JPH11155571A - Production of l-cysteine - Google Patents

Abstract

PROBLEM TO BE SOLVED: To stably produce L-cysteine by a microbial strain belonging to the genus Escherichia holding SAT (serine acetyltransferase) having decreased feedback inhibition with L-cysteine by lowering cysteine desulfhydrase activity in the cell. SOLUTION: L-cysteine is produced by a bacterial strain belonging to the genus Escherichia characterized by (A) the suppressed L-cysteine decomposition system and (B) the retention of SAT having decreased feedback inhibition by L-cysteine, e.g. a bacterial strain having an L-cysteine decomposition system suppressed e.g. by the lowering of cysteine desulfhydrase activity in the cell. The feedback inhibition by L-cysteine can be decreased by introducing e.g. a mutation to substitute the amino acid residue corresponding to the 256th methionine residue of the wild-type SAT with an amino acid residue other than lysine residue and leucine residue.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for producing L-cysteine, and more particularly to a novel Escherichia bacterium suitable for producing L-cysteine, and a method for producing L-cysteine using the same. L-cysteine and derivatives of L-cysteine are used in the pharmaceutical, cosmetic and food fields.

[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. Have been. Also,
Mass production of L-cysteine by an immobilized enzyme method using a novel enzyme is also planned. Furthermore, production of L-cysteine by a fermentation method using a microorganism has been attempted.

[0003] The biosynthesis of L-cysteine has been studied in detail in bacteria such as Escherichia coli (Kred).
ich, NM et al., J. Biol. Chem., 241, 4955-4965
(1966), Kredich, NM et al., 1987, Biosynthesis
of Cysteine. In: Neidhardt, FC, et al., (eds) Es
cherichia coli and Salmonella typhimurium: cellula
r and molecular biology, Vol. 1, American Society f
or Microbiology, Washington DC, 419-428), L-
It has been found that L-cysteine is produced from serine by a two-step reaction. In Escherichia coli, the first reaction is the activation of L-serine by acetyl-CoA, and serine acetyltransferase (serine a).
cetyltransferase (EC 2.3.1.30): Hereinafter, "SAT"
). The second reaction is a reaction in which L-cysteine is generated from O-acetylserine generated by the above reaction, and is catalyzed by O-acetylserine (thiol) lyase.

[0004] Gene encoding SAT (cysE)
Has been cloned in Escherichia coli from wild-type and L-cysteine secretion mutants (Den
k, D. and Bock, A., J. General Microbiol., 133, 51
5-525 (1987)). In addition, the base sequences of these cysEs were determined, and it has been reported that the SAT in which feedback inhibition by L-cysteine was reduced had the methionine residue at position 256 replaced with an isoleucine residue. Furthermore, a method for producing L-cysteine or the like using DNA encoding SAT in which feedback inhibition by L-cysteine is reduced by a mutation different from the above mutation is disclosed (WO 97/15673 International Publication Pamphlet). ). This SAT is derived from the amino acid residue at position 97 to 273
Mutation in the region up to the amino acid residue at position 227, or
It has a deletion of the C-terminal region from the amino acid residue at the position.

[0005]

As described above, a technique for producing L-cysteine using a gene encoding SAT with reduced feedback inhibition by L-cysteine is known, but there is still room for improvement. Is left. That is, the present inventor has proposed that L-
A study on cysteine biosynthesis revealed that SAT with reduced feedback inhibition by L-cysteine
Was found to be unstable in L-cysteine productivity. An object of the present invention is to provide an improved method for producing L-cysteine using a bacterium belonging to the genus Escherichia and a bacterium belonging to the genus Escherichia used in the method.

[0006]

Means for Solving the Problems The present inventors have conducted intensive studies in order to solve the above-mentioned problems, and as a result, the instability of L-cysteine production has shown that cysteine desulfhydrase activity in cells is reduced. The inventors have found that stabilization is achieved by lowering the content, and have completed the present invention.

[0007] That is, the present invention is a bacterium belonging to the genus Escherichia, which retains a serine acetyltransferase in which the L-cysteine degradation system is suppressed and feedback inhibition by L-cysteine is reduced. Examples of the Escherichia bacteria include bacteria belonging to the genus Escherichia in which the L-cysteine degradation system is suppressed due to a decrease in cysteine desulfhydrase (hereinafter, also referred to as “CDase”) activity in cells.

[0008] The serine acetyltransferase with reduced feedback inhibition by L-cysteine is the wild-type serine acetyltransferase.
A mutation that substitutes an amino acid residue corresponding to a methionine residue at position 56 with an amino acid residue other than a lysine residue and a leucine residue, or a C-terminal region from an amino acid residue corresponding to a methionine residue at position 256 Serine acetyltransferase having a mutation to be deleted is exemplified.

[0009] The present invention also provides a method for producing L-cysteine, comprising culturing the bacterium belonging to the genus Escherichia in a medium, producing and accumulating L-cysteine in the culture, and collecting L-cysteine from the culture. Provide a manufacturing method.

[0010]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below in detail.

<1> Escherichia bacterium of the present invention Examples of the Escherichia bacterium of the present invention include Escherichia coli (E. coli).

The bacterium belonging to the genus Escherichia of the present invention has a SAT in which the L-cysteine degradation system is suppressed and feedback inhibition by L-cysteine is reduced (hereinafter, also referred to as “mutant SAT”). , A bacterium belonging to the genus Escherichia in which the L-cysteine degradation system is suppressed (hereinafter, referred to as “
"L-cysteine-degrading strain-suppressed strain")
It can be obtained by retaining AT or by causing mutation in an Escherichia bacterium retaining mutant SAT to suppress the L-cysteine degradation system in cells. In addition, "inhibition of L-cysteine degradation system" means that at least one activity of enzymes involved in cysteine degradation is reduced or eliminated. Also,
The term “reduction of feedback inhibition” includes a case where feedback inhibition is substantially canceled, in addition to a case where reduced feedback inhibition remains. Enzymes involved in the decomposition of L-cysteine include CDase and cystathion β-lyase. A strain in which CDase activity is reduced or eliminated (hereinafter, also referred to as “CDase activity-reduced strain”) can be mutated to a bacterium belonging to the genus Escherichia holding wild-type CDase and grown on a medium containing L-cysteine as a sole nitrogen source. And can be obtained by selecting a mutant strain that can grow on a medium containing a normal nitrogen source. Mutation treatment includes UV irradiation or N-methyl-N'-nitro-N-nitrosoguanidine (NT
G) or treatment with a mutagen used in normal mutations such as nitrite. C of the obtained candidate strain
The decrease in Dase activity was confirmed by the method of Kredich et al. (J. Biol. Chem., 248,
6187-6196 (1973)) can be confirmed by measuring the CDase activity and comparing it with the CDase activity of the parent strain. Similarly, a strain with reduced cystation β-lyase activity can be obtained by mutagenizing a bacterium belonging to the genus Escherichia carrying wild-type cystathion β-lyase and selecting a strain having reduced or eliminated cystation β-lyase activity.

In order to allow the bacterium belonging to the genus Escherichia to carry the mutant SAT, the bacterium belonging to the genus Escherichia carrying the wild-type SAT is subjected to mutation treatment, and a mutant strain carrying the mutant SAT is selected, or the mutant SAT is encoded. DNA
(Hereinafter, also referred to as “mutant cysE”) into Escherichia bacteria. Mutated cysE can be obtained by introducing a mutation into wild-type cysE such that the feedback inhibition of L-cysteine of the encoded SAT is released. Such mutations include the acetyl-Co of the encoded SAT.
There is no particular limitation as long as it does not substantially impair the activity of catalyzing the activation of L-serine by A. Specifically, it corresponds to the methionine residue at position 256 of wild-type SAT or this methionine residue. Mutations in which an amino acid residue is substituted with an amino acid residue other than a lysine residue and a leucine residue, or mutation in which a C-terminal region is deleted from an amino acid residue corresponding to a methionine residue at position 256 are exemplified. Examples of the other amino acid residues include 17 types of amino acid residues excluding methionine residues, lysine residues, and leucine residues among amino acids constituting ordinary proteins. In addition, the mutation possessed by mutant cysE includes WO 9
It may be a mutation described in International Publication Pamphlet No. 7/15673.

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

[0015] Wild-type cysE can be prepared by the method reported by Denk et al. (Denk, D. and Bock, A., J. general).
Microbiol., 133, 515-525 (1987)) or PCR (polymerase chain) using an oligonucleotide prepared based on the cysE nucleotide sequence described in this report as a primer and chromosomal DNA of a bacterium belonging to the genus Escherichia as a template.・ Reaction ： White, TJ et al; Tren
ds Genet. 5,185 (1989)) by amplifying a DNA fragment containing cysE. Specific examples of the primer include oligonucleotides having the nucleotide sequences shown in SEQ ID NOS: 1 and 2. For reference, SEQ ID NOS: 5 and 6 show the nucleotide sequence and encoded amino acid sequence of wild-type cysE reported by Denk et al.

A method for introducing a desired mutation into the obtained cysE includes site-specific mutation. Examples of primers used for site-specific mutation include oligonucleotides having the nucleotide sequences shown in SEQ ID NOs: 3 and 4. A region including the site where the mutation was introduced was cut out from the cysE fragment into which the mutation was introduced, and the wild-type cysE fragment was cut out.
And by replacing the corresponding region, the mutant cy
sE can be obtained.

The mutated cysE obtained as described above
In order to introduce a DNA fragment containing E. coli into a bacterium belonging to the genus Escherichia, various vectors used for normal protein expression can be used. At this time, the mutant cysE gene inserted into a vector capable of autonomously replicating may be introduced into a host, and may be retained in the host Escherichia bacterium as extrachromosomal DNA such as a plasmid.
Gene, transduction, transposon (Ber
g, DE and Berg, CM, Bio / Technol., 1,417 (1983)),
Mu phage (JP-A-2-109985) or homologous recombination (Experiments in Molecular Genetics, Cold Sp
(ring Harbor Lab. (1972)) may be incorporated into the chromosome of the host Escherichia bacterium.

As the promoter, a cysE-specific promoter may be used. Alternatively, a cysE coding region may be ligated to a promoter contained in an expression vector. As an expression vector, pBluescrip
Commercially available vectors such as tII SK + (STRATAGENE), pGEMEX-1 (Promega), pUC (Takara Shuzo), pPROK (Clontech), and pKK233-2 (Clontech) can be used. it can.

In order to introduce an expression vector containing cysE into a bacterium belonging to the genus Escherichia, the method of DM Morrison (Method
s in Enzymology 68, 326 (1979)) or a method of treating recipient cells with calcium chloride to increase DNA permeability (Mandel, M. and Higa, A., J. Mol. Biol., 53, 159 (1970)). )
Etc. can be carried out by a method usually used for transformation of Escherichia bacteria.

<2> Method for Producing L-Cysteine As described above, cysteine desulfhydrase activity in cells is reduced or eliminated, and serine acetyltransferase having reduced L-cysteine feedback inhibition is retained. Escherichia bacteria are cultured in a suitable medium, L-cysteine is produced and accumulated in the culture, and L-cysteine is collected from the culture to efficiently and stably produce L-cysteine. be able to. In addition, L- produced by the method of the present invention.
Cysteine may include cystine in addition to reduced cysteine, and the subject of the production method of the present invention also includes cystine or a mixture of reduced cysteine and cystine.

The medium to be used includes a carbon source, a nitrogen source,
Examples of the medium include a usual medium containing a sulfur source, inorganic ions, and if necessary, other organic components. As a carbon source,
Sugars such as glucose, fructose, sucrose, molasses and starch hydrolysates, fumaric acid, citric acid,
Organic acids such as succinic acid can be used.

As the nitrogen source, inorganic ammonium salts such as ammonium sulfate, ammonium chloride and ammonium phosphate, organic nitrogen such as soybean hydrolysate, ammonia gas, aqueous ammonia and the like can be used.

Examples of the sulfur source include inorganic sulfur compounds such as sulfates, sulfites, sulfides, hyposulfites, and thiosulfates. Examples of organic trace nutrients include required substances such as vitamin B1 and yeast extract. Is desirably contained. In addition to these, small amounts of potassium phosphate, magnesium sulfate, iron ions, manganese ions and the like are added as necessary.

The cultivation is preferably carried out under aerobic conditions for 30 to 90 hours. The cultivation temperature is preferably controlled at 25 ° C. to 37 ° C., and the pH during cultivation is preferably controlled at 5 to 8. In addition, an inorganic or organic acidic or alkaline substance, furthermore, ammonia gas or the like can be used for pH adjustment. Collection of L-cysteine from the culture can be carried out by a combination of a conventional ion exchange resin method, a precipitation method and other known methods.

[0025]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below in detail.

<1> Construction of mutant cysE gene and plasmid for introducing the gene Escherichia coli JM240 (Hfr ProC47 Cys-54 supE4
A DNA fragment containing cysE is prepared by PCR using a chromosomal DNA prepared from 2) by a conventional method, using this as a template and oligonucleotides (SEQ ID NOS: 1 and 2) synthesized based on the known cysE nucleotide sequence as primers. Was amplified. Primers were designed so that the sequence to be amplified contained a coding region and a promoter sequence. The PCR reaction was performed using DNA Thermal Cycler PJ200 manufactured by Takara Shuzo Co., Ltd.
The procedure was performed using type 0 and using Taq DNA polymerase according to the method specified by the supplier.

TA cloning was performed on the amplified 1.2 kb DNA fragment using pBluescriptIISK + cut with EcoRV to obtain a 4.2 kb plasmid. This plasmid was named pCE. The nucleotide sequence of the obtained cloned fragment was determined, and it was confirmed that it contained the same nucleotide sequence as that of wild-type cysE.

In the cysE obtained as described above, a methionine residue at position 256 of the encoded SAT was added to the other one.
Mutations that substitute nine amino acid residues or termination codons were introduced by site-specific mutation using pCE as a template, using complementary oligonucleotide pairs having the nucleotide sequences shown in SEQ ID NOs: 3 and 4. CysE into which the mutation was introduced
Was digested with PstI and BstEII, and the resulting 0.31 kb fragment was replaced to obtain a plasmid containing mutant cysE (FIG. 1). The nucleotide sequence of the obtained plasmid was determined, and it was confirmed that a mutation was introduced into the target site of the mutant cysE and that no mismatch mutation occurred in a site other than the target site. However, 256
When the methionine residue at position 2 is replaced with a lysine residue,
There was a mismatch mutation in which the serine residue at position 24 was replaced with a proline residue. The series of plasmids thus obtained was generically named pCEM256 *. still,
“*” Represents a symbol representing an amino acid residue other than a methionine residue. For example, pCEM256A represents a plasmid containing a mutant cysE encoding SAT in which the methionine residue at position 256 has been substituted with an alanine residue. .

With the plasmid pCE and the pCEM256 * obtained as described above, the cysE-deficient strain Escherichia coli JM39 (F ⁺ cysE51 tfr-8) (Denk, D. a.
nd Bock, A., J. Gene.Microbiol, 133, 515-525 (198
7)), and the resulting transformants were each subjected to JM39.
(pCE) and JM39 (pCEM256 *).

LB (Bacto-T) containing 50 mg / L of ampicillin
ryptone 10g / L, Bacto-Yeast Extract 5g / L, NaCl 5g /
L, pH 7.0) One loopful of each transformant cultured on a plate medium at 30 ° C. for 24 hours was placed in a test tube containing 3 ml of the following liquid medium (composition is as described below) to which 50 mg / L of ampicillin was added. The cells were inoculated and cultured at 30 ° C. for 72 hours.
Culture was performed in duplicate for each transformant. After culture
Growth, pH and cysteine accumulation were examined. Growth was determined by diluting the culture 26-fold with 0.1N HCl and measuring the absorbance at 562 nm (OD ₅₆₂ ). The amount of L-cysteine accumulated was determined by diluting the culture solution with 0.5N HCl to dissolve the precipitated cystine, and using a bioassay using Leuconostoc mesenteroides (Tsunoda, T. et al., Amino). acids,
3, 7-13 (1961)) as the total amount of reduced cysteine and cystine. Table 1 shows the results. “256th” in the table indicates the amino acid residue at position 256. “Stop” indicates that position 256 was replaced with a stop codon.

Compared to JM39 (pCE) which carries a plasmid containing wild-type cysE, the methionine residue at position 256 is a leucine residue or a lysine residue in JM39 (pCEM256 *) which carries a plasmid containing a mutant cysE. (Except for JM39 (pCEM256L, JM39 (pCEM256K), the amount of cysteine accumulation increased significantly, but the results varied)
Cysteine productivity was unstable.

[0032] (Medium composition) Glucose 30 g / L of ammonium chloride _{10g / L KH 2 PO 4 2g} / L MgSO 4 · 7H 2 O 1g / L FeSO 4 · 7H 2 O 10mg / L MnCl 2 · 4H 2 O 10mg / L CaCO ₃ 20g / L

[0033]

[Table 1]

<2> Acquisition of CDase-lowering strain Escherichia coli JM39 was subjected to mutation treatment in a 0.1 mg / ml NTG solution for 15 minutes, and an ammonium chloride-substituted M9 medium (0.3 g / L) was substituted for L-cysteine (0.3 g / L). The composition is as follows)
Mutants, 39-8 strains, which cannot grow on the plate and grow on M9 medium containing 0.1 g / L of L-cysteine, were selected.

[0035] (Medium _{composition) Na 2 HPO 4 6g / L} KH 2 PO 4 3g / L NaCl 0.5g / L MgSO 4 · 7H 2 O 0.25g / L CaCl 2 · 4H 2 O 0.015mg / L glucose 4g / L Thiamine / HCl 0.001g / L

Next, the CDase activity of the obtained L-cysteine non-assimilating strain 39-8 was measured as follows. 39-8 strain was added to LB medium in an amount of 1 mg / L-cysteine-HCl.
After culturing at 37 ° C. for 8 hours in a medium containing ml, the cells were sonicated, centrifuged at 30,000 × g for 30 minutes, and the supernatant was collected as a crude enzyme solution. The crude enzyme solution was treated with Credich et al. (J. Biol. Chem., 248, 6187-6196 (1973)).
Dase activity was measured. That is, 0.1M Tris-HCl, 5
mM pyridoxal phosphate, 2 mM L-cysteine / HC
l, 1.5 ml of a reaction solution consisting of 0.3 ml crude enzyme solution at 37 ° C for 30 minutes.
The amount of pyruvic acid produced was quantified, and the amount produced per protein was defined as CDase activity. Table 2 shows the results. The CDase activity of the 39-8 strain was clearly lower than that of the parent strain.

[0037]

[Table 2]

<3> Mutant cys to CDAase reduced strain
Introduction of E gene and production of L-cysteine The CDase-reduced strain 39-8 obtained above was purified by pC
Transformed with E and pCEM256 *. The resulting transformants were named 39-8 (pCE) and 39-8 (pCEM256 *), respectively. These transformants were cultured in an LB plate medium containing 50 mg / L of ampicillin at 30 ° C. for 24 hours. One platinum loop of each transformant was added to 20 ml of C1 medium (composition is as described below) to which 50 mg / L of ampicillin was added. Were inoculated into Sakaguchi flasks, and cultured at 30 ° C. with shaking for 72 hours. Culture was performed in duplicate for each transformant. After the culture, growth, pH of the culture solution, residual sugar, accumulated amount of L-cysteine, and yield to sugar were examined. The growth and the amount of L-cysteine were performed as described above. Table 3 shows the results.

Table 4 shows the results obtained by culturing 39 (pCE) and 39 (pCEM256 *) in the same manner as described above and examining the amount of L-cysteine accumulated. From this result, the mutant cysE was converted to CD
It is apparent that the introduction into the ase-reduced strain increases L-cysteine production and shows stable productivity.

[0040]

[Table 3]

[0041]

[Table 4]

A 39-8 strain (E. coli 39-8 (p.) Carrying pCEM256E containing a mutant cysE encoding SAT in which the methionine residue at position 256 has been replaced with a glutamic acid residue.
CEM256), private number: AJ13391)
Was deposited with the National Institute of Advanced Industrial Science and Technology (Postal Code 305, 1-3 1-3 Higashi, Tsukuba, Ibaraki, Japan) under the accession number of FERM P-16527 from November 20, 1997. Have been. PCE from the deposited strain
By obtaining M256E and introducing a site-specific mutation, a mutant cysE encoding SAT in which the methionine residue at position 256 is substituted with a desired amino acid residue can be obtained.

<4> C into which mutant cysE gene has been introduced
Measurement of SAT activity of Dase-reduced strains 39-8 (pCE) and 39-8 (pCEM256 *) were combined with 50 mg of ampicillin.
20 ml of C1 medium (composition is as described below) supplemented with / L
Was inoculated into a Sakaguchi flask containing, and cultured with shaking at 30 ° C. for 48 hours. The cells were ice-cold 50 mM Tris-HC
1 × (pH 7.5), containing 2 mM dithiothreitol
The cells were suspended in 50 mM Tris-HCl (pH 7.5) and disrupted by sonication. The crushed product was centrifuged at 30,000 × g for 30 minutes, and the supernatant was used as a crude extract for measurement of SAT activity.

The SAT activity was measured using 50 mM Tris-HCl (pH 7.
5) 1 ml of a reaction solution containing 1 mM L-serine, 0.1 mM acetyl-CoA and a crude enzyme solution was reacted at 30 ° C. for 15 minutes,
232n by cleavage of thioester bond of acetyl-CoA
This was done by measuring the decrease in absorbance in m. The reaction solution from which L-serine was removed was used as a blank.
Further, 10 μM of L-cysteine was added to the reaction solution, and the reaction was carried out in the same manner, and the degree of feedback inhibition by L-cysteine was examined. The ε-value of acetyl-CoA was 6.5.
× 10 ³ M ^-1 cm ^-1 . Table 5 shows the SAT specific activity when L-cysteine was not added and the residual activity (%) when L-cysteine was added.

In all SATs in which the methionine residue at position 256 was substituted with another amino acid residue, feedback inhibition by L-cysteine was reduced. SAT in which the methionine residue at position 256 was substituted with a leucine residue or a lysine residue did not show SAT activity.

[0046]

[Table 5]

[0047]

The Escherichia bacterium of the present invention has the ability to produce L-cysteine with high efficiency and stability.

[0048]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 26 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic oligonucleotide Antisense: NO sequence GGGAATTCAT CGCTTCGGCG TTGAAA 26

SEQ ID NO: 2 Sequence length: 27 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic oligonucleotide Antisense: YES sequence GGCTCTAGAA GCGGTATTGA GAGATTA 27

SEQ ID NO: 3 Sequence length: 17 Sequence type: Number of nucleic acid strands: Single-stranded Topology: Linear Sequence type: Other nucleic acid Synthetic oligonucleotide Antisense: NO Sequence characteristics: Features Symbol to represent: Location: 8..10 Method for determining characteristics: Other information: NNN represents a codon encoding an amino acid other than methionine Sequence GCTGGTCNNN ATCCATT 17

SEQ ID NO: 4 Sequence length: 17 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic oligonucleotide Antisense: YES Sequence characteristics: Features Representation symbol: Location: 8..10 Method for determining characteristics: Other information: NNN represents a codon encoding an amino acid other than methionine Sequence AATGGATNNN GACCAGC 17

SEQ ID NO: 5 Sequence length: 1134 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: Genomic DNA Origin Organism name: Escherichia coli Strain name: JM39 Sequence characteristics symbols have the features: CDS existing position: 223..1044 method to determine the characteristics: S sequence 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 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 TAATGTCCTG 105 1 Asn Gly Ile Asn His Thr Phe Glu Tyr Gly Asp Gly Ile 265 270 TGATCGTGCC GGATGCGATG TAATCATCTA TCCGGCCTAC AGTAACTAAT CTCTCAATAC 1111 CGCTCCCGAT ACCCCAACTG TCG 1134

SEQ ID NO: 6 Sequence length: 272 Sequence type: amino acid Topology: linear Sequence type: protein sequence 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 T hr 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 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 the drawings]

FIG. 1 is a diagram showing the outline of the construction of pCEM256 *.

──────────────────────────────────────────────────の Continued on front page (51) Int.Cl. ⁶ Identification code FI (C12P 13/12 C12R 1:19)

Claims (4)

[Claims] 1. A bacterium belonging to the genus Escherichia which retains a serine acetyltransferase in which an L-cysteine degradation system is suppressed and feedback inhibition by L-cysteine is reduced. 2. The bacterium according to claim 1, wherein the suppression of the L-cysteine degradation system is due to a decrease in cysteine desulfhydrase activity in the cell. 3. The serine acetyltransferase having reduced feedback inhibition by L-cysteine is a wild-type serine acetyltransferase of 256.
Mutation in which the amino acid residue corresponding to the methionine residue at position 2 is replaced with an amino acid residue other than lysine residue and leucine residue, or the region at the C-terminal side is deleted from the amino acid residue corresponding to the methionine residue at position 256. 2. The bacterium of the genus Escherichia according to claim 1, which has a mutation to be eliminated. 4. The Escherichia bacterium according to any one of claims 1 to 3 is cultured in a medium, L-cysteine is produced and accumulated in the culture, and L-cysteine is collected from the culture. A method for producing L-cysteine, comprising: