cag ctt ccc tat ctg aag aag cta ggc atc 207 Phe Ala Gln Ala Lys Ala Gln Leu Pro Tyr Leu Lys Lys Leu Gly Ile 30 35 40 agc cac ctg tac ctc tcc cct att ttt acg gcc atg cca gat tcc aat 255 Ser His Leu Tyr Leu Ser Pro Ile Phe Thr Ala Met Pro Asp Ser Asn 45 50 55 cat ggc tac gat gtc att gat ccc acc gcc atc aat gaa gag ctc ggt 303 His Gly Tyr Asp Val Ile Asp Pro Thr Ala Ile Asn Glu Glu Leu Gly 60 65 70 ggc atg gag ggt ctt cga gat ctt gct gca gct aca cac gag ttg ggc 351 Gly Met Glu Gly Leu Arg Asp Leu Ala Ala Ala Thr His Glu Leu Gly 75 80 85 90 atg ggc atc atc att gat att gtt ccc aac cat tta ggt gtt gcc gtt 399 Met Gly Ile Ile Ile Asp Ile Val Pro Asn His Leu Gly Val Ala Val 95 100 105 cca cat ttg aat cct tgg tgg tgg gat gtt cta aaa aac ggc aaa gat 447 Pro His Leu Asn Pro Trp Trp Trp Asp Val Leu Lys Asn Gly Lys Asp 110 115 120 tcc gct ttt gag ttc tat ttc gat att gac tgg cac gaa gac aac ggt 495 Ser Ala Phe Glu Phe Tyr Phe Asp Ile Asp Trp His Glu Asp Asn Gly 125 130 135 tct ggt ggc aag ctg ggc atg ccg att ctg ggt gct gaa ggc gat gaa 543 Ser Gly Gly Lys Leu Gly Met Pro Ile Leu Gly Ala Glu Gly Asp Glu 140 145 150 gac aag ctg gaa ttc gcg gag ctt gat gga gag aaa gtg ctc aaa tat 591 Asp Lys Leu Glu Phe Ala Glu Leu Asp Gly Glu Lys Val Leu Lys Tyr 155 160 165 170 ttt gac cac ctc ttc cca atc gcg cct ggt acc gaa gaa ggg aca ccg 639 Phe Asp His Leu Phe Pro Ile Ala Pro Gly Thr Glu Glu Gly Thr Pro 175 180 185 caa gaa gtc tac aag cgc cag cat tac cgc ctg cag ttc tgg cgc gac 687 Gln Glu Val Tyr Lys Arg Gln His Tyr Arg Leu Gln Phe Trp Arg Asp 190 195 200 ggc gtg atc aac ttc cgt cgc ttc ttt tcc gtg aat acg ttg gct ggc 735 Gly Val Ile Asn Phe Arg Arg Phe Phe Ser Val Asn Thr Leu Ala Gly 205 210 215 atc agg caa gaa gat ccc ttg gtg ttt gaa cat act cat cgt ctg ctg 783 Ile Arg Gln Glu Asp Pro Leu Val Phe Glu His Thr His Arg Leu Leu 220 225 230 cgc gaa ttg gtg gcg gaa gac ctc att gac ggc gtg cgc gtc gat cac 831 Arg Glu Leu Val Ala Glu Asp Leu Ile Asp Gly Val Arg Val Asp His 235 240 245 250 ccc gac ggg ctt tcc gat cct ttt gga tat ctg cac aga ctc cgc gac 879 Pro Asp Gly Leu Ser Asp Pro Phe Gly Tyr Leu His Arg Leu Arg Asp 255 260 265 ctc att gga cct gac cgc tgg ctg atc atc gaa aag atc ttg agc gtt 927 Leu Ile Gly Pro Asp Arg Trp Leu Ile Ile Glu Lys Ile Leu Ser Val 270 275 280 gat gaa cca ctc gat ccc cgc ctg gcc gtt gat ggc acc act ggc tac 975 Asp Glu Pro Leu Asp Pro Arg Leu Ala Val Asp Gly Thr Thr Gly Tyr 285 290 295 gac ccc ctc cgt gaa ctc gac ggc gtg ttt atc tcc cga gaa tct gag 1023 Asp Pro Leu Arg Glu Leu Asp Gly Val Phe Ile Ser Arg Glu Ser Glu 300 305 310 gac aaa ttc tcc atg ttg gcg ctg acc cac agt gga tcc acc tgg gat 1071 Asp Lys Phe Ser Met Leu Ala Leu Thr His Ser Gly Ser Thr Trp Asp 315 320 325 330 gaa cgc gcc cta aaa tcc acg gag gaa agc ctc aaa cga gtc gtc gcg 1119 Glu Arg Ala Leu Lys Ser Thr Glu Glu Ser Leu Lys Arg Val Val Ala 335 340 345 caa caa gaa ctc gca gcc gaa atc tta agg ctc gcc cgc gcc atg cgc 1167 Gln Gln Glu Leu Ala Ala Glu Ile Leu Arg Leu Ala Arg Ala Met Arg 350 355 360 cgc gat aac ttc tcc acc gca ggc acc aac gtc acc gaa gac aaa ctt 1215 Arg Asp Asn Phe Ser Thr Ala Gly Thr Asn Val Thr Glu Asp Lys Leu 365 370 375 agc gaa acc atc atc gaa tta gtc gcc gcc atg ccc gtc tac cgc gcc 1263 Ser Glu Thr Ile Ile Glu Leu Val Ala Ala Met Pro Val Tyr Arg Ala 380 385 390 gac tac atc tcc ctc tca cgc acc acc gcc acc gtc atc gcg gag atg 1311 Asp Tyr Ile Ser Leu Ser Arg Thr Thr Ala Thr Val Ile Ala Glu Met 395 400 405 410 tcc aaa cgc ttc ccc tcc cgg cgc gac gca ctc gac ctc atc tcg gcc 1359 Ser Lys Arg Phe Pro Ser Arg Arg Asp Ala Leu Asp Leu Ile Ser Ala 415 420 425 gcc cta ctt ggc aat ggc gag gcc aaa atc cgc ttc gcc caa gtc tgc 1407 Ala Leu Leu Gly Asn Gly Glu Ala Lys Ile Arg Phe Ala Gln Val Cys 430 435 440 ggc gcc gtc atg gcc aaa ggt gtg gaa gac acc acc ttc tac cgc gca 1455 Gly Ala Val Met Ala Lys Gly Val Glu Asp Thr Thr Phe Tyr Arg Ala 445 450 455 tct agg ctc gtt gca ctg caa gaa gtc ggt ggc gcg ccg ggc agg ttc 1503 Ser Arg Leu Val Ala Leu Gln Glu Val Gly Gly Ala Pro Gly Arg Phe 460 465 470 ggc gtc tcc gct gca gaa ttc cac ttg ctg cag gaa gaa cgc agc ctg 1551 Gly Val Ser Ala Ala Glu Phe His Leu Leu Gln Glu Glu Arg Ser Leu 475 480 485 490 ctg tgg cca cgc acc atg acc acc ttg tcc acg cac gac acc aaa cgc 1599 Leu Trp Pro Arg Thr Met Thr Thr Leu Ser Thr His Asp Thr Lys Arg 495 500 505 ggc gaa gat acc cgc gcc cgc atc atc tcc ctg tcc gaa gtc ccc gat 1647 Gly Glu Asp Thr Arg Ala Arg Ile Ile Ser Leu Ser Glu Val Pro Asp 510 515 520 atg tac tcc gag ctg gtc aat cgt gtt ttc gca gtg ctc ccc gcg cca 1695 Met Tyr Ser Glu Leu Val Asn Arg Val Phe Ala Val Leu Pro Ala Pro 525 530 535 gac ggc gca acg ggc agt ttc ctc cta caa aac ctg ctg ggc gta tgg 1743 Asp Gly Ala Thr Gly Ser Phe Leu Leu Gln Asn Leu Leu Gly Val Trp 540 545 550 ccc gcc gac ggc gtg atc acc gat gcg ctg cgc gat cga ttc agg gaa 1791 Pro Ala Asp Gly Val Ile Thr Asp Ala Leu Arg Asp Arg Phe Arg Glu 555 560 565 570 tac gcc cta aaa gct atc cgc gaa gca tcc aca aaa acc acg tgg gtg 1839 Tyr Ala Leu Lys Ala Ile Arg Glu Ala Ser Thr Lys Thr Thr Trp Val 575 580 585 gac ccc aac gag tcc ttc gag gct gcg gtc tgc gat tgg gtg gaa gcg 1887 Asp Pro Asn Glu Ser Phe Glu Ala Ala Val Cys Asp Trp Val Glu Ala 590 595 600 ctt ttc gac gga ccc tcc acc tca tta atc acc gaa ttt gtc tcc cac 1935 Leu Phe Asp Gly Pro Ser Thr Ser Leu Ile Thr Glu Phe Val Ser His 605 610 615 atc aac cgt ggc tct gtg aat atc tcc tta ggt agg aaa ctg ctg caa 1983 Ile Asn Arg Gly Ser Val Asn Ile Ser Leu Gly Arg Lys Leu Leu Gln 620 625 630 atg gtg ggc gct gga atc ccc gac act tac caa gga act gag ttt tta 2031 Met Val Gly Ala Gly Ile Pro Asp Thr Tyr Gln Gly Thr Glu Phe Leu 635 640 645 650 gaa gac tcc ctg gta gat ccc gat aac cga cgc ttt gtt gat tac acc 2079 Glu Asp Ser Leu Val Asp Pro Asp Asn Arg Arg Phe Val Asp Tyr Thr 655 660 665 gcc aga gaa caa gtc ctg gag cgc ctg caa acc tgg gat tgg acg cag 2127 Ala Arg Glu Gln Val Leu Glu Arg Leu Gln Thr Trp Asp Trp Thr Gln 670 675 680 gtt aat tcg gta gaa gac ttg gtg gat aac gcc gac atc gcc aaa atg 2175 Val Asn Ser Val Glu Asp Leu Val Asp Asn Ala Asp Ile Ala Lys Met 685 690 695 gcc gtg gtc cat aaa tcc ctc gag ttg cgt gct gaa ttt cgt gca agc 2223 Ala Val Val His Lys Ser Leu Glu Leu Arg Ala Glu Phe Arg Ala Ser 700 705 710 ttt gtt ggt gga gat cat cag gca gta ttt ggc gaa ggt cgc gca gaa 2271 Phe Val Gly Gly Asp His Gln Ala Val Phe Gly Glu Gly Arg Ala Glu 715 720 725 730 tcc cac atc atg ggc atc gcc cgc ggt aca gac cga aac cac ctc aac 2319 Ser His Ile Met Gly Ile Ala Arg Gly Thr Asp Arg Asn His Leu Asn 735 740 745 atc att gct ctt gct acc cgt cga cca ctg atc ttg gaa gac cgt ggc 2367 Ile Ile Ala Leu Ala Thr Arg Arg Pro Leu Ile Leu Glu Asp Arg Gly 750 755 760 gga tgg tat gac acc acc gtc acg ctt cct ggt gga caa tgg gaa gac 2415 Gly Trp Tyr Asp Thr Thr Val Thr Leu Pro Gly Gly Gln Trp Glu Asp 765 770 775 agg ctc acc ggg caa cgc ttc agt ggt gtt gtc cca gcc acc gat ttg 2463 Arg Leu Thr Gly Gln Arg Phe Ser Gly Val Val Pro Ala Thr Asp Leu 780 785 790 ttc tca cat tta ccc gta tct ttg ttg gtt tta gta ccc gat agt gag 2511 Phe Ser His Leu Pro Val Ser Leu Leu Val Leu Val Pro Asp Ser Glu 795 800 805 810 ttt tgatccctgc acaggaaagt tagcggcgct actatgaacg atcgatatgt 2564 Phe ctgacaacac tctctcccaa tttggcagtt actaccacga attccgacgt gcccatccca 2624 tggccgacgt cgaattcctc ctagcaattg aagaattact cacagacggt ggtgtcacct 2684 tcgatcgcgt caccacacgc atcaaagaat ggtcaagcct gaaagccaag gctcgcaagc 2744 gtcgcgacga tggctcgttg atctaccctg atccgcgcaa agacatccac gacatgatcg 2804 gtgttcggat caccacgtac cactccacgg aaattcccgt ggccttaaaa gtgctccaag 2864 actccttcat cgtccacaaa tccgtagaca aagccgctga aactcgcatc tcaggcggct 2924 ttggttacgg ctcccaccac caaggattnt ag 2956 32 811 PRT Brevibacterium lactofermentum 32 Met Ala Arg Pro Ile Ser Ala Thr Tyr Arg Leu Gln Met Arg Gly Pro 1 5 10 15 Gln Ala Asp Ser Ala Gly Arg Phe Phe Gly Phe Ala Gln Ala Lys Ala 20 25 30 Gln Leu Pro Tyr Leu Lys Lys Leu Gly Ile Ser His Leu Tyr Leu Ser 35 40 45 Pro Ile Phe Thr Ala Met Pro Asp Ser Asn His Gly Tyr Asp Val Ile 50 55 60 Asp Pro Thr Ala Ile Asn Glu Glu Leu Gly Gly Met Glu Gly Leu Arg 65 70 75 80 Asp Leu Ala Ala Ala Thr His Glu Leu Gly Met Gly Ile Ile Ile Asp 85 90 95 Ile Val Pro Asn His Leu Gly Val Ala Val Pro His Leu Asn Pro Trp 100 105 110 Trp Trp Asp Val Leu Lys Asn Gly Lys Asp Ser Ala Phe Glu Phe Tyr 115 120 125 Phe Asp Ile Asp Trp His Glu Asp Asn Gly Ser Gly Gly Lys Leu Gly 130 135 140 Met Pro Ile Leu Gly Ala Glu Gly Asp Glu Asp Lys Leu Glu Phe Ala 145 150 155 160 Glu Leu Asp Gly Glu Lys Val Leu Lys Tyr Phe Asp His Leu Phe Pro 165 170 175 Ile Ala Pro Gly Thr Glu Glu Gly Thr Pro Gln Glu Val Tyr Lys Arg 180 185 190 Gln His Tyr Arg Leu Gln Phe Trp Arg Asp Gly Val Ile Asn Phe Arg 195 200 205 Arg Phe Phe Ser Val Asn Thr Leu Ala Gly Ile Arg Gln Glu Asp Pro 210 215 220 Leu Val Phe Glu His Thr His Arg Leu Leu Arg Glu Leu Val Ala Glu 225 230 235 240 Asp Leu Ile Asp Gly Val Arg Val Asp His Pro Asp Gly Leu Ser Asp 245 250 255 Pro Phe Gly Tyr Leu His Arg Leu Arg Asp Leu Ile Gly Pro Asp Arg 260 265 270 Trp Leu Ile Ile Glu Lys Ile Leu Ser Val Asp Glu Pro Leu Asp Pro 275 280 285 Arg Leu Ala Val Asp Gly Thr Thr Gly Tyr Asp Pro Leu Arg Glu Leu 290 295 300 Asp Gly Val Phe Ile Ser Arg Glu Ser Glu Asp Lys Phe Ser Met Leu 305 310 315 320 Ala Leu Thr His Ser Gly Ser Thr Trp Asp Glu Arg Ala Leu Lys Ser 325 330 335 Thr Glu Glu Ser Leu Lys Arg Val Val Ala Gln Gln Glu Leu Ala Ala 340 345 350 Glu Ile Leu Arg Leu Ala Arg Ala Met Arg Arg Asp Asn Phe Ser Thr 355 360 365 Ala Gly Thr Asn Val Thr Glu Asp Lys Leu Ser Glu Thr Ile Ile Glu 370 375 380 Leu Val Ala Ala Met Pro Val Tyr Arg Ala Asp Tyr Ile Ser Leu Ser 385 390 395 400 Arg Thr Thr Ala Thr Val Ile Ala Glu Met Ser Lys Arg Phe Pro Ser 405 410 415 Arg Arg Asp Ala Leu Asp Leu Ile Ser Ala Ala Leu Leu Gly Asn Gly 420 425 430 Glu Ala Lys Ile Arg Phe Ala Gln Val Cys Gly Ala Val Met Ala Lys 435 440 445 Gly Val Glu Asp Thr Thr Phe Tyr Arg Ala Ser Arg Leu Val Ala Leu 450 455 460 Gln Glu Val Gly Gly Ala Pro Gly Arg Phe Gly Val Ser Ala Ala Glu 465 470 475 480 Phe His Leu Leu Gln Glu Glu Arg Ser Leu Leu Trp Pro Arg Thr Met 485 490 495 Thr Thr Leu Ser Thr His Asp Thr Lys Arg Gly Glu Asp Thr Arg Ala 500 505 510 Arg Ile Ile Ser Leu Ser Glu Val Pro Asp Met Tyr Ser Glu Leu Val 515 520 525 Asn Arg Val Phe Ala Val Leu Pro Ala Pro Asp Gly Ala Thr Gly Ser 530 535 540 Phe Leu Leu Gln Asn Leu Leu Gly Val Trp Pro Ala Asp Gly Val Ile 545 550 555 560 Thr Asp Ala Leu Arg Asp Arg Phe Arg Glu Tyr Ala Leu Lys Ala Ile 565 570 575 Arg Glu Ala Ser Thr Lys Thr Thr Trp Val Asp Pro Asn Glu Ser Phe 580 585 590 Glu Ala Ala Val Cys Asp Trp Val Glu Ala Leu Phe Asp Gly Pro Ser 595 600 605 Thr Ser Leu Ile Thr Glu Phe Val Ser His Ile Asn Arg Gly Ser Val 610 615 620 Asn Ile Ser Leu Gly Arg Lys Leu Leu Gln Met Val Gly Ala Gly Ile 625 630 635 640 Pro Asp Thr Tyr Gln Gly Thr Glu Phe Leu Glu Asp Ser Leu Val Asp 645 650 655 Pro Asp Asn Arg Arg Phe Val Asp Tyr Thr Ala Arg Glu Gln Val Leu 660 665 670 Glu Arg Leu Gln Thr Trp Asp Trp Thr Gln Val Asn Ser Val Glu Asp 675 680 685 Leu Val Asp Asn Ala Asp Ile Ala Lys Met Ala Val Val His Lys Ser 690 695 700 Leu Glu Leu Arg Ala Glu Phe Arg Ala Ser Phe Val Gly Gly Asp His 705 710 715 720 Gln Ala Val Phe Gly Glu Gly Arg Ala Glu Ser His Ile Met Gly Ile 725 730 735 Ala Arg Gly Thr Asp Arg Asn His Leu Asn Ile Ile Ala Leu Ala Thr 740 745 750 Arg Arg Pro Leu Ile Leu Glu Asp Arg Gly Gly Trp Tyr Asp Thr Thr 755 760 765 Val Thr Leu Pro Gly Gly Gln Trp Glu Asp Arg Leu Thr Gly Gln Arg 770 775 780 Phe Ser Gly Val Val Pro Ala Thr Asp Leu Phe Ser His Leu Pro Val 785 790 795 800 Ser Leu Leu Val Leu Val Pro Asp Ser Glu Phe 805 810 33 30 DNA Artificial Sequence Synthetic DNA 33 ccaaaatcga taacatcaat cgagatcggg 30 34 30 DNA Artificial Sequence Synthetic DNA 34 cttgatcgat taaaaacgct cgacgagccg 30

Claims

What is claimed is:

1. A coryneform bacterium having L-glutamic acid producing ability, wherein trehalose synthesis ability is decreased or deleted in the bacterium.

2. The coryneform bacteria according to claim 1, wherein the trehalose synthesis ability is decreased or deleted by introducing a mutation into a chromosomal gene coding for an enzyme in trehalose systhesis pathway or disrupting the gene.

3. The coryneform bacteria according to claim 2, wherein the gene coding for the enzyme in trehalose synthesis pathway consists of a gene coding for trehalose-6-phosphate synthase, a gene coding for maltooligosyltrehalose synthase, or both of these genes.

4. The coryneform bacteria according to claim 3, wherein the gene coding for trehalose-6-phosphate synthase codes for the amino acid sequence of SEQ ID NO: 30, and the gene coding for maltooligosyltrehalose synthase codes for the amino acid sequence of SEQ ID NO: 32.

5. A method for producing L-glutamic acid comprising the steps of culturing a coryneform bacterium according to any one of claims 1-4 in a medium to produce and accumulate L-glutamic acid in the medium, and collecting the L-glutamic acid from the medium.

6. A DNA coding for a protein defined in the following (A) or (B):

(A) a protein having the amino acid sequence of SEQ ID NO: 30,

(B) a protein having the amino acid sequence of SEQ ID NO: 30 including substitution, deletion, insertion or addition of one or several amino acid residues and having trehalose-6-phosphate synthase activity.

7. A DNA according to claim 6, which is a DNA defined in the following (a) or (b):

8. A DNA coding for a protein defined in the following (A) or (B):

(A) a protein having the amino acid sequence of SEQ ID NO: 32,

(B) a protein having the amino acid sequence of SEQ ID NO: 32 including substitution, deletion, insertion or addition of one or several amino acid residues and having maltooligosyltrehalose synthase activity.

9. A DNA according to claim 8, which is a DNA defined in the following (a) or (b):