JP2003169668A - L-cysteine-producing microorganism and method for producing l-cysteine - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new method for the production of L-cysteine by finding a yeast having cysteine desulfuhydrase activity and its gene in Escherichia bacteria and using the gene for the breeding of an L-crysteine producing strain. <P>SOLUTION: A bacterial strain of the genus Escherichia having L-crysteine productivity and lowered or depleted cystathionine-β-lyase activity, or cystathionine-β-lyase activity and tryptophanase activity is cultured in a medium to produce and accumulate L-cysteine in the medium and the accumulated L-cysteine is separated from the medium. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for producing L-cysteine, and more particularly to a microorganism suitable for producing L-cysteine, and a method for producing L-cysteine using the same. L-cysteine and derivatives of L-cysteine are
It is used in the fields of medicine, cosmetics and food.

[0002]

2. Description of the Related Art Conventionally, L-cysteine has been obtained by extraction from keratin-containing substances such as hair, horns and feathers, or by microbial enzyme conversion using DL-2-aminothiazoline-4-carboxylic acid as a precursor. Has been. Also,
Large-scale production of L-cysteine by an immobilized enzyme method using a novel enzyme is also planned.

Further, L- by a fermentation method using microorganisms
Attempts have also been made to produce cysteine. For example, the inventors of the present invention have suppressed the L-cysteine degradation system and reduced the feedback inhibition by L-cysteine, which is serine acetyltransfera.
se (EC 2.3.1.30): Hereinafter, a method for producing L-cysteine using a bacterium belonging to the genus Escherichia that holds se (EC) is disclosed (Patent Document 1). As a means for suppressing the L-cysteine degradation system, it is disclosed that cysteine desulfhydrase (hereinafter, also referred to as “CD”) activity in cells is reduced. Similarly, SAs with certain mutations have reduced feedback inhibition by L-cysteine.
A method for producing L-cysteine using a microorganism having a cysteine substance metabolism deregulated by a DNA sequence encoding T is known (Patent Document 2).

Non-Patent Document 1 discloses a method for producing L-cysteine using Escherichia coli into which a gene encoding a SAT isozyme derived from Arabidopsis thaliana that is not subject to feedback inhibition by L-cysteine is introduced. .

On the other hand, there has been reported a method for producing L-cysteine using a microorganism which overexpresses a gene encoding a protein suitable for releasing an antibiotic or a substance toxic to the microorganism directly from cells (Patent Reference 3).

[0006] As described above, many studies have been conducted on the enhancement of the activity of L-cysteine biosynthetic enzymes such as SAT and the L-cysteine producing bacterium in which the L-cysteine excretion system is modified. On the other hand, the L-cysteine degrading system has not been studied in detail, particularly in bacteria belonging to the genus Escherichia.

In Escherichia coli
As an enzyme having CD activity, cystathionine-β-lyase (metC product; hereinafter, also referred to as “CBL”) (Non-patent document 2), and tryptophanase (tnaA product, hereinafter, “TN
(also referred to as “ase”) (Non-Patent Document 3).
However, CBL is an enzyme that catalyzes the reaction of converting cystathionine to homocysteine, and TNase is an enzyme that catalyzes the reaction of degrading tryptophan.
-It was not known whether it was an enzyme responsible for the cysteine degradation system. Further, Patent Document 1 describes a mutant strain having reduced CD activity, but does not report on the main body of the enzyme responsible for CD activity.

[0008]

[Patent Document 1] JP-A-11-155571

[Patent Document 2] Special Table No. 2000-504926

[Patent Document 3] Japanese Patent Laid-Open No. 11-56381

[Non-Patent Document 1] FEMS Microbiol. Lett., 179 (1999)
453-459

[Non-patent document 2] Chandra et. Al., Biochemistry, 21.
(1982) 3064-3069

[Non-Patent Document 3] Austin Newton et. Al., J. Biol. Ch.
em. 240 (1965) 1211-1218

[0009]

DISCLOSURE OF THE INVENTION The present invention clarifies an enzyme responsible for CD activity and its gene, and utilizes the same gene for breeding L-cysteine-producing bacteria to provide a new method for producing L-cysteine. This is an issue.

[0010]

[Means for Solving the Problems] As a result of intensive studies to solve the above problems, the present inventors have found that CBL and TNase are the enzymes responsible for the major CD activity of Escherichia coli. It was found that the ability to produce L-cysteine can be improved by reducing or eliminating the enzyme activity of, and thus the present invention has been completed. That is, the present invention is as follows.

(1) A bacterium belonging to the genus Escherichia, which has the ability to produce L-cysteine and has been modified so that the cystathionine-β-lyase activity is reduced or deleted. (2) A bacterium belonging to the genus Escherichia, which has L-cysteine producing ability and has been modified so that ciscystathionine-β-lyase activity and tryptophanase activity are reduced or deleted. (3) The Escherichia bacterium of (1), in which the gene encoding cystathionine-β-lyase is disrupted. (4) The bacterium belonging to the genus Escherichia of (2), in which the gene encoding cis-cystathionine-β-lyase and the gene encoding tryptophanase are disrupted. (5) The bacterium of the genus Escherichia according to any one of (1) to (4), which is further modified so that the enzyme activity of the L-cysteine biosynthesis system is enhanced. (6) The Escherichia bacterium according to (5), which has been modified to enhance serine acetyltransferase. (7) Retains serine acetyltransferase that is insensitive to feedback inhibition by L-cysteine
(6) Escherichia bacterium. (8) The Escherichia bacterium according to any one of (1) to (7), which is Escherichia coli. (9) A method for producing L-cysteine, which comprises culturing the Escherichia bacterium according to any one of (1) to (8) in a medium, producing and accumulating L-cysteine in the medium, and collecting L-cysteine from the medium. . (10) cis-cystathionine-β of Escherichia bacterium
-A method for producing an L-cysteine-producing bacterium, which comprises increasing the L-cysteine-producing ability of the bacterium by reducing or eliminating lyase activity or tryptophanase activity, or both of these activities.

In the present invention, the L-cysteine producing ability means the ability to accumulate in the medium an amount of L-cysteine that can be recovered from the medium when the Escherichia bacterium of the present invention is cultured in the medium. In addition, "insensitive to feedback inhibition by L-cysteine" includes a case where feedback inhibition by L-cysteine is reduced or canceled and a case where feedback inhibition is not originally caused. In the present invention, L-cysteine refers to reduced L-cysteine or L-cystine or a mixture thereof unless otherwise specified.

[0013]

BEST MODE FOR CARRYING OUT THE INVENTION The first embodiment of the bacterium belonging to the genus Escherichia of the present invention has L-cysteine producing ability and CBL.
It is a bacterium belonging to the genus Escherichia modified so that the activity is reduced or deleted. The second form of the Escherichia bacterium of the present invention is an Escherichia bacterium which has L-cysteine producing ability and has been modified so that CBL activity and TNase activity are reduced or deleted. The Escherichia bacterium of the present invention is the Escherichia bacterium modified in the first form or the second form so that the L-cysteine biosynthesis enzyme activity is further enhanced. Such an Escherichia bacterium has a LBL in the Escherichia bacterium in which CBL activity or CBL activity and TNase activity is reduced or deleted.
-It is obtained by enhancing the enzyme activity of cysteine biosynthesis system. In addition, in Escherichia bacterium with enhanced L-cysteine biosynthesis enzyme activity, CBL activity or CBL
The Escherichia bacterium of the present invention can also be obtained by reducing or deleting the activity and TNase activity.

<1> Reduction or elimination of CBL activity or TNase activity To reduce or eliminate CBL activity or TNase activity of Escherichia bacterium, for example, Escherichia bacterium is irradiated with ultraviolet rays or N-methyl-N′-nitro-. Examples include a method of treating with a mutagen such as N-nitrosoguanidine (NTG) or nitrous acid, which is usually used for mutagenesis, and selecting a mutant having a decreased CBL activity or TNase activity. Also,
Bacteria belonging to the genus Escherichia with reduced CBL activity or TNase activity can be obtained by gene disruption as well as mutation treatment. In order to surely reduce or eliminate CBL activity or TNase activity, a method by gene disruption is preferable.
In Escherichia coli, CBL is metC gene, TNase is tn
Each is encoded by the aA gene.

Escherichia coli metC gene (GenBan
k accession M12858, Proc. Natl. Acad. Sci. USA 8
3, 867-871 (1986)) and tnaA gene (GenBank accessi
onK00032, J. Bacteriol. 147, 787-796 (1981), J. Ba
cteriol. 151, 942-951 (1982)) has been reported, using primers synthesized on the basis of the respective sequences and using Escherichia coli chromosomal DNA as a template.
PCR (PCR: polymerase chain reaction; White, TJ e
t al., Trends Genet. 5, 185 (1989)),
A DNA fragment containing each gene can be obtained. In addition, the inside of the gene encoding CBL was deleted, and the inside of the gene encoding TNase was deleted by deleting the metC gene (deleting type metC gene) modified so as not to produce a normally functioning CBL. The tnaA gene (deletion type tnaA gene) modified so that it does not produce TNase that functions as described above can be obtained by PCR using the primers shown in the examples. For reference, the nucleotide sequence of the metC gene and the encoded amino acid sequence are shown in SEQ ID NOs: 10 and 11. The nucleotide sequence of the tnaA gene and the encoded amino acid sequence are shown in SEQ ID NOs: 12 and 13.

The method for destroying the gene encoding CBL will be described below, but the gene encoding TNase can be similarly destroyed. DN containing the metC gene (deleted metC gene) modified so that the inside of the gene encoding CBL is deleted and does not produce a normally functioning CBL
By transforming a bacterium belonging to the genus Escherichia with A and causing recombination between the deleted metC gene and the metC gene on the chromosome, the metC gene on the chromosome can be destroyed. Gene disruption by gene replacement utilizing such homologous recombination has already been established, and there are methods such as a method using linear DNA and a method using a plasmid containing a temperature-sensitive replication origin.

The deletion type metC gene is replaced with metC on the host chromosome.
To replace with a gene, for example, the following may be performed. Recombinant DNA obtained by inserting a temperature-sensitive origin of replication, a deletion type metC gene, and a marker gene resistant to a drug such as ampicillin or chloramphenicol into a vector
And transforming Escherichia bacterium with this recombinant DNA, culturing the transformant strain at a temperature at which the temperature-sensitive origin of replication does not function, and then culturing this in a medium containing a drug to give the recombinant DNA. A transformant in which is integrated into the chromosomal DNA is obtained.

The strain in which the recombinant DNA has been integrated into the chromosome thus undergoes recombination with the metC gene sequence originally existing on the chromosome, and two fusion genes of the chromosomal metC gene and the deletion type metC gene are recombined. It is inserted into the chromosome with the other part of the DNA (vector part, temperature-sensitive origin of replication and drug resistance marker) sandwiched. Therefore, in this condition, the normal metC gene is dominant,
The transformant expresses normal metC.

Next, in order to leave only the deleted metC gene on the chromosomal DNA, the two metC genes were recombined to 1
Copy metC gene along with vector part (including temperature sensitive origin of replication and drug resistance marker) on chromosome DN
Remove from A. At that time, there are cases where the normal metC gene is left on the chromosomal DNA and the deletion type metC gene is excised, and conversely, the deletion type metC gene is left on the chromosomal DNA and the normal metC gene is excised. is there. In either case, the excised DNA is retained inside the cell in the form of a plasmid if it is cultured at a temperature at which the temperature-sensitive replication origin functions. Then, when the cells are cultured at a temperature at which the temperature-sensitive replication origin does not function, the metC gene on the plasmid is shed from the cells together with the plasmid. Then, by selecting a strain in which the deleted metC gene remained on the chromosome by PCR or Southern hybridization, metC
A strain in which the gene has been disrupted can be obtained.

The fact that the CBL activity of the gene-disrupted strain or mutant strain is reduced or eliminated means that the cell extract of the candidate strain was subjected to the method of Guggenheim, S. (Methods Enzymol., 17, 439).
-442 (1971) and other methods to measure CBL activity,
It can be confirmed by comparison with BL activity.

As a plasmid containing a temperature-sensitive replication origin for Escherichia coli, for example, pMAN031 (Yasued
a, H. et al, Appl. Microbiol. Biotechnol., 36, 211
(1991)), pMAN997 (WO 99/03988), and pEL3 (K.
A. Armstrong et. Al., J. Mol. Biol. (1984) 175, 33
1-347).

A tnaA gene-disrupted strain can be obtained in the same manner as described above. TN of tnaA gene disrupted strain or mutant strain
The decrease or disappearance of the ase activity indicates that the bacterial cell extract of the candidate strain was subjected to the method of Newton, WA et al. (J. Biol.
hem., 240, 1211-1218 (1965)) and the like, and can be confirmed by comparing the TNase activity with that of the parent strain. The deletion-type metC gene and the deletion-type tnaA gene used for gene disruption may have homology enough to cause homologous recombination with the metC gene and tnaA gene on the chromosomal DNA of the target Escherichia bacterium. .
Such homology is preferably 70% or more, more preferably 80% or more, and particularly preferably 90% or more.
In addition, homologous recombination can occur as long as the DNAs can hybridize under stringent conditions.

<2> Enhancement of SAT activity Enhancement of SAT activity in Escherichia bacterium cells is achieved by increasing the copy number of the gene encoding SAT. For example, a gene fragment encoding SAT, a vector that functions in Escherichia bacterium, preferably a multi-copy type vector is ligated to produce a recombinant DNA, which is introduced into a host Escherichia bacterium and transformed. Good.

As the SAT gene, both a gene derived from a bacterium belonging to the genus Escherichia and a gene derived from another organism can be used. As a gene encoding SAT of Escherichia coli, cysE was cloned from a wild strain and an L-cysteine secretion mutant strain, and the nucleotide sequence has been revealed (Denk, D. and Boeck, A., J. General Microbio.
l., 133, 515-525 (1987)). Therefore, the SAT gene can be obtained by PCR using a primer prepared based on the nucleotide sequence and PCR using the chromosomal DNA of Escherichia bacterium as a template (see Japanese Patent Laid-Open No. 11-155571). Genes encoding SAT of other organisms can be obtained in the same manner. For reference, SEQ ID NOS: 14 and 15
, The wild-type cysE nucleotide sequence and the encoded SAT amino acid sequence (Denk, D. and Bock, A., J. g.
eneral Microbiol., 133, 515-525 (1987)). SA
As the T gene, in addition to the wild-type SAT gene, acetyl-
It has an amino acid sequence containing substitution, deletion, insertion, addition, or inversion of one or several amino acid residues that does not substantially impair the activity of CoA for catalyzing the activation of L-serine. May be. Here, "several" means
The number of amino acid residues is preferably 2 to 50, more preferably 2 to 40, and particularly preferably 2 to 30, though it depends on the position and type of the protein in the three-dimensional structure.

As a DNA encoding a protein substantially the same as the SAT as described above, a base sequence consisting of base numbers 223 to 1047 of SEQ ID NO: 14 or a probe which can be prepared from the same base sequence and stringent conditions are used. And a DNA encoding a protein having the same activity as SAT. The “stringent conditions” are conditions under which so-called specific hybrid is formed and non-specific hybrid is not formed. Although it is difficult to quantify this condition clearly, as an example, DNAs having high homology, for example, DNAs having 50% or more homology, are hybridized, and DNAs having lower homology 60 ° C, 1 x SSC, 0.1, which is a condition that does not hybridize with each other, or is a condition for washing in ordinary Southern hybridization.
% SDS, preferably 0.1 × SSC, 0.1% SD
The conditions under which hybridization occurs at a salt concentration corresponding to S are mentioned.

Chromosomal DNA can be obtained from bacteria that are DNA donors, for example, by the method of Saito and Miura (H. Saito and K. Miu).
ra, Biochem. Biophys. Acta, 72, 619 (1963), Biotechnology Experiment Book, Japan Society for Biotechnology, pages 97-98, Baifukan,
1992) and the like.

D containing the SAT gene amplified by the PCR method
To introduce the NA fragment into a bacterium belonging to the genus Escherichia, various vectors used for ordinary protein expression can be used. Such vectors include pUC19, pUC
18, pHSG299, pHSG399, pHSG398, RSF1010, pBR322, pA
Examples include CYC184 and pMW219.

To introduce a recombinant vector containing the SAT gene into Escherichia bacteria, the method of DA Morrison (Me
thods in Enzymology 68, 326 (1979)) or a method of increasing the permeability of DNA by treating recipient cells with calcium chloride (Mandel, M. and Higa, A., J. Mol. Biol., 53, 159 (197).
0)) and the like, which are commonly used for transformation of Escherichia bacteria.

Increasing the copy number of the SAT gene is called SAT
It can also be achieved by allowing multiple copies of the gene to be present on the chromosomal DNA of Escherichia bacteria. To introduce multiple copies of the SAT gene into the chromosomal DNA of Escherichia bacterium, homologous recombination is carried out using a sequence present in multiple copies on the chromosomal DNA as a target. Repetitive DNA and inverted repeats present at the ends of transposable elements can be used as the sequences present in multiple copies on the chromosomal DNA. Alternatively, as disclosed in Japanese Patent Application Laid-Open No. 2-109985, it is also possible to incorporate the SAT gene into a transposon, transfer it, and introduce multiple copies of it into chromosomal DNA.

In addition to the above-mentioned gene amplification, the enhancement of SAT activity can also be achieved by substituting a strong expression control sequence such as a promoter of the SAT gene on the chromosomal DNA or on a plasmid (Japanese Patent Laid-Open No. Hei 1). -See 215280). For example, lac promoter, trp promoter, tr
The c promoter and the like are known as strong promoters. The replacement of the expression control sequence can also be carried out by gene replacement using, for example, a temperature sensitive plasmid.

Further, as disclosed in WO00 / 18935, it is also possible to introduce a base substitution of several bases into the promoter region of the SAT gene to modify it into a stronger one. SA by replacing or modifying these promoters
Expression of the T gene is enhanced and SAT activity is enhanced. Modification of these expression control sequences may be combined with increasing the copy number of the SAT gene.

Further, when there is a mechanism for suppressing the expression of the SAT gene, the expression of the SAT gene can also be expressed by modifying the expression control sequence or the gene involved in the suppression so that the suppression is canceled or reduced. Can be enhanced.

The SAT activity in Escherichia bacterial cells is
It can also be increased by allowing a bacterium belonging to the genus Escherichia to hold SAT in which feedback inhibition by L-cysteine is reduced or canceled (hereinafter, also referred to as “mutant SAT”). As the mutant SAT, 256 of the wild-type SAT
A mutation that substitutes an amino acid residue corresponding to the methionine residue at the position with an amino acid residue other than a lysine residue and a leucine residue, or lacks a region on the C-terminal side from the amino acid residue corresponding to the methionine residue at position 256. SAT with a mutation that causes it to disappear
Is mentioned. Examples of the amino acid residues other than the lysine residue and the leucine residue include 17 kinds of amino acid residues excluding the methionine residue, the lysine residue, and the leucine residue among the amino acids constituting a normal protein. More specifically, an isoleucine residue can be mentioned. Wild type SA
Examples of the method for introducing a desired mutation into the T gene include site-specific mutation. As a mutant SAT gene, a mutant cysE encoding a mutant SAT of Escherichia coli
Are known (see WO 97/15673, International Publication Pamphlet, JP-A-11-155571). The Escherichia coli JM39-8 strain (E. coli JM39-8 (pCEM256
E), private number: AJ13391)
From the 20th of March, FERM to Institute of Biotechnology, Institute of Biotechnology (now National Institute of Advanced Industrial Science and Technology, Patent Biological Depository Center, 1-chome, 1-chome, 1-chome, Higashi Tsukuba, Ibaraki, 305-8566, Japan) It was deposited under the accession number of P-16527, transferred to an international deposit under the Budapest Treaty on July 8, 2002, and is given accession number FERM BP-8112.

It is known that SAT of Arabidopsis thaliana is not subject to feedback inhibition by L-cysteine and can be preferably used in the present invention. As a plasmid containing SAT gene derived from Arabidopsis thaliana, pEAS-
m (FEMS Microbiol. Lett., 179 (1999) 453-459) is known.

The mutant SAT includes, in addition to the above-mentioned mutation that reduces feedback inhibition by L-cysteine,
Those having an amino acid sequence containing substitution, deletion, insertion, addition, or inversion of one or several amino acid residues that do not substantially impair the activity of catalyzing the activation of L-serine by acetyl-CoA May be In the SAT having such a mutation, the position of the methionine residue at the 256th position may be changed, but even in such a case, the amino acid residue corresponding to the methionine residue at the 256th position is changed to lysine. By substituting amino acid residues other than the residue and leucine residue, mutant SAT with reduced feedback inhibition by L-cysteine can be obtained.

Further, in order for Escherichia bacteria to retain a mutant SAT, S encoded by the intracellular SAT gene is used.
This can be done by introducing a mutation that eliminates the feedback inhibition of AT by L-cysteine. The mutation was introduced by ultraviolet irradiation or N-methyl-N'-
It can be carried out by treatment with mutating agents such as nitro-N-nitrosoguanidine (NTG) or nitrous acid, which are commonly used for mutation.

<3> Production of L-Cysteine The Escherichia bacterium of the present invention obtained as described above is cultivated in a suitable medium, L-cysteine is produced and accumulated in the culture, and L-cysteine is produced from the culture. -L-cysteine can be efficiently and stably produced by collecting cysteine. The L-cysteine produced by the method of the present invention may contain cystine in addition to the reduced cysteine, but the subject of the production method of the present invention is cystine or reduced cysteine and cystine. Mixtures of are also included.

The medium used is a carbon source, a nitrogen source,
There may be mentioned conventional media containing a sulfur source, inorganic ions and optionally other organic components. As a carbon source,
Glucose, fructose, sucrose, sugars such as molasses and starch hydrolysates, fumaric acid, citric acid,
Organic acids such as succinic acid can be used.

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 hydrolysate, ammonia gas and aqueous ammonia.

Examples of sulfur sources include inorganic sulfur compounds such as sulfates, sulfites, sulfides, hyposulfites, and thiosulfates. Organic trace nutrient sources include required substances such as vitamin B1 and yeast extract. It is desirable to contain an appropriate amount. In addition to these, small amounts of potassium phosphate, magnesium sulfate, iron ions, manganese ions and the like are added as necessary.

Cultivation is preferably carried out under aerobic conditions for 30 to 90 hours, preferably at a culture temperature of 25 to 37 ° C. and at a pH of 5 to 8 during the culture. Inorganic or organic acidic or alkaline substances, ammonia gas, etc. can be used for pH adjustment. Collection of L-cysteine from the culture can be carried out by combining a conventional ion exchange resin method, precipitation method and other known methods.

[0042]

EXAMPLES The present invention will be described in more detail below with reference to examples.

<1> Identification of enzyme responsible for CD activity in Escherichia coli (1) Electrophoresis of Escherichia coli cell extract and CD
SAT-deficient strain of Escherichia coli
Kori JM39 (F ⁺ cysE51tfr-8) (Denk, D. and Bock,
A., J. Gene. Microbiol, 133, 515-525 (1987)) and a cell extract of a CD activity-decreasing strain JM39-8 (Japanese Patent Laid-Open No. 11-155571) were subjected to non-denaturing polyacrylamide gel electrophoresis ( Na
tive-PAGE), CD activity staining was performed, and the main components of CD activity were analyzed. The JM39-8 strain is a mutant strain isolated as an L-cysteine non-assimilating strain by treating the JM39 strain with NTG (JP-A-11-155571).

LB medium with or without addition of 0.3% L-cysteine (tryptone 10 g / L, Yeast Extra
The cells were inoculated into ct 5g / L, NaCl 5g / L (pH 7.0)) and cultured at 37 ° C overnight. The bacterial cells were collected by centrifugation, washed with 0.85% NaCl, and the resulting wet bacterial cells were treated with about 3 ml of Buffer for cell destruction (10 mM Tris-HCl (pH
8.6), 10 μM PLP, 100 μM DTT) and suspend it in COSMOBIO B
Ultrasonic cell destruction was performed for 30 minutes with an ioruptor. 12,000 rpm, 1
After centrifugation for 0 minute, the obtained supernatant was used as a cell extract. Prote
The protein concentration was quantified using in Assay Kit (Bio-Rad).

Each bacterial cell extract (30 μg protein each)
2 x Native-PAGE buffer (Glycine14.43g / L, Tris
It was suspended in 3.0 g / L, pH 8.6 (HCl)) and subjected to non-denaturing polyacrylamide gel electrophoresis (Native-PAGE) according to a standard method. CD activity staining was performed using the obtained electrophoretic gel. Reaction solution (Tris ・ HCl 100 mM, EDTA 10 mM, PLP (Py
ridoxal-5-phosphate) 20 μM, L-Cys 50 mM, adjusted to pH 8.6, filled up to 100 ml, and bismuth chloride (BiCl ₃ ).
Lanes at both ends of the gel were cut out and dipped, and the mixture was shaken at room temperature and allowed to stand until bands were visible, and two main bands were detected (Fig. 1A). The lane in the center of the gel was used for amino acid sequence determination. In the JM39-8 strain, the one having the larger molecular weight was weaker than the JM39 strain in the above two bands.

(2) Identification of CBL as an enzyme responsible for CD activity As an enzyme having CD activity in Escherichia coli,
CBL (metC product) has been reported (Chandra et. Al., B
iochemistry, 21 (1982) 3064-3069). The band above is CB
To verify the possibility of L, an experiment similar to the above was carried out using a metC-deficient strain and Escherichia coli in which the metC gene was amplified with a multicopy plasmid.

MetC-deficient strain EZ5 (metC: Tn10Tet ^s rpsL add-
uid-man uraA: Tn10), and plasmid p carrying metC
IP29 (Granted by Dr. Saint-Girons, Institute of Pasteur, France. Belfaiza et. Al., Proc. Natl. Aca
d. Sci. 83 (1986) 867-871) was introduced into EZ5, and Native-PAGE and CD activity staining were performed.
As a result, it was revealed that in the metC-deficient strain, the band with the smaller molecular weight was deleted, and that the band was greatly amplified by the introduction of pIP29 (FIG. 1B). From this result, it was concluded that the band with the smaller molecular weight was CBL.

(3) Identification of TNase as an enzyme responsible for the CD activity The protein corresponding to the band having the higher molecular weight of the two bands observed by the activity staining was purified. From the electrophoretic gel, the band with the larger molecular weight was cut out from the gel, extracted and concentrated by a conventional method. Got
The fractions with CD activity were subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and PVDF was used without staining.
Blotting was performed on the membrane (Bio-Rad). The transferred film was washed with distilled water for 5 minutes and stained with 40% methanol containing 0.025% CBB (Coomassie Brilliant Blue) R-250 for 5 minutes. Decolorization was performed with 50% methanol, and the confirmed target enzyme band was cut off to remove Protein Sequencer 476A.
The N-terminal amino acid sequence was determined by the automated Edman degradation method using (Applied Biosystems).

The results are shown in SEQ ID NO: 1. This sequence has been reported to have some CD activity (Austin New
ton et. al., J. Biol. Chem. 240 (1965) 1211-1218)
When compared with the amino acid sequence of TNase of Escherichia coli, 14 out of 15 residues were in agreement. The methionine at position 10 in the amino acid sequence of SEQ ID NO: 1 was proline in the reported amino acid sequence. From this result, it was considered that the band with the larger molecular weight is likely to be TNase. Moreover, from this result, it was estimated that the expression of TNase was reduced in the JM39-8 strain.

<2> Construction of tnaA-deficient strain and metC-deficient strain of Escherichia coli As described above, CBL and TNase were identified as enzymes responsible for the CD activity in Escherichia coli. -To examine the effect on cysteine production, disrupted strains of the gene encoding metBL or the gene encoding tNAA encoding TNase, and disrupted strains of both of these genes were prepared (Fig. 2). Specifically, it was performed as follows.

(1) Construction of tnaA deficient strain Using the chromosomal DNA of Escherichia coli JM109 strain as a template, a primer having the nucleotide sequences shown in SEQ ID NOs: 2 and 3 and a primer having the nucleotide sequences shown in SEQ ID NOs: 4 and 5 were used. Was used to perform PCR. Next, set each PCR product to Hi
After cutting with ndIII, ligation was performed. PCR was carried out using the reaction solution as a template and the primers having the nucleotide sequences shown in SEQ ID NOS: 2 and 5 to obtain a fragment of about 1.6 kb. This fragment was digested with BamHI and the temperature sensitive plasmid pEL3 (KA Armstrong et. Al., J. Mol. Biol. (198
4) Incorporated into the BamHI site of 175, 331-347) to construct plasmid pEL3ΔtnaA for destroying tnaA.

The JM39 strain was transformed with the plasmid pEL3ΔtnaA to obtain a clone showing ampicillin resistance at 30 ° C. Appropriately dilute the culture solution of this clone on LB agar medium containing ampicillin, incubate at 42 ° C overnight, and pEL3
A strain in which ΔtnaA was integrated into the JM39 strain chromosome was obtained. Next, this strain was subcultured at 37 ° C. in ampicillin-free LB medium to isolate ampicillin-sensitive clones. For isolated clones, tna on the chromosome was analyzed by genomic PCR.
We analyzed around the A gene and did not have wild type tnaA.
A strain having only the disrupted tnaA gene cloned on EL3ΔtnaA was selected. Thus, tnaA derived from JM39 strain
A gene disruption strain JM39ΔtnaA was obtained.

(2) Construction of metC-deficient strain Using the chromosomal DNA of the Escherichia coli JM109 strain as a template, primers having the nucleotide sequences shown in SEQ ID NOs: 6 and 7 and primers having the nucleotide sequences shown in SEQ ID NOs: 8 and 9 were prepared. PCR was carried out respectively. Next, Kp each PCR product
It was ligated after cutting with nI. PCR was carried out using the reaction solution as a template and the primers having the nucleotide sequences shown in SEQ ID NOs: 6 and 9 to obtain a fragment of about 1.4 kb. This fragment was digested with BamHI and integrated into the BamHI site of pEL3 to construct the plasmid pEL3ΔmetC for disrupting metC.

JM39 strain or JM39ΔtnaA was added to plasmid pEL3
Transformation was performed with ΔmetC to obtain a clone showing ampicillin resistance at 30 ° C. The culture solution of this clone was appropriately diluted, applied on LB agar medium containing ampicillin, and cultured overnight at 42 ° C. to obtain a strain in which pEL3ΔmetC was integrated into the JM39 strain chromosome. Then, this strain was added to ampicillin-free LB medium.
After subculture at 37 ° C., ampicillin-sensitive clones were isolated. The isolated clone was analyzed around the metC gene on the chromosome by genomic PCR, and it had no wild-type metC and was cloned on pEL3ΔmetC.
A strain having only the metC gene was selected. Thus, JM39
Strain-derived metC gene-disrupted strains JM39ΔmetC and JM39ΔtnaA
A metC gene-disrupted strain JM39ΔtnaAΔmetC derived from the strain was obtained.

(3) Confirmation of Gene Disruption Next, the JM39ΔtnaA strain and JM39ΔmetC strain were examined for the presence or absence of a band having CD activity by electrophoresis and activity staining (FIG. 3). As a result, the tnaA-disrupted strain had a larger molecular weight band, the metC-disrupted strain had a smaller molecular weight band, and both tnaA / metC-disrupted strains had both bands completely disappeared.

<3> Introduction of feedback inhibition insensitive SAT gene A feedback inhibition insensitive SAT gene derived from Arabidopsis was introduced into each of the gene-disrupted strains obtained above.

SAT gene-containing plasmid pEAS-m derived from Arabidopsis thaliana (FEMS Microbiol. Lett., 179 (1999) 45
3-459) was used. The plasmid pES-m is described in JP-A-11-155.
Based on the plasmid pCE used to introduce the cysE gene derived from E. coli described in Japanese Patent No. 571,
The CYsE gene is replaced with an Arabidopsis thaliana-derived feedback inhibition-insensitive SAT gene, which can be constructed by the following method. First, FEMS Microbio
l. Lett., 179 (1999) 453-459.
A plasmid pCE (NcoI) having an NcoI site introduced immediately before the start codon and immediately after the stop codon of the cysE gene of E is constructed. The cysE gene is removed by NcoI digestion of this plasmid, and instead, the SAT gene of Arabidopsis thaliana with an NcoI linker added is introduced. SA of Arabidopsis
The T gene has a known structure (Plant Molecular Bio
logy, 30, 1041-1049, 1996), chemical synthesis, PCR or the like. pEAS-m is Plant Molecula
r Biology, 30, 1041-1049, 1996, obtained by introducing a DNA fragment with NcoI sites added before and after the sequence at positions 189-1201 in the nucleotide sequence shown in Fig. 2 into pCE NcoI site. It is a thing.

JM39, JM39ΔtnaA, JM39ΔmetC, and JM39
ΔtnaAΔmetC was transformed with the above plasmid pEAS-m. Each of the obtained transformants was cultured in an LB plate medium containing 50 mg / L of ampicillin at 30 ° C. for 24 hours, and then a platinum loop of cells 1 was added to C1 medium (glucose 30 g / L, glucose 30 g / L, _{NH 4 Cl 10g / L, KH} 2 PO 4 2g / L, MgSO 4 · 7H 2 O 1
_{g / L, FeSO 4 · 7H} 2 O 10mg / L, MnCl 2 · 4H 2 O 10mg / L, CaCO 3 2
0 g / L) 20 ml was inoculated into a Sakaguchi flask and cultured at 30 ° C. for 48 hours and 72 hours. When the L-cysteine content of the supernatant after culturing was quantified by a bioassay, it was confirmed that all the disrupted strains exceeded the wild strain, and that disruption of tnaA and metC was effective in improving L-cysteine productivity. (Fig. 4).

[0059]

INDUSTRIAL APPLICABILITY According to the present invention, L of Escherichia bacteria is
-It is possible to improve cysteine productivity.

[0060]

[Sequence list]                                SEQUENCE LISTING <110> Ajinomoto Co., Inc. <120> L-Cysteine-producing bacterium and method for producing L-cysteine (L-Cysteine-       Producing Bacterium and Method for Producing L-Cysteine) <130> P-B0293 <140> <141> 2002-09-18 <150> JP 2001-302008 <151> 2001-09-28 <160> 15 <170> PatentIn Ver. 2.0 <210> 1 <211> 15 <212> PRT <213> Escherichia coli <400> 1 Met Glu Asn Phe Lys His Leu Pro Glu Met Phe Arg Ile Arg Val   1 5 10 15 <210> 2 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 2 cgcggatcca agccgcattc tgactg 26 <210> 3 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 3 cccaagcttc tgactcgggc taacgca 27 <210> 4 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 4 cccaagcttg ccggtttcac tggcaa 26 <210> 5 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 5 ctatggatcc ttatagccac tctgtag 27 <210> 6 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 6 cgcggatcca acagagcttc tgcgatacc 29 <210> 7 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 7 cggggtacca ctagcatgaa tattcgcgg 29 <210> 8 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 8 cggggtacct accgcctata taaccagcc 29 <210> 9 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 9 aatatgagga tccgccagc 19 <210> 10 <211> 1880 <212> DNA <213> Escherichia coli <220> <221> CDS <222> (499) .. (1686) <400> 10 aagcttttgc taccaaaatc agcggcgata tcgttggcct ggtttaagga acgcgcttca 60 gccagcagtt gctgctcgcg cttaagggac gcttctgatt gaagaactct acgctcttac 120 tgaagaagat tgcccaggtg actacggagg ccaaaataag cccaatcatc acgcacttaa 180 cgacaatatc ggcgtgctga tacatacccc agacggaaag gtccgtctgc attaaattat 240 tacccactgt gtatctccag gacgcaagtc acaaaatctg cgcataataa tatcaaaacg 300 acgtcgaatt gatagtcgtt ctcattacta tttgcatact gccgtacctt tgctttcttt 360 tccttgcgtt tacgcagtaa aaaagtcacc agcacgccat ttgcgaaaat tttctgcttt 420 atgccaattc ttcaggatgc gcccgcgaat attcatgcta gtttagacat ccagacgtat 480 aaaaacagga atcccgac atg gcg gac aaa aag ctt gat act caa ctg gtg 531                     Met Ala Asp Lys Lys Leu Asp Thr Gln Leu Val                       1 5 10 aat gca gga cgc agc aaa aaa tac act ctc ggc gcg gta aat agc gtg 579 Asn Ala Gly Arg Ser Lys Lys Tyr Thr Leu Gly Ala Val Asn Ser Val              15 20 25 att cag cgc gct tct tcg ctg gtc ttt gac agt gta gaa gcc aaa aaa 627 Ile Gln Arg Ala Ser Ser Leu Val Phe Asp Ser Val Glu Ala Lys Lys          30 35 40 cac gcg aca cgt aat cgc gcc aat gga gag ttg ttc tat gga cgg cgc 675 His Ala Thr Arg Asn Arg Ala Asn Gly Glu Leu Phe Tyr Gly Arg Arg      45 50 55 gga acg tta acc cat ttc tcc tta caa caa gcg atg tgt gaa ctg gaa 723 Gly Thr Leu Thr His Phe Ser Leu Gln Gln Ala Met Cys Glu Leu Glu  60 65 70 75 ggt ggc gca ggc tgc gtg cta ttt ccc tgc ggg gcg gca gcg gtt gct 771 Gly Gly Ala Gly Cys Val Leu Phe Pro Cys Gly Ala Ala Ala Val Ala                  80 85 90 aat tcc att ctt gct ttt atc gaa cag ggc gat cat gtg ttg atg acc 819 Asn Ser Ile Leu Ala Phe Ile Glu Gln Gly Asp His Val Leu Met Thr              95 100 105 aac acc gcc tat gaa ccg agt cag gat ttc tgt agc aaa atc ctc agc 867 Asn Thr Ala Tyr Glu Pro Ser Gln Asp Phe Cys Ser Lys Ile Leu Ser         110 115 120 aaa ctg ggc gta acg aca tca tgg ttt gat ccg ctg att ggt gcc gat 915 Lys Leu Gly Val Thr Thr Ser Trp Phe Asp Pro Leu Ile Gly Ala Asp     125 130 135 atc gtt aag cat ctg cag cca aac act aaa atc gtg ttt ctg gaa tcg 963 Ile Val Lys His Leu Gln Pro Asn Thr Lys Ile Val Phe Leu Glu Ser 140 145 150 155 cca ggc tcc atc acc atg gaa gtc cac gac gtt ccg gcg att gtt gcc 1011 Pro Gly Ser Ile Thr Met Glu Val His Asp Val Pro Ala Ile Val Ala                 160 165 170 gcc gta cgc agt gtg gtg ccg gat gcc atc att atg atc gac aac acc 1059 Ala Val Arg Ser Val Val Pro Asp Ala Ile Ile Met Ile Asp Asn Thr             175 180 185 tgg gca gcc ggt gtg ctg ttt aag gcg ctg gat ttt ggc atc gat gtt 1107 Trp Ala Ala Gly Val Leu Phe Lys Ala Leu Asp Phe Gly Ile Asp Val         190 195 200 tct att caa gcc gcc acc aaa tat ctg gtt ggg cat tca gat gcg atg 1155 Ser Ile Gln Ala Ala Thr Lys Tyr Leu Val Gly His Ser Asp Ala Met     205 210 215 att ggc act gcc gtg tgc aat gcc cgt tgc tgg gag cag cta cgg gaa 1203 Ile Gly Thr Ala Val Cys Asn Ala Arg Cys Trp Glu Gln Leu Arg Glu 220 225 230 235 aat gcc tat ctg atg ggc cag atg gtc gat gcc gat acc gcc tat ata 1251 Asn Ala Tyr Leu Met Gly Gln Met Val Asp Ala Asp Thr Ala Tyr Ile                 240 245 250 acc agc cgt ggc ctg cgc aca tta ggt gtg cgt ttg cgt caa cat cat 1299 Thr Ser Arg Gly Leu Arg Thr Leu Gly Val Arg Leu Arg Gln His His             255 260 265 gaa agc agt ctg aaa gtg gct gaa tgg ctg gca gaa cat ccg caa gtt 1347 Glu Ser Ser Leu Lys Val Ala Glu Trp Leu Ala Glu His Pro Gln Val         270 275 280 gcg cga gtt aac cac cct gct ctg cct ggc agt aaa ggt cac gaa ttc 1395 Ala Arg Val Asn His Pro Ala Leu Pro Gly Ser Lys Gly His Glu Phe     285 290 295 tgg aaa cga gac ttt aca ggc agc agc ggg cta ttt tcc ttt gtg ctt 1443 Trp Lys Arg Asp Phe Thr Gly Ser Ser Gly Leu Phe Ser Phe Val Leu 300 305 310 315 aag aaa aaa ctc aat aat gaa gag ctg gcg aac tat ctg gat aac ttc 1491 Lys Lys Lys Leu Asn Asn Glu Glu Leu Ala Asn Tyr Leu Asp Asn Phe                 320 325 330 agt tta ttc agc atg gcc tac tcg tgg ggc ggg tat gaa tcg ttg atc 1539 Ser Leu Phe Ser Met Ala Tyr Ser Trp Gly Gly Tyr Glu Ser Leu Ile             335 340 345 ctg gca aat caa cca gaa cat atc gcc gcc att cgc cca caa ggc gag 1587 Leu Ala Asn Gln Pro Glu His Ile Ala Ala Ile Arg Pro Gln Gly Glu         350 355 360 atc gat ttt agc ggg acc ttg att cgc ctg cat att ggt ctg gaa gat 1635 Ile Asp Phe Ser Gly Thr Leu Ile Arg Leu His Ile Gly Leu Glu Asp     365 370 375 gtc gac gat ctg att gcc gat ctg gac gcc ggt ttt gcg cga att gta 1683 Val Asp Asp Leu Ile Ala Asp Leu Asp Ala Gly Phe Ala Arg Ile Val 380 385 390 395 taa cattgccact tttggacaat tttgcagaca ttttattgtg aaaagtctta 1736 aattgttgcg tccgggatca aggcgtcccg gacgattcag gagtacaata ggcagataaa 1796 ggcttaaacg ctgttccaca ggaaagtcca tggctgttat tcaagatatc atcgctgcgc 1856 tctggcaaca cgactttgcc gcgc 1880 <210> 11 <211> 395 <212> PRT <213> Escherichia coli <400> 11 Met Ala Asp Lys Lys Leu Asp Thr Gln Leu Val Asn Ala Gly Arg Ser   1 5 10 15 Lys Lys Tyr Thr Leu Gly Ala Val Asn Ser Val Ile Gln Arg Ala Ser              20 25 30 Ser Leu Val Phe Asp Ser Val Glu Ala Lys Lys His Ala Thr Arg Asn          35 40 45 Arg Ala Asn Gly Glu Leu Phe Tyr Gly Arg Arg Gly Thr Leu Thr His      50 55 60 Phe Ser Leu Gln Gln Ala Met Cys Glu Leu Glu Gly Gly Ala Gly Cys  65 70 75 80 Val Leu Phe Pro Cys Gly Ala Ala Ala Val Ala Asn Ser Ile Leu Ala                  85 90 95 Phe Ile Glu Gln Gly Asp His Val Leu Met Thr Asn Thr Ala Tyr Glu             100 105 110 Pro Ser Gln Asp Phe Cys Ser Lys Ile Leu Ser Lys Leu Gly Val Thr         115 120 125 Thr Ser Trp Phe Asp Pro Leu Ile Gly Ala Asp Ile Val Lys His Leu     130 135 140 Gln Pro Asn Thr Lys Ile Val Phe Leu Glu Ser Pro Gly Ser Ile Thr 145 150 155 160 Met Glu Val His Asp Val Pro Ala Ile Val Ala Ala Val Arg Ser Val                 165 170 175 Val Pro Asp Ala Ile Ile Met Ile Asp Asn Thr Trp Ala Ala Gly Val             180 185 190 Leu Phe Lys Ala Leu Asp Phe Gly Ile Asp Val Ser Ile Gln Ala Ala         195 200 205 Thr Lys Tyr Leu Val Gly His Ser Asp Ala Met Ile Gly Thr Ala Val     210 215 220 Cys Asn Ala Arg Cys Trp Glu Gln Leu Arg Glu Asn Ala Tyr Leu Met 225 230 235 240 Gly Gln Met Val Asp Ala Asp Thr Ala Tyr Ile Thr Ser Arg Gly Leu                 245 250 255 Arg Thr Leu Gly Val Arg Leu Arg Gln His His Glu Ser Ser Leu Lys             260 265 270 Val Ala Glu Trp Leu Ala Glu His Pro Gln Val Ala Arg Val Asn His         275 280 285 Pro Ala Leu Pro Gly Ser Lys Gly His Glu Phe Trp Lys Arg Asp Phe     290 295 300 Thr Gly Ser Ser Gly Leu Phe Ser Phe Val Leu Lys Lys Lys Leu Asn 305 310 315 320 Asn Glu Glu Leu Ala Asn Tyr Leu Asp Asn Phe Ser Leu Phe Ser Met                 325 330 335 Ala Tyr Ser Trp Gly Gly Tyr Glu Ser Leu Ile Leu Ala Asn Gln Pro             340 345 350 Glu His Ile Ala Ala Ile Arg Pro Gln Gly Glu Ile Asp Phe Ser Gly         355 360 365 Thr Leu Ile Arg Leu His Ile Gly Leu Glu Asp Val Asp Asp Leu Ile     370 375 380 Ala Asp Leu Asp Ala Gly Phe Ala Arg Ile Val 385 390 395 <210> 12 <211> 2083 <212> DNA <213> Escherichia coli <220> <221> CDS <222> (539) .. (1954) <400> 12 gtaaaccgcg catacagccg cattctgact gtcagatgcg cttcgcttca ttgttaccgc 60 tcctgttatt cctcaaccct ttttttaaac attaaaattc ttacgtaatt tataatcttt 120 aaaaaaagca tttaatattg ctccccgaac gattgtgatt cgattcacat ttaaacaatt 180 tcagaataga caaaaactct gagtgtaata atgtagcctc gtgtcttgcg aggataagtg 240 cattatgaat atcttacata tatgtgtgac ctcaaaatgg ttcaatattg acaacaaaat 300 tgtcgatcac cgcccttgat ttgcccttct gtagccatca ccagagccaa accgattgat 360 tcaatgtgtt ctatttgttt gctatatctt aattttgcct tttgcaaagg tcatctctcg 420 tttatttact tgttttagta aatgatggtg cttgcatata tatctggcga attaatcggt 480 atagcagatg taatattcac agggatcact gtaattaaaa taaatgaagg attatgta 538 atg gaa aac ttt aaa cat ctc cct gaa ccg ttc cgc att cgt gtt att 586 Met Glu Asn Phe Lys His Leu Pro Glu Pro Phe Arg Ile Arg Val Ile   1 5 10 15 gag cca gta aaa cgt acc act cgc gct tat cgt gaa gag gca att att 634 Glu Pro Val Lys Arg Thr Thr Arg Ala Tyr Arg Glu Glu Ala Ile Ile              20 25 30 aaa tcc ggt atg aac ccg ttc ctg ctg gat agc gaa gat gtt ttt atc 682 Lys Ser Gly Met Asn Pro Phe Leu Leu Asp Ser Glu Asp Val Phe Ile          35 40 45 gat tta ctg acc gac agc ggc acc ggg gcg gtg acg cag agc atg cag 730 Asp Leu Leu Thr Asp Ser Gly Thr Gly Ala Val Thr Gln Ser Met Gln      50 55 60 gct gcg atg atg cgc ggc gac gaa gcc tac agc ggc agt cgt agc tac 778 Ala Ala Met Met Arg Gly Asp Glu Ala Tyr Ser Gly Ser Arg Ser Tyr  65 70 75 80 tat gcg tta gcc gag tca gtg aaa aat atc ttc ggt tat caa tac acc 826 Tyr Ala Leu Ala Glu Ser Val Lys Asn Ile Phe Gly Tyr Gln Tyr Thr                  85 90 95 att ccg act cac cag ggc cgt ggc gca gag caa atc tat att ccg gta 874 Ile Pro Thr His Gln Gly Arg Gly Ala Glu Gln Ile Tyr Ile Pro Val             100 105 110 ctg att aaa aaa cgc gag cag gaa aaa ggc ctg gat cgc agc aaa atg 922 Leu Ile Lys Lys Arg Glu Gln Glu Lys Gly Leu Asp Arg Ser Lys Met         115 120 125 gtg gcg ttc tct aac tat ttc ttt gat acc acg cag ggc cat agc cag 970 Val Ala Phe Ser Asn Tyr Phe Phe Asp Thr Thr Gln Gly His Ser Gln     130 135 140 atc aac ggc tgt acc gtg cgt aac gtc tat atc aaa gaa gcc ttc gat 1018 Ile Asn Gly Cys Thr Val Arg Asn Val Tyr Ile Lys Glu Ala Phe Asp 145 150 155 160 acg ggc gtg cgt tac gac ttt aaa ggc aac ttt gac ctt gag gga tta 1066 Thr Gly Val Arg Tyr Asp Phe Lys Gly Asn Phe Asp Leu Glu Gly Leu                 165 170 175 gaa cgc ggt att gaa gaa gtt ggt ccg aat aac gtg ccg tat atc gtt 1114 Glu Arg Gly Ile Glu Glu Val Gly Pro Asn Asn Val Pro Tyr Ile Val             180 185 190 gca acc atc acc agt aac tct gca ggt ggt cag ccg gtt tca ctg gca 1162 Ala Thr Ile Thr Ser Asn Ser Ala Gly Gly Gln Pro Val Ser Leu Ala         195 200 205 aac tta aaa gcg atg tac agc atc gcg aag aaa tac gat att ccg gtg 1210 Asn Leu Lys Ala Met Tyr Ser Ile Ala Lys Lys Tyr Asp Ile Pro Val     210 215 220 gta atg gac tcc gcg cgc ttt gct gaa aac gcc tat ttc att aag cag 1258 Val Met Asp Ser Ala Arg Phe Ala Glu Asn Ala Tyr Phe Ile Lys Gln 225 230 235 240 cgt gaa gca gaa tac aaa gac tgg acc atc gag cag atc acc cgc gaa 1306 Arg Glu Ala Glu Tyr Lys Asp Trp Thr Ile Glu Gln Ile Thr Arg Glu                 245 250 255 acc tac aaa tat gcc gat atg ctg gcg atg tcc gcc aag aaa gat gcg 1354 Thr Tyr Lys Tyr Ala Asp Met Leu Ala Met Ser Ala Lys Lys Asp Ala             260 265 270 atg gtg ccg atg ggc ggc ctg ctg tgc atg aaa gac gac agc ttc ttt 1402 Met Val Pro Met Gly Gly Leu Leu Cys Met Lys Asp Asp Ser Phe Phe         275 280 285 gat gtg tac acc gag tgc aga acc ctt tgc gtg gtg cag gaa ggc ttc 1450 Asp Val Tyr Thr Glu Cys Arg Thr Leu Cys Val Val Gln Glu Gly Phe     290 295 300 ccg aca tat ggc ggc cta gaa ggc ggc gcg atg gag cgt ctg gcg gta 1498 Pro Thr Tyr Gly Gly Leu Glu Gly Gly Ala Met Glu Arg Leu Ala Val 305 310 315 320 ggt ctg tat gac ggc atg aat ctc gac tgg ctg gct tat cgt atc gcg 1546 Gly Leu Tyr Asp Gly Met Asn Leu Asp Trp Leu Ala Tyr Arg Ile Ala                 325 330 335 cag gta cag tat ctg gtc gat ggt ctg gaa gag att ggc gtt gtc tgc 1594 Gln Val Gln Tyr Leu Val Asp Gly Leu Glu Glu Ile Gly Val Val Cys             340 345 350 cag cag gcg ggc ggt cac gcg gca ttc gtt gat gcc ggt aaa ctg ttg 1642 Gln Gln Ala Gly Gly His Ala Ala Phe Val Asp Ala Gly Lys Leu Leu         355 360 365 ccg cat atc ccg gca gac cag ttc ccg gca aca ggc ctg gcc tgc gag 1690 Pro His Ile Pro Ala Asp Gln Phe Pro Ala Thr Gly Leu Ala Cys Glu     370 375 380 ctg tat aaa gtc gcc ggt atc cgt gcg gta gaa att ggc tct ttc ctg 1738 Leu Tyr Lys Val Ala Gly Ile Arg Ala Val Glu Ile Gly Ser Phe Leu 385 390 395 400 tta ggc cgc gat ccg aaa acc ggt aaa caa ctg cca tgc ccg gct gaa 1786 Leu Gly Arg Asp Pro Lys Thr Gly Lys Gln Leu Pro Cys Pro Ala Glu                 405 410 415 ctg ctg cgt tta acc att ccg cgc gca aca tat act caa aca cat atg 1834 Leu Leu Arg Leu Thr Ile Pro Arg Ala Thr Tyr Thr Gln Thr His Met             420 425 430 gac ttc att att gaa gcc ttt aaa cat gtg aaa gag aac gcg gcg aat 1882 Asp Phe Ile Ile Glu Ala Phe Lys His Val Lys Glu Asn Ala Ala Asn         435 440 445 att aaa gga tta acc ttt acg tac gaa ccg aaa gta ttg cgt cac ttc 1930 Ile Lys Gly Leu Thr Phe Thr Tyr Glu Pro Lys Val Leu Arg His Phe     450 455 460 acc gca aaa ctt aaa gaa gtt taa ttaatactac agagtggcta taaggatgtt 1984 Thr Ala Lys Leu Lys Glu Val 465 470 agccactctc ttaccctaca tcctcaataa caaaaatagc cttcctctaa aggtggcatc 2044 atgactgttc aagctgaaaa aaagcactct gcattttgg 2083 <210> 13 <211> 471 <212> PRT <213> Escherichia coli <400> 13 Met Glu Asn Phe Lys His Leu Pro Glu Pro Phe Arg Ile Arg Val Ile   1 5 10 15 Glu Pro Val Lys Arg Thr Thr Arg Ala Tyr Arg Glu Glu Ala Ile Ile              20 25 30 Lys Ser Gly Met Asn Pro Phe Leu Leu Asp Ser Glu Asp Val Phe Ile          35 40 45 Asp Leu Leu Thr Asp Ser Gly Thr Gly Ala Val Thr Gln Ser Met Gln      50 55 60 Ala Ala Met Met Arg Gly Asp Glu Ala Tyr Ser Gly Ser Arg Ser Tyr  65 70 75 80 Tyr Ala Leu Ala Glu Ser Val Lys Asn Ile Phe Gly Tyr Gln Tyr Thr                  85 90 95 Ile Pro Thr His Gln Gly Arg Gly Ala Glu Gln Ile Tyr Ile Pro Val             100 105 110 Leu Ile Lys Lys Arg Glu Gln Glu Lys Gly Leu Asp Arg Ser Lys Met         115 120 125 Val Ala Phe Ser Asn Tyr Phe Phe Asp Thr Thr Gln Gly His Ser Gln     130 135 140 Ile Asn Gly Cys Thr Val Arg Asn Val Tyr Ile Lys Glu Ala Phe Asp 145 150 155 160 Thr Gly Val Arg Tyr Asp Phe Lys Gly Asn Phe Asp Leu Glu Gly Leu                 165 170 175 Glu Arg Gly Ile Glu Glu Val Gly Pro Asn Asn Val Pro Tyr Ile Val             180 185 190 Ala Thr Ile Thr Ser Asn Ser Ala Gly Gly Gln Pro Val Ser Leu Ala         195 200 205 Asn Leu Lys Ala Met Tyr Ser Ile Ala Lys Lys Tyr Asp Ile Pro Val     210 215 220 Val Met Asp Ser Ala Arg Phe Ala Glu Asn Ala Tyr Phe Ile Lys Gln 225 230 235 240 Arg Glu Ala Glu Tyr Lys Asp Trp Thr Ile Glu Gln Ile Thr Arg Glu                 245 250 255 Thr Tyr Lys Tyr Ala Asp Met Leu Ala Met Ser Ala Lys Lys Asp Ala             260 265 270 Met Val Pro Met Gly Gly Leu Leu Cys Met Lys Asp Asp Ser Phe Phe         275 280 285 Asp Val Tyr Thr Glu Cys Arg Thr Leu Cys Val Val Gln Glu Gly Phe     290 295 300 Pro Thr Tyr Gly Gly Leu Glu Gly Gly Ala Met Glu Arg Leu Ala Val 305 310 315 320 Gly Leu Tyr Asp Gly Met Asn Leu Asp Trp Leu Ala Tyr Arg Ile Ala                 325 330 335 Gln Val Gln Tyr Leu Val Asp Gly Leu Glu Glu Ile Gly Val Val Cys             340 345 350 Gln Gln Ala Gly Gly His Ala Ala Phe Val Asp Ala Gly Lys Leu Leu         355 360 365 Pro His Ile Pro Ala Asp Gln Phe Pro Ala Thr Gly Leu Ala Cys Glu     370 375 380 Leu Tyr Lys Val Ala Gly Ile Arg Ala Val Glu Ile Gly Ser Phe Leu 385 390 395 400 Leu Gly Arg Asp Pro Lys Thr Gly Lys Gln Leu Pro Cys Pro Ala Glu                 405 410 415 Leu Leu Arg Leu Thr Ile Pro Arg Ala Thr Tyr Thr Gln Thr His Met             420 425 430 Asp Phe Ile Ile Glu Ala Phe Lys His Val Lys Glu Asn Ala Ala Asn         435 440 445 Ile Lys Gly Leu Thr Phe Thr Tyr Glu Pro Lys Val Leu Arg His Phe     450 455 460 Thr Ala Lys Leu Lys Glu Val 465 470 <210> 14 <211> 1134 <212> DNA <213> Escherichia coli <220> <221> CDS <222> (223) .. (1047) <400> 14 tccgcgaact ggcgcatcgc ttcggcgttg aaatgccaat aaccgaggaa atttatcaag 60 tattatattg cggaaaaaac gcgcgcgagg cagcattgac tttactaggt cgtgcacgca 120 aggacgagcg cagcagccac taaccccagg gaacctttgt taccgctatg acccggcccg 180 cgcagaacgg gccggtcatt atctcatcgt gtggagtaag ca atg tcg tgt gaa 234                                                Met Ser Cys Glu                                                  1 gaa ctg gaa att gtc tgg aac aat att aaa gcc gaa gcc aga acg ctg 282 Glu Leu Glu Ile Val Trp Asn Asn Ile Lys Ala Glu Ala Arg Thr Leu   5 10 15 20 gcg gac tgt gag cca atg ctg gcc agt ttt tac cac gcg acg cta ctc 330 Ala Asp Cys Glu Pro Met Leu Ala Ser Phe Tyr His Ala Thr Leu Leu                  25 30 35 aag cac gaa aac ctt ggc agt gca ctg agc tac atg ctg gcg aac aag 378 Lys His Glu Asn Leu Gly Ser Ala Leu Ser Tyr Met Leu Ala Asn Lys              40 45 50 ctg tca tcg cca att atg cct gct att gct atc cgt gaa gtg gtg gaa 426 Leu Ser Ser Pro Ile Met Pro Ala Ile Ala Ile Arg Glu Val Val Glu          55 60 65 gaa gcc tac gcc gct gac ccg gaa atg atc gcc tct gcg gcc tgt gat 474 Glu Ala Tyr Ala Ala Asp Pro Glu Met Ile Ala Ser Ala Ala Cys Asp      70 75 80 att cag gcg gtg cgt acc cgc gac ccg gca gtc gat aaa tac tca acc 522 Ile Gln Ala Val Arg Thr Arg Asp Pro Ala Val Asp Lys Tyr Ser Thr  85 90 95 100 ccg ttg tta tac ctg aag ggt ttt cat gcc ttg cag gcc tat cgc atc 570 Pro Leu Leu Tyr Leu Lys Gly Phe His Ala Leu Gln Ala Tyr Arg Ile                 105 110 115 ggt cac tgg ttg tgg aat cag ggg cgt cgc gca ctg gca atc ttt ctg 618 Gly His Trp Leu Trp Asn Gln Gly Arg Arg Ala Leu Ala Ile Phe Leu             120 125 130 caa aac cag gtt tct gtg acg ttc cag gtc gat att cac ccg gca gca 666 Gln Asn Gln Val Ser Val Thr Phe Gln Val Asp Ile His Pro Ala Ala         135 140 145 aaa att ggt cgc ggt atc atg ctt gac cac gcg aca ggc atc gtc gtt 714 Lys Ile Gly Arg Gly Ile Met Leu Asp His Ala Thr Gly Ile Val Val     150 155 160 ggt gaa acg gcg gtg att gaa aac gac gta tcg att ctg caa tct gtg 762 Gly Glu Thr Ala Val Ile Glu Asn Asp Val Ser Ile Leu Gln Ser Val 165 170 175 180 acg ctt ggc ggt acg ggt aaa tct ggt ggt gac cgt cac ccg aaa att 810 Thr Leu Gly Gly Thr Gly Lys Ser Gly Gly Asp Arg His Pro Lys Ile                 185 190 195 cgt gaa ggt gtg atg att ggc gcg ggc gcg aaa atc ctc ggc aat att 858 Arg Glu Gly Val Met Ile Gly Ala Gly Ala Lys Ile Leu Gly Asn Ile             200 205 210 gaa gtt ggg cgc ggc gcg aag att ggc gca ggt tcc gtg gtg ctg caa 906 Glu Val Gly Arg Gly Ala Lys Ile Gly Ala Gly Ser Val Val Leu Gln         215 220 225 ccg gtg ccg ccg cat acc acc gcc gct ggc gtt ccg gct cgt att gtc 954 Pro Val Pro Pro His Thr Thr Ala Ala Gly Val Pro Ala Arg Ile Val     230 235 240 ggt aaa cca gac agc gat aag cca tca atg gat atg gac cag cat ttc 1002 Gly Lys Pro Asp Ser Asp Lys Pro Ser Met Asp Met Asp Gln His Phe 245 250 255 260 aac ggt att aac cat aca ttt gag tat ggg gat ggg atc taa tgt 1047 Asn Gly Ile Asn His Thr Phe Glu Tyr Gly Asp Gly Ile Cys                 265 270 275 cctgtgatcg tgccggatgc gatgtaatca tctatccggc ctacagtaac taatctctca 1107 ataccgctcc cgatacccca actgtcg 1134 <210> 15 <211> 273 <212> PRT <213> Escherichia coli <400> 15 Met Ser Cys Glu Glu Leu Glu Ile Val Trp Asn Asn Ile Lys Ala Glu   1 5 10 15 Ala Arg Thr Leu Ala Asp Cys Glu Pro Met Leu Ala Ser Phe Tyr His              20 25 30 Ala Thr Leu Leu Lys His Glu Asn Leu Gly Ser Ala Leu Ser Tyr Met          35 40 45 Leu Ala Asn Lys Leu Ser Ser Pro Ile Met Pro Ala Ile Ala Ile Arg      50 55 60 Glu Val Val Glu Glu Ala Tyr Ala Ala Asp Pro Glu Met Ile Ala Ser  65 70 75 80 Ala Ala Cys Asp Ile Gln Ala Val Arg Thr Arg Asp Pro Ala Val Asp                  85 90 95 Lys Tyr Ser Thr Pro Leu Leu Tyr Leu Lys Gly Phe His Ala Leu Gln             100 105 110 Ala Tyr Arg Ile Gly His Trp Leu Trp Asn Gln Gly Arg Arg Ala Leu         115 120 125 Ala Ile Phe Leu Gln Asn Gln Val Ser Val Thr Phe Gln Val Asp Ile     130 135 140 His Pro Ala Ala Lys Ile Gly Arg Gly Ile Met Leu Asp His Ala Thr 145 150 155 160 Gly Ile Val Val Gly Glu Thr Ala Val Ile Glu Asn Asp Val Ser Ile                 165 170 175 Leu Gln Ser Val Thr Leu Gly Gly Thr Gly Lys Ser Gly Gly Asp Arg             180 185 190 His Pro Lys Ile Arg Glu Gly Val Met Ile Gly Ala Gly Ala Lys Ile         195 200 205 Leu Gly Asn Ile Glu Val Gly Arg Gly Ala Lys Ile Gly Ala Gly Ser     210 215 220 Val Val Leu Gln Pro Val Pro Pro His Thr Thr Ala Ala Gly Val Pro 225 230 235 240 Ala Arg Ile Val Gly Lys Pro Asp Ser Asp Lys Pro Ser Met Asp Met                 245 250 255 Asp Gln His Phe Asn Gly Ile Asn His Thr Phe Glu Tyr Gly Asp Gly             260 265 270 Ile

[Brief description of drawings]

[Figure 1] Escherichia coli cell extract Native-PAGE
The figure which shows the result of CD activity staining. A: SAT-deficient strain JM39 and CD activity-reduced strain JM39-8B: metC-deficient strain EZ5 and metC
EZ5 with plasmid pIP29

FIG. 2 is a diagram showing the construction process of Escherichia coli metC-disrupted strain, tnaA-disrupted strain, and metC / tnaA-disrupted strain.
“A” and “B” indicate the terminal region of the gene, and “X” indicates the deleted region.

FIG. 3 is a photograph showing the results of electrophoresis and CD activity staining for confirming gene disruption of metC-disrupted strain, tnaA-disrupted strain, and metC / tnaA-disrupted strain.

FIG. 4 shows L-cysteine productivity of metC-disrupted strain, tnaA-disrupted strain, and metC / tnaA-disrupted strain.

   ─────────────────────────────────────────────────── ─── Continued front page    (72) Inventor Shigeru Nakamori             38-7 Kanejojima, Matsuoka-cho, Yoshida-gun, Fukui Prefecture B-             103 F term (reference) 4B064 AE14 CA02 CA19 CC24 DA01                       DA20                 4B065 AA26X AA89Y AB01 AC14                       BA02 BA23 BA25 CA17

Claims (10)

[Claims] 1. A bacterium belonging to the genus Escherichia, which has L-cysteine producing ability and has been modified so as to have a decreased or deleted cystathionine-β-lyase activity. 2. A bacterium belonging to the genus Escherichia, which has an L-cysteine producing ability and has been modified so that the ciscystathionine-β-lyase activity and the tryptophanase activity are reduced or deleted. 3. The Escherichia bacterium according to claim 1, wherein the gene encoding cystathionine-β-lyase is disrupted. 4. The Escherichia bacterium according to claim 2, wherein the gene encoding cis-cystathionine-β-lyase and the gene encoding tryptophanase are disrupted. 5. The bacterium belonging to the genus Escherichia according to any one of claims 1 to 4, which is further modified so that the enzyme activity of the L-cysteine biosynthesis system is enhanced. 6. The bacterium of the genus Escherichia according to claim 5, which has been modified so that serine acetyltransferase is enhanced. 7. The Escherichia bacterium according to claim 6, which retains a serine acetyltransferase that is insensitive to feedback inhibition by L-cysteine. 8. The method according to claim 1, which is Escherichia coli.
The bacterium belonging to the genus Escherichia according to any one of 1. 9. An Escherichia bacterium according to any one of claims 1 to 8 is cultured in a medium, L-cysteine is produced and accumulated in the medium, and L-cysteine is collected from the medium. Method for producing cysteine. 10. A method for enhancing L-cysteine production ability of a bacterium belonging to the genus Escherichia by reducing or eliminating cis-cystathionine-β-lyase activity or tryptophanase activity or both of these activities. A method for producing an L-cysteine-producing bacterium of Escherichia bacterium.