JP4186564B2 - L-cysteine producing bacterium and method for producing L-cysteine - Google Patents

Description

【図面の簡単な説明】
【図１】 エシェリヒア・コリ細胞抽出液Native-PAGEのCD活性染色の結果を示す図。
Ａ：SAT欠損株JM39、及び、CD活性低下株JM39-8
Ｂ：metC欠損株EZ5、及びmetCを搭載したプラスミドpIP29を導入したEZ5
【図２】 エシェリヒア・コリのmetC破壊株、tnaA破壊株、及びmetC/tnaA破壊株の構築過程を示す図。「Ａ」、「Ｂ」は遺伝子の末端領域、「Ｘ」は欠失領域を示す。
【図３】 metC破壊株、tnaA破壊株、及びmetC/tnaA破壊株の遺伝子破壊を確認する電気泳動及びCD活性染色の結果を示す写真。
【図４】 metC破壊株、tnaA破壊株、及びmetC/tnaA破壊株のＬ−システイン生産性を示す図。[0001]
BACKGROUND OF THE INVENTION
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 L-cysteine derivatives are used in the fields of pharmaceuticals, cosmetics and foods.
[0002]
[Prior 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. In addition, mass production of L-cysteine by an immobilized enzyme method using a novel enzyme is also planned.
[0003]
Furthermore, production of L-cysteine by fermentation using microorganisms has also been attempted. For example, the present inventors have found that serine acetyltransferase (EC 2.3.1.30: hereinafter also referred to as “SAT”) in which the L-cysteine decomposition system is suppressed and feedback inhibition by L-cysteine is reduced. Has disclosed a method for producing L-cysteine using Escherichia bacteria that retains (Patent Document 1). As a means for suppressing the L-cysteine degradation system, it is disclosed to reduce cysteine desulfhydrase (hereinafter also referred to as “CD”) activity in cells. Similarly, a method for producing L-cysteine using a microorganism having cysteine substance metabolism deregulated by a DNA sequence encoding SAT having a specific mutation with reduced feedback inhibition by L-cysteine is known. (Patent Document 2).
[0004]
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 that is not subject to feedback inhibition by L-cysteine is introduced.
[0005]
On the other hand, a method for producing L-cysteine using a microorganism that overexpresses a gene encoding a protein that is suitable for directly releasing antibiotics or substances that are toxic to microorganisms from cells has been reported (Patent Document 3). .
[0006]
As described above, many studies have been made on the enhancement of the activity of L-cysteine biosynthetic enzymes such as SAT and the L-cysteine producing bacteria having a modified L-cysteine excretion system. On the other hand, the L-cysteine decomposition system has not been studied in detail, particularly in bacteria belonging to the genus Escherichia.
[0007]
As an enzyme having some CD activity in Escherichia coli, cystathionine-β-lyase (metC product; hereinafter also referred to as “CBL”) (Non-patent Document 2) and tryptophanase (tnaA product; hereinafter referred to as “TNase”. (Non-Patent Document 3) has also been reported. However, CBL is considered to be an enzyme that catalyzes a reaction that converts cystathionine to homocysteine, and TNase is an enzyme that catalyzes a reaction that degrades tryptophan. Was not known. Further, Patent Document 1 describes a mutant strain having decreased CD activity, but no report has been made regarding the main body of the enzyme responsible for CD activity.
[0008]
[Patent Document 1]
JP 11-155571 A
[Patent Document 2]
Special table 2000-504926
[Patent Document 3]
JP-A-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. Chem. 240 (1965) 1211-1218
[0009]
[Problems to be solved by the invention]
An object of the present invention is to clarify an enzyme responsible for CD activity and its gene, and to use the same gene for breeding L-cysteine-producing bacteria to provide a new method for producing L-cysteine.
[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 enzymes responsible for the major CD activity of Escherichia coli, and reduce or eliminate these enzyme activities. Thus, it was found that the ability to produce L-cysteine can be improved, and the present invention has been completed.
That is, the present invention is as follows.
[0011]
(1) A bacterium belonging to the genus Escherichia having an ability to produce L-cysteine and modified so that cystathionine-β-lyase activity is reduced or deleted.
(2) A bacterium belonging to the genus Escherichia having an ability to produce L-cysteine and modified so that ciscystathionine-β-lyase activity and tryptophanase activity are reduced or deleted.
(3) The Escherichia bacterium according to (1), wherein a gene encoding cystathionine-β-lyase is disrupted.
(4) The Escherichia bacterium according to (2), wherein a gene encoding ciscystathionine-β-lyase and a gene encoding tryptophanase are disrupted.
(5) The bacterium belonging to the genus Escherichia according to any one of (1) to (4), which is further modified to enhance the L-cysteine biosynthesis enzyme activity.
(6) The Escherichia bacterium according to (5), which is modified so that serine acetyltransferase is enhanced.
(7) The Escherichia bacterium according to (6), which retains a serine acetyltransferase insensitive to feedback inhibition by L-cysteine.
(8) The Escherichia bacterium according to any one of (1) to (7), which is Escherichia coli.
(9) A method for producing L-cysteine, wherein the bacterium belonging to the genus Escherichia of any one of (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. .
(10) It is characterized by increasing the L-cysteine-producing ability of the bacterium by decreasing or eliminating the ciscystathionine-β-lyase activity or tryptophanase activity of the bacterium belonging to the genus Escherichia, or both of these activities. A method for producing an L-cysteine-producing bacterium.
[0012]
In the present invention, the ability to produce L-cysteine refers to 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 eliminated and a case where feedback inhibition is not originally received.
In the present invention, L-cysteine refers to reduced L-cysteine or L-cystine or a mixture thereof unless otherwise specified.
[0013]
DETAILED DESCRIPTION OF THE INVENTION
The first form of the genus Escherichia of the present invention is an Escherichia bacterium that has L-cysteine-producing ability and has been modified so that CBL activity is reduced or eliminated. The second form of the genus Escherichia of the present invention is an Escherichia bacterium that has L-cysteine-producing ability and has been modified so that CBL activity and TNase activity are reduced or deleted. Further, the Escherichia bacterium of the present invention is an 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 can be obtained by enhancing L-cysteine biosynthesis enzyme activity in an Escherichia bacterium in which CBL activity, or CBL activity and TNase activity are reduced or deleted. Further, the Escherichia bacterium of the present invention can also be obtained by reducing or deleting CBL activity, CBL activity and TNase activity in an Escherichia bacterium having enhanced L-cysteine biosynthesis enzyme activity.
[0014]
<1> Decrease or disappearance of CBL activity or TNase activity
In order to reduce or eliminate the CBL activity or TNase activity of Escherichia bacteria, for example, the Escherichia bacteria are irradiated with ultraviolet rays or subjected to normal mutation treatment such as N-methyl-N′-nitro-N-nitrosoguanidine (NTG) or nitrous acid. And a method of selecting a mutant strain having a decreased CBL activity or TNase activity by treatment with the mutagen used in the above. In addition, Escherichia bacteria having reduced CBL activity or TNase activity can be obtained by gene disruption in addition to mutation treatment. In order to reliably reduce or eliminate CBL activity or TNase activity, a gene disruption method is preferred. In Escherichia coli, CBL is encoded by the metC gene and TNase is encoded by the tnaA gene.
[0015]
Escherichia coli metC gene (GenBank accession M12858, Proc. Natl. Acad. Sci. USA 83, 867-871 (1986)) and tnaA gene (GenBank accession K00032, J. Bacteriol. 147, 787-796 (1981), The base sequence of J. Bacteriol. 151, 942-951 (1982) has been reported. PCR using primers synthesized based on the respective sequences and PCR using Escherichia coli chromosomal DNA as a template (PCR: polymerase chain reaction) White, TJ et al., Trends Genet. 5, 185 (1989)), DNA fragments containing each gene can be obtained. In addition, the inside of the gene encoding CBL has been deleted, and the inside of the gene encoding TNase that has been modified so that it does not produce CBL that functions normally (deleted metC gene) and TNase is deleted. The tnaA gene (deleted tnaA gene) modified so as not to produce a functional TNase 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, respectively. In addition, the nucleotide sequence of the tnaA gene and the encoded amino acid sequence are shown in SEQ ID NOs: 12 and 13, respectively.
[0016]
In the following, a method for destroying a gene encoding CBL will be described, but a gene encoding TNase can be similarly disrupted.
The Escherichia bacterium is transformed with DNA containing the metC gene (deleted metC gene) that has been modified so that it does not produce CBL that functions normally by deleting the CBL-encoding gene, and the deleted metC gene And the metC gene on the chromosome can be recombined to destroy the metC gene on the chromosome. Gene disruption by gene replacement using such homologous recombination has already been established, and there are a method using linear DNA and a method using a plasmid containing a temperature-sensitive replication origin.
[0017]
In order to replace the deleted metC gene with the metC gene on the host chromosome, for example, the following may be performed. A recombinant DNA is prepared by inserting a temperature-sensitive replication origin, a deletion-type metC gene, and a marker gene showing resistance to drugs such as ampicillin or chloramphenicol into a vector, and Escherichia bacteria using this recombinant DNA And culturing the transformed strain at a temperature at which the temperature-sensitive replication origin does not function, and then culturing the transformed strain in a medium containing a drug to obtain a transformed strain in which the recombinant DNA is incorporated into the chromosomal DNA. It is done.
[0018]
In this way, a strain in which the recombinant DNA is integrated into the chromosome undergoes recombination with the original metC gene sequence on the chromosome, and two fusion genes of the chromosome metC gene and the deleted metC gene are present in addition to the recombinant DNA. (A vector part, a temperature sensitive replication origin and a drug resistance marker) are inserted into the chromosome. Therefore, since the normal metC gene is dominant in this state, the transformed strain expresses normal metC.
[0019]
Next, in order to leave only the deleted metC gene on the chromosomal DNA, one copy of the metC gene is recombined by recombination of the two metC genes together with the vector portion (including the temperature-sensitive replication origin and drug resistance marker). Remove from DNA. At that time, there is a case where the normal metC gene is left on the chromosomal DNA and the deleted metC gene is excised, and conversely, the deleted metC gene is left on the chromosomal DNA and the normal metC gene is excised. is there. In either case, if the cells are cultured at a temperature at which the temperature-sensitive replication origin functions, the excised DNA is retained in the cell in the form of a plasmid. Next, when cultured at a temperature at which the temperature-sensitive replication origin does not function, the metC gene on the plasmid is removed from the cell together with the plasmid. Then, a strain in which the metC gene is disrupted can be obtained by selecting a strain in which the deletion-type metC gene remains on the chromosome by PCR or Southern hybridization.
[0020]
The fact that the CBL activity of the gene-disrupted strain or mutant strain is reduced or eliminated is that the method of Guggenheim, S. (Methods Enzymol., 17, 439-442 (1971), etc.) Can be confirmed by measuring CBL activity and comparing with the CBL activity of the parent strain.
[0021]
Examples of plasmids containing a temperature sensitive replication origin for Escherichia coli include pMAN031 (Yasueda, H. et al, Appl. Microbiol. Biotechnol., 36, 211 (1991)), pMAN997 (WO 99/03988), and pEL3 (KA Armstrong et. al., J. Mol. Biol. (1984) 175, 331-347).
[0022]
In the same manner as described above, a tnaA gene-disrupted strain can be obtained. The fact that the TNase activity of the tnaA gene disrupted strain or mutant strain is reduced or eliminated is determined by the method of Newton, WA et al. (J. Biol. Chem., 240, 1211-1218 (1965 )) Method can be used to measure the TNase activity and compare it with the TNase activity of the parent strain.
Deletion-type metC gene and deletion-type tnaA gene used for gene disruption only have to have homology to the extent that homologous recombination occurs 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 if the DNAs can hybridize under stringent conditions.
[0023]
<2> Enhancement of SAT activity
Enhancement of SAT activity in Escherichia bacterial cells is achieved by increasing the copy number of the gene encoding SAT. For example, if a gene fragment encoding SAT is ligated with a vector that functions in an Escherichia bacterium, preferably a multicopy-type vector, a recombinant DNA is prepared, and this is introduced into the host Escherichia bacterium and transformed. Good.
[0024]
As the SAT gene, any of genes derived from bacteria belonging to the genus Escherichia and genes derived from other organisms can be used. As a gene encoding SAT of Escherichia coli, cysE has been cloned from a wild type strain and an L-cysteine secreting mutant, and its nucleotide sequence has been clarified (Denk, D. and Boeck, A., J. General Microbiol. , 133, 515-525 (1987)). Therefore, the SAT gene can be obtained by PCR using a chromosomal DNA of an Escherichia bacterium as a template using a primer prepared based on the base sequence (see JP-A-11-155571). Genes encoding SATs of other organisms can be obtained in the same manner.
For reference, SEQ ID NOs: 14 and 15 include the reported nucleotide sequence of wild-type cysE and the encoded amino acid sequence of SAT (Denk, D. and Bock, A., J. general Microbiol., 133, 515-525 (1987)).
As the SAT gene, in addition to the wild-type SAT gene, substitution, deletion or insertion of one or several amino acid residues that do not substantially impair the activity of catalyzing the activation of L-serine by acetyl-CoA It may have an amino acid sequence containing an addition or inversion. Here, “several” varies depending on the position and type of the amino acid residue protein in the three-dimensional structure, but is preferably 2 to 50, more preferably 2 to 40, particularly preferably 2 to 30. is there.
[0025]
The DNA encoding the protein substantially the same as SAT as described above is hybridized under stringent conditions with a base sequence consisting of base numbers 223 to 1047 of SEQ ID NO: 14 or a probe that can be prepared from the base sequence. And a DNA encoding a protein having the same activity as SAT. “Stringent conditions” refers to conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed. Although it is difficult to quantify these conditions clearly, for example, DNAs with high homology, for example, DNAs having 50% or more homology hybridize and DNA with lower homology than that. A salt corresponding to 60 ° C., 1 × SSC, 0.1% SDS, preferably 0.1 × SSC, 0.1% SDS, which does not hybridize with each other, or is a condition for washing of ordinary Southern hybridization Conditions for hybridizing at a concentration are mentioned.
[0026]
Chromosomal DNA is obtained from bacteria that are DNA donors, for example, the method of Saito and Miura (H. Saito and K. Miura, Biochem. Biophys. Acta, 72, 619 (1963), Biotechnology Experiments, Nippon Biotechnology, Ed., Pp. 97-98, Bafukan, 1992).
[0027]
In order to introduce a DNA fragment containing the SAT gene amplified by the PCR method into a bacterium belonging to the genus Escherichia, various vectors used for normal protein expression can be used. Examples of such vectors include pUC19, pUC18, pHSG299, pHSG399, pHSG398, RSF1010, pBR322, pACYC184, pMW219 and the like.
[0028]
In order to introduce a recombinant vector containing the SAT gene into a bacterium belonging to the genus Escherichia, the method of DA Morrison (Methods in Enzymology 68, 326 (1979)) or the method of increasing the DNA permeability by treating recipient cells with calcium chloride ( Mandel, M. and Higa, A., J. Mol. Biol., 53, 159 (1970)) and the like, can be performed by a method usually used for transformation of bacteria belonging to the genus Escherichia.
[0029]
Increasing the copy number of the SAT gene can also be achieved by making multiple copies of the SAT gene on the chromosomal DNA of Escherichia bacteria. In order to introduce multiple copies of the SAT gene into the chromosomal DNA of an Escherichia bacterium, homologous recombination is performed using a sequence present in multiple copies on the chromosomal DNA as a target. As a sequence present in multiple copies on chromosomal DNA, repetitive DNA and inverted repeats present at the end of a transposable element can be used. Alternatively, as disclosed in Japanese Patent Application Laid-Open No. 2-109985, it is possible to mount a SAT gene on a transposon, transfer it, and introduce multiple copies onto chromosomal DNA.
[0030]
In addition to the gene amplification described above, the enhancement of SAT activity can also be achieved by replacing expression control sequences such as the promoter of the SAT gene on chromosomal DNA or plasmid with a strong one (JP-A-1-215280). See the official gazette). For example, lac promoter, trp promoter, trc promoter and the like are known as strong promoters. The replacement of the expression regulatory sequence can also be performed, for example, by gene replacement using a temperature sensitive plasmid.
[0031]
Further, as disclosed in International Publication WO00 / 18935, it is possible to introduce a base substitution of several bases into the promoter region of the SAT gene and modify it to be more powerful. These promoter substitutions or modifications enhance SAT gene expression and enhance SAT activity. Modification of these expression regulatory sequences may be combined with increasing the copy number of the SAT gene.
[0032]
Furthermore, if there is a suppression mechanism for the expression of SAT gene, the expression of SAT gene can be enhanced by modifying the expression regulatory sequence or the gene involved in the suppression so that the suppression is released or reduced. Can do.
[0033]
The SAT activity in Escherichia bacteria cells can also be increased by retaining SAT (hereinafter also referred to as “mutant SAT”) in which feedback inhibition by L-cysteine is reduced or eliminated in Escherichia bacteria. . The mutant SAT is a mutation in which the amino acid residue corresponding to the methionine residue at position 256 of wild-type SAT is substituted with an amino acid residue other than the lysine residue and leucine residue, or corresponds to the methionine residue at position 256. And SAT having a mutation that deletes the C-terminal region from the amino acid residue. Examples of amino acid residues other than the lysine residue and leucine residue include 17 types of amino acid residues other than methionine residue, lysine residue and leucine residue among amino acids constituting normal proteins. More specifically, an isoleucine residue is mentioned. Examples of a method for introducing a desired mutation into the wild-type SAT gene include site-specific mutation. As a mutant SAT gene, a mutant cysE encoding a mutant SAT of Escherichia coli is known (see WO 97/15673 International Publication Pamphlet, JP-A-11-155571). Escherichia coli JM39-8 strain (E. coli JM39-8 (pCEM256E) carrying a plasmid pCEM256E containing a mutant cysE encoding a mutant SAT in which the methionine residue at position 256 is substituted with a glutamic acid residue, private number: AJ13391) is the National Institute of Biotechnology, National Institute of Advanced Industrial Science and Technology (currently the National Institute of Advanced Industrial Science and Technology, Japan) on November 20, 1997. 1 center 6), deposited under the deposit number of FERM P-16527, transferred to the international deposit under the Budapest Treaty on July 8, 2002, and assigned the deposit number FERM BP-8112.
[0034]
Moreover, SAT of Arabidopsis thaliana is known not to be subjected to feedback inhibition by L-cysteine, and can be suitably used in the present invention. As an SAT gene-containing plasmid derived from Arabidopsis thaliana, pEAS-m (FEMS Microbiol. Lett., 179 (1999) 453-459) is known.
[0035]
The mutant SAT includes one or several amino acids that do not substantially impair the activity of catalyzing the activation of L-serine by acetyl-CoA in addition to the mutation that reduces the feedback inhibition by L-cysteine. It may have an amino acid sequence including residue substitution, deletion, insertion, addition, or inversion. In the SAT having such a mutation, the position of the methionine residue at position 256 may be changed. Even in such a case, the amino acid residue corresponding to the methionine residue at position 256 is changed to lysine. By substituting amino acid residues other than residues and leucine residues, mutant SATs with reduced feedback inhibition by L-cysteine can be obtained.
[0036]
In addition, the Escherichia bacterium can retain the mutant SAT by introducing into the intracellular SAT gene a mutation that cancels feedback inhibition of the encoded SAT by L-cysteine. Mutation can be introduced by ultraviolet irradiation or treatment with a mutagen used for normal mutation such as N-methyl-N′-nitro-N-nitrosoguanidine (NTG) or nitrous acid.
[0037]
<3> Production of L-cysteine
By culturing the Escherichia bacterium of the present invention obtained as described above in a suitable medium, producing and accumulating L-cysteine in the culture, and collecting L-cysteine from the culture, L-cysteine is obtained. Can be produced efficiently and stably. The L-cysteine produced by the method of the present invention may contain cystine in addition to the reduced cysteine, but the object of the production method of the present invention includes cystine or reduced cysteine and cystine. A mixture of
[0038]
Examples of the medium to be used include a normal medium containing a carbon source, a nitrogen source, a sulfur source, inorganic ions, and other organic components as required.
As the carbon source, saccharides such as glucose, fructose, sucrose, molasses and starch hydrolysate, and organic acids such as fumaric acid, citric acid and succinic acid can be used.
[0039]
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.
[0040]
Examples of the sulfur source include inorganic sulfur compounds such as sulfate, sulfite, sulfide, hyposulfite, and thiosulfate.
As an organic trace nutrient source, it is desirable to contain a required substance such as vitamin B1 or a yeast extract. In addition to these, potassium phosphate, magnesium sulfate, iron ions, manganese ions and the like are added in small amounts as necessary.
[0041]
Cultivation is preferably carried out under aerobic conditions for 30 to 90 hours. The culture temperature is preferably controlled at 25 ° C. to 37 ° C., and the pH during culture is preferably controlled at 5 to 8. In addition, an inorganic or organic acidic or alkaline substance, 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 ordinary ion exchange resin method, precipitation method and other known methods.
[0042]
【Example】
Hereinafter, the present invention will be described more specifically with reference to examples.
[0043]
<1> Identification of enzymes responsible for CD activity in Escherichia coli
(1) Electrophoresis and CD activity staining of Escherichia coli cell extract
Escherichia coli JM39 (F), an SAT-deficient strain of Escherichia coli ⁺ cysE51 tfr-8) (Denk, D. and Bock, A., J. Gene. Microbiol, 133, 515-525 (1987)) and CD activity-reduced strain JM39-8 (Japanese Patent Laid-Open No. 11-155571) The cell extract was subjected to non-denaturing polyacrylamide gel electrophoresis (Native-PAGE), stained for CD activity, and analyzed for CD activity. The JM39-8 strain is a mutant strain obtained by treating the JM39 strain with NTG and isolated as an L-cysteine non-assimilating strain (Japanese Patent Laid-Open No. 11-155571).
[0044]
Each strain was inoculated into 0.3% L-cysteine-added or non-added LB medium (tryptone 10g / L, Yeast Extract 5g / L, NaCl 5g / L (pH7.0)), and then cultured at 37 ° C overnight. did. The bacterial cells are collected by centrifugation, washed with 0.85% NaCl, and the resulting wet cells are suspended in approximately 3 ml of a cell disruption buffer (10 mM Tris-HCl (pH 8.6), 10 μM PLP, 100 μM DTT). Ultrasonic cell destruction was performed with Bioruptor, Inc. for 30 minutes. Centrifugation was performed at 12,000 rpm for 10 minutes, and the resulting supernatant was used as a cell extract. Protein concentration was quantified using Protein Assay Kit (Bio-Rad).
[0045]
Suspend each bacterial cell extract (30 μg protein each) in 2 × Native-PAGE buffer (Glycine 14.43 g / L, Tris 3.0 g / L, pH 8.6 (HCl)) Electrophoresis (Native-PAGE) was performed. Using the obtained electrophoresis gel, CD activity staining was performed. Prepare the reaction solution (Tris · HCl 100 mM, EDTA 10 mM, PLP (Pyridoxal-5-phosphate) 20 μM, L-Cys 50 mM, pH 8.6, fill up to 100 ml, and add bismuth chloride (BiCl _Three ) Was added, and only the lanes at both ends of the gel were cut out and soaked. The gel was shaken at room temperature and allowed to stand until the bands were visible. Two main bands were detected (FIG. 1A). The lane at the center of the gel was used for amino acid sequencing. In the JM39-8 strain, of the two bands described above, the band with the higher molecular weight was weaker than the JM39 strain.
[0046]
(2) Identification of CBL as an enzyme responsible for CD activity
CBL (metC product) has been reported as an enzyme having CD activity in Escherichia coli (Chandra et. Al., Biochemistry, 21 (1982) 3064-3069). In order to verify the possibility that the band was CBL, an experiment similar to the above was performed using a metC-deficient strain and Escherichia coli obtained by amplifying the metC gene with a multicopy plasmid.
[0047]
metC deficient strain EZ5 (metC: Tn10Tet ^s rpsL add-uid-man uraA: Tn10) and plasmid pIP29 carrying metC (provided by Dr. Saint-Girons of the Pasteur Institute, France. Belfaiza et. al., Proc. Natl. Acad. Sci. 83 (1986) 867-871) was used to carry out Native-PAGE and CD activity staining using a strain introduced into EZ5. As a result, it was found that the band having the smaller molecular weight was deleted in the metC-deficient strain, and that the band was greatly amplified by introduction of pIP29 (FIG. 1B). From this result, it was concluded that the lower molecular weight band was CBL.
[0048]
(3) Identification of TNase as an enzyme responsible for CD activity
The protein corresponding to the band with the larger molecular weight of the two bands observed by activity staining was purified. From the electrophoresis gel, the band having the larger molecular weight was cut out from the gel, extracted and concentrated by a conventional method. The obtained fraction having CD activity was subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and blotting was performed on a PVDF membrane (Bio-Rad) without staining. The membrane after the transfer 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, the band of the confirmed target enzyme was cut out, and the N-terminal amino acid sequence was determined by an automated Edman degradation method using Protein Sequencer 476A (Applied Biosystems).
[0049]
The result is shown in SEQ ID NO: 1. This sequence has been reported to have some CD activity (Austin Newton et. Al., J. Biol. Chem. 240 (1965) 1211-1218) and compared with the amino acid sequence of Escherichia coli TNase. , 14 of 15 residues matched. 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 higher molecular weight is likely to be TNase. From this result, it was estimated that the expression of TNase was reduced in the JM39-8 strain.
[0050]
<2> Construction of Escherichia coli tnaA-deficient and metC-deficient strains
As described above, CBL and TNase were identified as enzymes responsible for CD activity in Escherichia coli. In order to examine the effect of these decreased activities on L-cysteine production, the gene metC or TNase encoding CBL was used. A disrupted strain of the encoded gene tnaA and a disrupted strain of both of these genes were prepared (FIG. 2). Specifically, it was performed as follows.
[0051]
(1) Construction of tnaA deficient strain
PCR was carried out using the chromosomal DNA of Escherichia coli JM109 strain as a template and primers having the nucleotide sequences shown in SEQ ID NOs: 2 and 3 and primers having the nucleotide sequences shown in SEQ ID NOs: 4 and 5, respectively. Next, each PCR product was cleaved with HindIII and ligated. Using this reaction solution as a template, PCR was performed using primers having the nucleotide sequences shown in SEQ ID NOs: 2 and 5, and a fragment of about 1.6 kb was obtained. This fragment was digested with BamHI and incorporated into the BamHI site of a temperature sensitive plasmid pEL3 (KA Armstrong et. Al., J. Mol. Biol. (1984) 175, 331-347) to construct a plasmid pEL3ΔtnaA for destroying tnaA. .
[0052]
The JM39 strain was transformed with the plasmid pEL3ΔtnaA, and a clone showing ampicillin resistance at 30 ° C. was obtained. The culture solution of this clone was appropriately diluted and applied to ampicillin-added LB agar medium and cultured overnight at 42 ° C. to obtain a strain in which pEL3ΔtnaA was integrated into the JM39 strain chromosome. Subsequently, this strain was subcultured at 37 ° C. in an ampicillin-free LB medium to isolate ampicillin-sensitive clones. The isolated clone was analyzed around the tnaA gene on the chromosome by genomic PCR, and a strain having no wild-type tnaA but having only the disrupted tnaA gene cloned on pEL3ΔtnaA was selected. In this way, a tnaA gene disruption strain JM39ΔtnaA derived from the JM39 strain was obtained.
[0053]
(2) Construction of metC deficient strain
PCR was carried out using the chromosomal DNA of Escherichia coli JM109 strain as a template and 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, respectively. Next, each PCR product was cleaved with KpnI and then ligated. Using this reaction solution as a template, PCR was carried out using primers having the nucleotide sequences shown in SEQ ID NOs: 6 and 9, and a fragment of about 1.4 kb was obtained. This fragment was digested with BamHI and incorporated into the BamHI site of pEL3 to construct a plasmid pEL3ΔmetC for disrupting metC.
[0054]
JM39 strain or JM39ΔtnaA was transformed with plasmid pEL3ΔmetC to obtain a clone showing ampicillin resistance at 30 ° C. The culture solution of this clone was appropriately diluted and applied to an LB agar medium supplemented with ampicillin, and cultured overnight at 42 ° C. to obtain a strain in which pEL3ΔmetC was integrated into the JM39 strain chromosome. Subsequently, this strain was subcultured at 37 ° C. in an ampicillin-free LB medium to isolate ampicillin-sensitive clones. The isolated clone was analyzed around the metC gene on the chromosome by genomic PCR, and a strain having no wild-type metC but having only the disrupted metC gene cloned on pEL3ΔmetC was selected. Thus, the metC gene-disrupted strain JM39ΔmetC derived from the JM39 strain and the metC gene-disrupted strain JM39ΔtnaAΔmetC derived from the JM39ΔtnaA strain were obtained.
[0055]
(3) Confirmation of gene disruption
Next, JM39ΔtnaA and JM39ΔmetC strains were examined for the presence or absence of a band having CD activity by electrophoresis and activity staining (FIG. 3). As a result, the band with the larger molecular weight was completely lost in the tnaA-disrupted strain, the band with the smaller molecular weight was lost in the metC-disrupted strain, and both bands were completely lost in the tnaA / metC-disrupted strain.
[0056]
<3> Introduction of feedback inhibition insensitive SAT gene
A feedback inhibition insensitive SAT gene derived from Arabidopsis thaliana was introduced into each of the gene-disrupted strains obtained above.
[0057]
The SAT gene-containing plasmid pEAS-m (FEMS Microbiol. Lett., 179 (1999) 453-459) derived from Arabidopsis thaliana was used. The plasmid pES-m is based on the plasmid pCE used for the introduction of the cysE gene derived from E. coli described in JP-A-11-155571, and is insensitive to feedback inhibition from Arabidopsis instead of the cysE gene derived from E. coli. The SAT gene is inserted and can be constructed by the following method. First, by the method described in FEMS Microbiol. 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 pCE is constructed. . The cysE gene is removed by NcoI digestion of this plasmid, and an Arabidopsis SAT gene added with an NcoI linker is introduced instead. The structure of the SAT gene of Arabidopsis thaliana is known (Plant Molecular Biology, 30, 1041-1049, 1996), and can be obtained by chemical synthesis or PCR. pEAS-m is a DNA fragment in which NcoI sites are added before and after the sequence of positions 189-1201 in the base sequence described in Fig. 2 of Plant Molecular Biology, 30, 1041-1049, 1996. It was obtained by introduction.
[0058]
JM39, JM39ΔtnaA, JM39ΔmetC, and JM39ΔtnaAΔmetC were transformed with the 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 1 platinum loop of cells was added to C1 medium (glucose 30 g / L, glucose, supplemented with 50 mg / L of ampicillin). NH _Four Cl 10g / L, KH ₂ PO _Four 2g / L, MgSO _Four ・ 7H ₂ O 1g / L, FeSO _Four ・ 7H ₂ O 10mg / L, MnCl ₂ ・ 4H ₂ O 10mg / L, CaCO _Three 20 g / L) 20 ml of Sakaguchi flask was inoculated and cultured at 30 ° C. for 48 hours and 72 hours. When the L-cysteine content of the supernatant after culturing was quantified by bioassay, it was confirmed that the disruption of tnaA and metC was effective in improving L-cysteine productivity in all disrupted strains over the wild strain. (FIG. 4).
[0059]
【The invention's effect】
According to the present invention, L-cysteine productivity of an Escherichia bacterium can be improved.
[0060]
[Sequence Listing]

[Brief description of the drawings]
FIG. 1 is a diagram showing the results of CD activity staining of Escherichia coli cell extract Native-PAGE.
A: SAT-deficient strain JM39 and CD activity-reduced strain JM39-8
B: EZ5 into which metC-deficient strain EZ5 and plasmid pIP29 carrying metC were introduced
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 terminal regions of the gene, and “X” indicates a deletion 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 is a diagram showing L-cysteine productivity of metC disrupted strain, tnaA disrupted strain, and metC / tnaA disrupted strain.

Claims (4)

Culturing a bacterium belonging to the genus Escherichia in a culture medium, L- cysteine is produced and accumulated in the medium, and collecting the L- cysteine from the medium, a method of manufacturing a L- cysteine, the Escherichia bacteria, L- cysteine A bacterium belonging to the genus Escherichia that has a production ability, retains serine acetyltransferase insensitive to feedback inhibition by L-cysteine, and is modified so that cystathionine-β-lyase activity and tryptophanase activity are reduced or eliminated A method characterized in that The method according to claim 1, wherein the Escherichia bacterium is an Escherichia bacterium in which a gene encoding cystathionine-β-lyase and a gene encoding tryptophanase are disrupted . The method according to claim 1 or 2, wherein the Escherichia bacterium is Escherichia coli. Escherichia bacteria Sutachionin -β- lyase activity or tryptophanase activity, or both of these activities is reduced or eliminated, by Rukoto to hold insensitive serine acetyltransferase to feedback inhibition by addition L- cysteine, A method for producing an L-cysteine-producing bacterium of the genus Escherichia characterized by increasing the L-cysteine-producing ability of the bacterium.

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