# Patent

## Title
L-cysteine producing microorganism and a method for producing L-cysteine

## Abstract

L-cysteine is produced by culturing an Escherichia bacterium having L-cysteine producing ability and containing a gene encoding an O-acetylserine sulphydrylase B or MalY regulatory protein that is modified so that cysteine desulfhydrase activity is reduced or eliminated. The bacterium is cultured in a medium to produce and cause accumulation of L-cysteine in the medium, and collecting L-cysteine from the medium.

## Background
This application is a divisional under 35 U.S.C. §120 of U.S. patent application Ser. No. 11/070,084, filed Mar. 3, 2005, now abandoned, which claims priority under 35 U.S.C. §119 to Japanese Patent Application No. 2004-060483, filed on Mar. 4, 2004, which are incorporated in their entireties by reference. The Sequence Listing in electronic format filed herewith is also hereby incorporated by reference in its entirety (File Name: US-214D_Seq_List; File Size: 28 KB; Date Created: Dec. 10, 2009).


BACKGROUND OF THE INVENTION
1. Technical Field
The present invention relates to a method for producing L-cysteine, and a microorganism suitable for the production of L-cysteine. L-cysteine and derivatives thereof are used in the fields of pharmaceuticals, cosmetics, foods and the like.
2. Background Art
L-cysteine is conventionally obtained by extraction from keratin-containing substances such as hair, horns, and feathers, or by conversion of precursor DL-2-aminothiazoline-4-carboxylic acid using a microbial enzyme. Large scale production of L-cysteine has been attempted using an immobilized enzyme method with a novel enzyme.
Furthermore, production of L-cysteine has also been attempted by fermentation utilizing a microorganism. A method of producing L-cysteine using a microorganism has been reported, wherein said microorganism contains a DNA encoding serine acetyltransferase (SAT) with a mutation which prevents feedback inhibition by L-cysteine (WO 97/15673). A method of producing L-cysteine using a strain of Escherichia coli which contains a gene encoding SAT isozyme of Arabidopsis thaliana is disclosed in FEMS Microbiol. Lett., vol. 179 (1999) p 453-459. This SAT isozyme gene is resistant to feedback inhibition by L-cysteine. Also, a method of producing L-cysteine using a microorganism which overexpresses a gene encoding a protein that excretes an antibiotic or a toxic substance is disclosed in JP11-56381A.
Furthermore, the inventors of the present invention have disclosed a method of producing L-cysteine using a strain of Escherichia coli which contains serine acetyltransferase with reduced feedback inhibition by L-cysteine, and in which the L-cysteine-decomposing system is attenuated (JP11-155571A). The L-cysteine-decomposing system of the bacterium is attenuated by reduction of the intracellular activity of cysteine desulfhydrase (hereinafter, also referred to as “CD”).
Enzymes which have been reported to have CD activity in Escherichia coli include cystathionine-β-lyase (metC gene product, hereinafter, also referred to as “CBL”) (Chandra et. al., Biochemistry, vol. 21 (1982) p 3064-3069) and tryptophanase (tnaA gene product, hereinafter, also referred to as “TNase”) (Austin Newton, et al., J. Biol. Chem. vol. 240 (1965) p 1211-1218). A method of producing L-cysteine using an Escherichia coli strain which has reduced activities of cystathionine-(3-lyase and tryptophanase is disclosed in JP2003-169668A (EP1,298,200). However, no enzymes other than these have been previously reported to have CD activity.
SUMMARY OF THE INVENTION
An object of the present invention is to identify a gene encoding a protein having CD activity, and utilize the gene for breeding L-cysteine-producing microorganism.
In order to attain the above-mentioned object, the inventors of the present invention made extensive studies and as a result, have found that the enzymes O-acetylserine sulphydrylase B (OASS-B) and MalY regulatory protein (MalY) have CD activity in Escherichia coli. The inventors also found that reducing CD activity by modifying these genes leads to improvement in the production of L-cysteine.
It is an object of the present invention to provide an Escherichia bacterium having L-cysteine-producing ability, wherein said bacterium contains a gene encoding O-acetylserine sulphydrylase B, and wherein said gene is modified so that cysteine desulfhydrase activity is reduced or eliminated.
It is a further object of the present invention to provide an Escherichia bacterium having L-cysteine-producing ability, wherein said bacterium contains a gene encoding MalY regulatory protein, and wherein said gene is modified so that cysteine desulfhydrase activity is reduced or eliminated.
It is a further object of the present invention to provide an Escherichia bacterium as described above, wherein said gene encoding O-acetylserine sulphydrylase B is disrupted.
It is a further object of the present invention to provide an Escherichia bacterium as described above, wherein said gene encoding MalY regulatory protein is disrupted.
It is a further object of the present invention to provide an Escherichia bacterium as described above, wherein activity of an L-cysteine biosynthetic enzyme is enhanced.
It is a further object of the present invention to provide an Escherichia bacterium as described above, wherein said L-cysteine biosynthetic enzyme is serine acetyltransferase.
It is a further object of the present invention to provide an Escherichia bacterium as described above, wherein said serine acetyltransferase is resistant to feedback inhibition by L-cysteine.
It is a further object of the present invention to provide an Escherichia bacterium as described above, wherein said Escherichia bacterium is Escherichia coli. 
It is a further object of the present invention to provide a method of producing L-cysteine comprising culturing the Escherichia bacterium as described above in a medium, and collecting L-cysteine from the medium.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows the results of CD activity staining of Escherichia coli cell extracts on Native-PAGE.
FIG. 2 shows primers used in gene disruption.
FIG. 3 shows L-cysteine-producing ability of the control strain and each CD gene-disrupted strain; JM39 (●), JM39ΔtnaA (▪), JM39ΔmetC (▴), JM39Δcysm (*), JM39ΔmalY (+), and JM39ΔtnaAΔmetCΔmalYΔcysM (♦).



DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
Hereinafter, the present invention will be explained in detail. In the present invention, unless otherwise described, L-cysteine refers to a reduced-type of L-cysteine, L-cystine, or a mixture thereof.
The Escherichia bacterium of the present invention has L-cysteine-producing ability and contains a gene encoding O-acetylserine sulphydrylase B (OASS-B) or MalY regulatory protein, wherein the gene is modified so that the cysteine desulfhydrase (CD) activity of the bacterium is reduced or eliminated. The Escherichia bacterium of the present invention may have L-cysteine producing-ability and may contain both of the genes encoding OASS-B and MalY regulatory protein which are modified so that the CD activity of the bacterium is reduced or eliminated. In the Escherichia bacterium of the present invention, one or both of the genes encoding tryptophanase (TNase) and cystathionine-β-lyase (CBL) may also be modified so that the CD activity of the bacterium is further reduced.
The term “L-cysteine-producing ability” as used herein refers to an ability of the Escherichia bacterium of the present invention to cause accumulation of L-cysteine in a culture medium to such a degree that L-cysteine can be collected from the medium when the bacterium is cultured in the medium. The L-cysteine-producing ability may be imparted to a parent strain of an Escherichia bacterium by a mutation technique or a recombinant DNA technique. The recombinant DNA technique includes introduction of a gene encoding an L-cysteine biosynthetic enzyme. Alternatively, bacteria having native L-cysteine-producing ability may also be used. Furthermore, a bacterium imparted with an L-cysteine-producing ability by modification of a gene encoding O-acetylserine sulphydrylase B (OASS-B) or MalY regulatory protein may be used.
The Escherichia bacteria which can be used as a parent strain include those described in Neidhardt, F. C. et al. (Escherichia coli and Salmonella Typhimurium, American Society for Microbiology, Washington D.C., 1208, table 1), and Escherichia coli is preferably used. Wild-type strains of Escherichia coli include K12 strain, or mutants thereof such as Escherichia coli MG1655 strain (ATCC No. 47076) and W3110 strain (ATCC No. 27325). These bacteria strains can be obtained from the American Type Culture Collection (ATCC, Address: P.O. Box 1549, Manassas, Va. 20108, United States of America).
The Escherichia bacteria of the present invention can be obtained by modifying a gene encoding OASS-B or MalY regulatory protein in a parent strain so that CD activity of the strain is reduced or eliminated, and then imparting an L-cysteine-producing ability to the modified strain. The bacteria of the present invention can also be obtained by imparting an L-cysteine-producing ability to a parent strain, and then modifying a gene encoding OASS-B or MalY regulatory protein so that CD activity of the strain is reduced or eliminated. One or both of the genes encoding TNase and CBL may be further modified.
The method of obtaining the Escherichia bacteria of the present invention will be explained in detail.
<1> Modification of a Gene Encoding OASS-B or MalY Regulatory Protein
Examples of the methods of modifying a gene encoding OASS-B or MalY regulatory protein so that the CD activity of the Escherichia bacteria is reduced or eliminated include a mutation treatment method and a gene disruption method. Examples of the mutation treatment method include treating Escherichia bacteria with ultraviolet ray irradiation or with a mutagen used in ordinary mutation treatments, such as N-methyl-N′-nitro-N-nitrosoguanidine (NTG) or nitrous acid, and selecting mutants which contain a mutation reducing the CD activity in a gene encoding OASS-B or MalY regulatory protein. To reduce or eliminate the CD activity of OASS-B or MalY regulatory protein with high accuracy, it is preferable to disrupt a gene encoding OASS-B or MalY regulatory protein.
In Escherichia coli, OASS-B is encoded by the cysM gene, and MalY regulatory protein is encoded by the malY gene. The nucleotide sequences of these genes have been already reported (see for cysM; GenBank accession M32101 (SEQ ID NO: 33), J. Bacteriol. 172 (6), 3351-3357 (1990), and for malY; GenBank accession M60722 (SEQ ID NO: 35), J. Bacteriol. 173 (15), 4862-4876 (1991)). Accordingly, DNA fragments which can be used to disrupt the genes can be obtained by PCR using primers based on the nucleotide sequences from a chromosomal DNA of Escherichia coli. More specifically, the cysM gene deletion mutant (deletion-type cysM gene) and the malY gene deletion mutant (deletion-type malY gene) can be obtained by PCR using the primers shown in FIG. 2. DNA fragments for gene disruption are not limited to those derived from Escherichia coli, and may be DNAs derived from other organisms or synthetic DNAs as long as they can cause homologous recombination with a chromosomal DNA of a host bacterium. For example, DNAs having 80% or more, preferably 90% or more, more preferably 95% or more homology to the cysM gene or malY gene of Escherichia coli may be used. Homology of the DNA sequences can be determined using the algorithm BLAST (Pro. Natl. Acad. Sci. USA, 90, and 5873 (1993)) and FASTA (Methods Enzymol., 183, and 63 (1990)) by Karlin and Altschul. The BLASTN and BLASTX programs have been developed based on this algorithm BLAST. (refer to http://www.ncbi.nlm.nih.gov). Furthermore, DNAs able to hybridize with the cysM gene or malY gene of Escherichia coli under stringent conditions may also be used. “Stringent conditions” as used herein are conditions under which a so-called specific hybrid is formed, and a non-specific hybrid is not formed. It is difficult to clearly express this condition by using any numerical value. However, examples of stringent conditions include, those under which DNAs having high homology to each other, for example, DNAs having a homology of not less than 50%, hybridize to each other, and DNAs having homology lower than 50% do not hybridize to each other, and those under which DNAs hybridize to each other at a salt concentration with washing typical of Southern hybridization, i.e., washing once or preferably 2-3 times under 1×SSC, 0.1% SDS at 60° C., preferably 0.1×SSC, 0.1% SDS at 60° C., more preferably 0.1×SSC, 0.1% SDS at 68° C.
Hereinafter, a method of disrupting the gene encoding OASS-B will be explained. The gene encoding MalY regulatory protein can be disrupted or mutated in a similar manner.
A chromosomal cysM gene can be disrupted by transforming an Escherichia bacterium with a DNA containing a cysM gene which has part of its sequence deleted, and subsequent loss of normal OASS-B protein function (deletion-type cysM gene), and causing recombination between the deletion-type cysM gene and the chromosomal cysM gene. Examples of the deletion-type cysM gene used in transformation include genes having part of a sequence of the cysM gene deleted, genes having an corresponding expression regulatory region such as a promoter deleted or mutated so that of the expression of the cysM gene decreases, and genes into which a site-specific mutation is introduced so that the CD activity of a protein encoded by the cysM gene decreases.
The gene disruption technique using homologous recombination has already been established and examples thereof include using a linear DNA or a plasmid containing a temperature-sensitive replication origin. 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 (K. A. Armstrong, et al., J. Mol. Biol. (1984) 175, 331-347).
A cysM gene on a host chromosome can be replaced with the deletion-type cysM gene, for example, as follows. That is, a recombinant DNA is prepared by inserting into a vector a temperature-sensitive replication origin, a deletion-type cysM gene, and a marker gene conferring resistance to a drug such as ampicillin or chloramphenicol. Then, an Escherichia bacterium is transformed with the recombinant DNA. Furthermore, the transformant strain is cultured at a temperature at which the temperature-sensitive replication origin does not function. Then the transformant strain is cultured in a medium containing the drug to obtain the transformant strain in which the recombinant DNA is incorporated into the chromosomal DNA.
In the strain in which the recombinant DNA is incorporated into the chromosomal DNA as described above, the deletion-type cysM gene is recombined with the native cysM, and the two fusion genes of the chromosomal cysM gene and the deletion-type cysM gene are inserted into the chromosome so that the other portions of the recombinant DNA (vector segment, temperature-sensitive replication origin and drug resistance marker) are present between the two fusion genes. Therefore, the transformant strain expresses normal OASS-B because the normal cysM gene is dominant in this state.
Then, in order to leave only the deletion-type cysM gene on the chromosomal DNA, one copy of the cysM gene is eliminated along with the vector segment (including the temperature-sensitive replication origin and the drug resistance marker) from the chromosomal DNA by recombination of two of the cysM genes. In this case, the normal cysM gene is left on the chromosomal DNA and the deletion-type cysM gene is excised from the chromosomal DNA, or to the contrary, the deletion-type cysM gene is left on the chromosomal DNA and the normal cysM gene is excised from the chromosomal DNA. In both cases, the excised DNA may be harbored in the cell as a plasmid when the cell is cultured at a temperature which allows the temperature-sensitive replication origin to function. Subsequently, if the cell is cultured at a temperature which does not allow the temperature-sensitive replication origin to function, the cysM gene on the plasmid is eliminated with the plasmid from the cell. Then, a strain having the disrupted cysM gene left in the chromosome can be selected by PCR, Southern hybridization, or the like.
CD activity is reduced or eliminated in the cysM gene-disrupted strain or mutant strain obtained as described above. Reduction or elimination of the CD activity in the cysM gene-disrupted strain or mutant strain can be confirmed by measuring the CD activity of a cell extract of a candidate strain by CD activity staining or quantification of hydrogen sulfide as described in the Examples, and comparing it with the CD activity of the parent or non-modified strain.
The bacteria of the present invention may be strains in which one or both of the genes encoding tryptophanase (TNase) and cystathionine-β-lyase (CBL) are modified so that CD activity of the strain is further reduced. The method of modifying those genes (tnaA gene or metC gene) is disclosed in detail in JP-A 2003-169668 (EP1,298,200).
<2> Enhancing L-Cysteine Biosynthetic Enzyme Activity
L-cysteine-producing ability may be imparted to a bacterium by enhancing an activity of an L-cysteine biosynthetic enzyme. Enhancing an L-cysteine biosynthetic enzyme can be performed by enhancing, for example, an activity of serine acetyltransferase (SAT). Enhancing the SAT activity in cells of an Escherichia bacterium can be attained by increasing a copy number of a SAT gene. For example, a recombinant DNA can be prepared by ligating a gene fragment encoding SAT to a vector that functions in Escherichia bacteria, preferably a multi-copy type vector, and transforming a host Escherichia bacterium with the vector.
The SAT gene of the present invention may be derived from Escherichia bacteria or from any other organism. The cysE SAT gene has been cloned from a wild-type Escherichia coli strain and an L-cysteine-secretion mutant strain, and the nucleotide sequence has been elucidated (Denk, D. and Boeck, A., J. General Microbiol., 133, 515-525 (1987)). Therefore, a SAT gene can be obtained by PCR utilizing primers based on the nucleotide sequence (SEQ ID NO: 31) from a chromosomal DNA of Escherichia bacterium (see JP11-155571A). Genes encoding SAT derived from other microorganisms can also be obtained in a similar manner. The SAT gene may be able to hybridize to a DNA having the nucleotide sequence of SEQ ID NO: 31 under stringent conditions, and also may encode a protein having SAT activity, which catalyzes the activation of L-serine by acetyl-CoA.
A chromosomal DNA can be prepared from a bacterium, which is a DNA donor, by the method of Saito and Miura (refer to H. Saito and K. Miura, Biochem. Biophys. Acta, 72, 619 (1963); Text for Bioengineering Experiments, Edited by the Society for Bioscience and Bioengineering, Japan, pp. 97-98, Baifukan, 1992).
In order to introduce the PCR-amplified DNA fragment containing a SAT gene into an Escherichia bacterium, vectors typically used for protein expression can be used. Examples of such vectors include pUC19, pUC18, pHSG299, pHSG399, pHSG398, RSF1010, pBR322, pACYC184, pMW219, and so forth.
Introduction of a recombinant vector containing the SAT gene into Escherichia bacterium can be attained by methods typically used for transformation of Escherichia bacteria, for example, the method of D. A. Morrison (Methods in Enzymology, 68, 326 (1979)), a method of treating recipient cells with calcium chloride so as to increase the permeability for DNA (Mandel, M. and Higa, A., J. Mol. Biol., 53, 159 (1970)), and so forth.
Increasing a copy number of the SAT gene can also be achieved by introducing multiple copies the gene into the chromosomal DNA of an Escherichia bacterium. To introduce multiple copies of the SAT gene into the chromosomal DNA of an Escherichia bacterium, homologous recombination may be carried out by targeting a sequence which exists on a chromosomal DNA in multiple copies. As sequences which exist on a chromosomal DNA in multi-copies, repetitive DNA or an inverted repeat which exists at the ends of a transposable element can be used. Furthermore, as disclosed in J2-109985A, it is also possible to incorporate a SAT gene into a transposon, and allow it to be transferred so that multiple copies of the gene are introduced into the chromosomal DNA.
Besides the aforementioned gene amplification technique, amplification of the SAT activity can also be attained by replacing an expression regulatory sequence such as a promoter of the SAT gene on a chromosomal DNA or on a plasmid with a stronger one (JP1-215280A). For example, lac promoter, trp promoter, trc promoter, and so forth are known as strong promoters. Substitution of an expression regulatory sequence can also be attained by, for example, gene substitution utilizing a temperature-sensitive plasmid.
Furthermore, it is also possible to substitute several nucleotides in the promoter region of the SAT gene, resulting in modification of the promoter to make it stronger as disclosed in WO00/18935. Expression of the SAT gene is enhanced by such substitution or modification of a promoter, and thereby the SAT activity is enhanced. These modifications of expression regulatory sequence may be combined with the increase of a copy number of SAT gene.
Furthermore, when a suppression mechanism exists for SAT gene expression, enhancing the expression can also be enhanced by modifying an expression regulatory sequence or a gene involved in the suppression so to eliminate or reduce the suppression.
The intracellular SAT activity of an Escherichia bacterium can also be increased by modifying an Escherichia bacterium to harbor SAT which has reduced or eliminated feedback inhibition by L-cysteine (henceforth also referred to as “mutant-type SAT”). Examples of the mutant-type SAT include SAT having a mutation replacing the methionine at a position 256 of wild-type SAT (SEQ ID 32) with an amino acid other than lysine and leucine, or a mutation deleting a C-terminal region of SAT from the methionine at a position 256 and thereafter. Examples of the amino acid other than lysine and leucine include the 17 kinds of amino acid residues which constitute ordinary proteins with the exceptions of methionine, lysine, and leucine. Preferably, isoleucine can be mentioned. A site-specific mutagenesis technique can be used to introduce a desired mutation into a wild-type SAT gene. As a mutant-type SAT gene, a mutant-type cysE encoding a mutant-type SAT of Escherichia coli is known (WO97/15673 and JP11-155571A). Escherichia coli JM39-8 strain harboring plasmid pCEM256E, which contains a mutant-type cysE encoding a mutant-type SAT in which the methionine at a position 256 is replaced with glutamic acid (E. coli JM39-8(pCEM256E), private number: AJ13391), has been deposited at the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology (currently, National Institute of Advanced Industrial Science and Technology, International Patent Organism Depositary, Central 6, 1-1, Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, 305-8566, Japan) on Nov. 20, 1997 under the accession number of FERM P-16527. The original deposit was converted to an international deposit in accordance with the Budapest Treaty on Jul. 8, 2002, and given the accession number of FERM BP-8112.
Furthermore, an Escherichia bacterium can be modified to contain a mutant-type SAT by introducing a mutation into a chromosomal SAT gene which prevents feedback inhibition by L-cysteine. The mutation can be introduced by ultraviolet irradiation or a mutagenizing agent used for usual mutagenesis treatment such as N-methyl-N′-nitro-N-nitrosoguanidine (NTG) or nitrous acid.
SAT which is resistant to feedback inhibition by L-cysteine used in the present invention may be a SAT protein modified to be resistant to feedback inhibition, and may also be a SAT protein with a native resistance to feedback inhibition. SAT of Arabidopsis thaliana is known not to suffer from feedback inhibition by L-cysteine and can be suitably used in the present invention. pEAS-m is known (FEMS Microbiol. Lett., 179 453-459 (1999)) as a plasmid containing SAT gene derived from Arabidopsis thaliana. 
<3> Production of L-Cysteine
L-Cysteine can be efficiently produced by culturing the Escherichia bacterium of the present invention obtained as described above in a suitable medium to cause accumulation of L-cysteine in the culture medium, and collecting the L-cysteine from the culture medium. Although L-cysteine produced by the method of the present invention may contain cystine in addition to reduced-type cysteine, the target substances produced by the method of the present invention include cystine and a mixture of reduced-type cysteine and cystine.
As culture media, ordinary media containing a carbon source, nitrogen source, sulfur source, inorganic ions, and other organic components, if required, can be used. As carbon sources, saccharides such as glucose, fructose, sucrose, molasses, and starch hydrolysate, organic acids such as fumaric acid, citric acid and succinic acid can be used. As nitrogen sources, inorganic ammonium salts such as ammonium sulfate, ammonium chloride and ammonium phosphate, organic nitrogen such as soybean hydrolysate, ammonia gas, aqueous ammonia, and so forth can be used. As sulfur sources, inorganic sulfur compounds, such as sulfates, sulfites, sulfides, hyposulfites, and thiosulfates can be used. As organic trace amount nutrients, it is desirable to add required substances such as vitamin B1, yeast extract, and so forth in appropriate amounts. In addition to these components, potassium phosphate, magnesium sulfate, iron ions, manganese ions, and so forth may be added in small amounts if required.
The culture is preferably performed under aerobic conditions for 30 to 90 hours. The culture temperature is preferably controlled at 25° C. to 37° C., and pH is preferably controlled at 5 to 8 during the culture. To adjust the pH, inorganic or organic, acidic or alkaline substances, ammonia gas, and so forth can be used. Collecting L-cysteine from the culture medium can be attained by, for example, an ordinary ion exchange resin method, precipitation, and other known methods, or combinations thereof.
EXAMPLES
Hereinafter, the present invention will be explained in detail by the following non-limiting examples.
Strains
cysE-deficient Escherichia coli JM39 (F+cysE51 tfr-8) (Denk, D. and Bock, A., J. Gene. Microbiol., 133, 515-525 (1987)) was used to identify a gene encoding a protein having CD activity.
To evaluate L-cysteine productivity of the CD-gene-disrupted strains, the following strains were used: JM39ΔtnaA, JM39ΔmetC, JM39ΔcysM, JM39ΔmalY, and JM39ΔcysK as a single-CD-gene-disrupted strain; JM39ΔtnaAΔmetC and JM39ΔcysKΔcysM as a double-CD-gene-disrupted strain; JM39ΔtnaAΔmetCΔcysMΔmalY as a quadruple-CD-gene-disrupted strain; and JM39ΔtnaAΔmetCΔcysKΔcysMΔmalY as a quintuple-CD-gene-disrupted strain. In the production of L-cysteine, a total of six strains, including JM39, single-CD-gene-disrupted strains of JM39ΔtnaA, JM39ΔmetC, JM39ΔcysM, and JM39ΔmalY, and quadruple-CD-gene-disrupted strain JM39ΔtnaAΔmetCΔcysMΔmalY, all of which harbors pEAS-m, a plasmid containing SAT gene of Arabidopsis thaliana (FEMS Microbiol. Lett., 179 (1999) 453-459) were used.
Plasmids
A plasmid library containing 4,388 kinds of genes (whole ORF fragments) of E. coli was used to identify a gene encoding a protein having CD activity (4,388 kinds of plasmids were respectively dispensed into the wells of forty eight 96-well plates). The plasmid library covers all of the 4,388 kinds of ORF fragments of E. coli located downstream to the lac promoter in the pCA24N vector and the expression of each ORF is induced by IPTG. For gene disruption, plasmid pEL3 (K. A. Armstrong et al., J. Mol. Biol. (1984) 175, 331-347) was used to construct pEL3gdtnaA, pEL3gdmetC, pEL3gdcysM, pEL3gdcysK, and pEL3gdmalY. The construction of the plasmids will be described below.
Culture Media
For transformation and culture of E. coli, LB medium was used as a complete medium, and M9 medium (6 g/L Na2HPO4, 3 g/L KH2PO4, 0.5 g/L NaCl, 0.25 g/L MgSO4.7H2O, 0.015 mg/L CaCl2.4H2O, 4 g/L glucose, and 0.001 g/L thiamine hydrochloride) was used as a minimum medium. Ampicillin (Amp) was added if necessary. In some experiments, LB liquid medium to which 10 to 30 mM cysteine was added was used. Unless otherwise described, the culture was performed at 37° C. For the culture of cysteine production (30 g/L glucose, 10 g/L NH4Cl, 2 g/L KH2PO4, 1 g/L MgSO4.7H2O, 10 mg/L FeSO4.7H2O, 10 mg/L MnCl2.4H2O, and 20 g/L CaCO3) sodium thiosulfate was added to the culture. The same medium was used to determine the quantity of cysteine.
Preparation of Cell Extract
The preparation of the cell extract from the cultured cells was performed by sonication. The composition of the buffer used for the sonication was 100 mM Tris-HCL (pH 8.6), 100 mM DTT ((±)-Dithiothreitol), and 10 mM PLP (pyridoxal phosphate).
Composition of Native-PAGE Gel and Procedure of Native-PAGE (Polyacrylamide Gel Electrophoresis Under Undenatured Conditions)
Since it was necessary to separate proteins in the cell extract under an undenatured state, Native-PAGE gel containing no SDS was prepared for the purpose of identifying and ascertaining a protein having CD activity, confirming the construction of the CD-gene-disrupted strains, and so on, by CD activity staining described hereinbelow. The composition of the Native-PAGE gel for three gel sheets was 6.4 ml of Acrylamide/Bisacrylamide/amide (37:5:1), 6.7 ml of 1 M Tris-HCl (pH 8.7), 6.8 ml of dH2O, 100 μl of 10% APS (Ammonium persulfate), and 10 μl of TEMED (N,N,N,N′-Tetra-methyl-ethylenediamine) for 12.5% gel, and 5.1 ml of Acrylamide/Bisacrylamide/amide (37:5:1), 6.7 ml of 1 M Tris-HCl (pH 8.7), 8.1 ml of dH2O, 100 μl of 10% APS, and 10 μl of TEMED for 10% gel. The concentrated gel was 4.5% and its composition for three gel sheets was (0.7 ml of Acrylamide/Bisacrylamide/amide (37:5:1), 0.75 ml of 1 M Tris-HCl (pH 6.8), 4.52 ml of dH2O, 30 μl of 10% APS, and 5 μl of TEMED. The Native-PAGE was performed using a mini-slab electrophoretic apparatus (AEV-6500, manufactured by ATTO), and a mixture of 30 μg to 50 μg of cell extract and 2-fold Native-PAGE buffer was applied to the gel. The electrophoresis was performed at 200 V and 20 mA/gel for 2 hours to 4 hours. The composition of 1 liter of the electrophoresis buffer was 14.43 g of L-glycine and 3.0 g of Tris, and the buffer was adjusted to pH 8.6.
CD Activity Staining
A CD activity staining method was used for specifically visualizing and detecting the existence of a protein having CD activity. As described in section 1-5, after proteins in the cell extract had been separated by electrophoresis, the gel was immersed in the CD activity staining solution and left to stand at room temperature from several hours to overnight with shaking to detect the protein band having CD activity. The composition of 100 ml of the CD activity staining solution was 1.21 g of Tris, 0.372 g of EDTA, 0.605 g of L-cysteine, 50 mg of BiCl3 (bismuth chloride), and 200 μl of 10 ml PLP, and the solution was adjusted to pH 8.6. The CD activity staining was performed based on the principle that cysteine contained in the CD activity staining solution is degraded into pyruvic acid, ammonia, and H2S at the site where a protein having CD activity separated with Native-PAGE exists on the gel. The generated H2S reacts with bismuth chloride (BiCl3) contained in the CD activity staining solution to form bismuth sulfide (Bi2S3), which exhibits a black color band.
Identification of a Gene Encoding a Protein Having CD Activity Using a Plasmid Library Containing E. coli Whole Genes
The forty-eight 96-well plates on which respective plasmids were dispensed were grouped into 5 plates such as 1 to 5, 6 to 10 . . . , and nine kinds of mixed plasmid solutions obtained from five plates (each containing 480 kinds of plasmids) were prepared. The mixed plasmid solutions were used to transform JM39 strains and about 10,000 colonies of transformants were stocked in glycerol. The nine kinds of glycerol-stock solutions were inoculated into LB medium containing chloramphenicol (Cm) and 0.01 mM IPTG and cultured. Then, cell extract was prepared and subjected to Native-PAGE. CD activity staining was performed to detect which mixed plasmid solution contained a candidate gene encoding a protein having CD activity. The population containing a candidate gene presumed to encode a protein having CD activity was downsized to a population of 480 kinds of plasmids, and then, further downsizing of the population to that of 96 kinds of plasmids was performed. 480 kinds of the plasmids were divided into five groups of 96 to prepare five kinds of mixed plasmid solutions. JM39 strains were transformed with the mixed plasmid solutions and about 6,000 colonies of the transformants were stocked in glycerol. Thereafter, the transformants were cultured and CD activity staining was performed to confirm if the mixed plasmid solution contains a candidate gene encoding a protein having CD activity. After the population containing a candidate gene presumed to encode a protein having CD activity was downsized to 96 kinds of plasmids, the population was further reduced to 8 kinds of plasmids. Finally, eight proteins were each expressed from the 8 kinds of plasmids and CD activity staining was performed to confirm if they are the target protein having CD activity.
Construction of Plasmids for CD Gene Disruption
To disrupt each CD gene, five kinds of plasmids for gene disruption, i.e., pEL3gdtnaA, pEL3gdmetC, pEL3gdcysM, pEL3gdcysK, and pEL3dgmalY were constructed using plasmid pEL3 having a temperature-sensitive replication origin. The preparation methods for these plasmids are described below. That is, using the genome of E. coli JM39 as a template, two kinds of 300 to 700 bp DNA fragments each covering a part of the respective CD gene was amplified by PCR. The DNA fragments were designated homologous region DNA fragments-A and -B, respectively. The primers used are described in FIG. 2. For the amplification of the homologous region DNA fragment-A, CD gene disruption primers-1 and -2 were used, and for the amplification of the homologous region DNA fragment-B, CD gene disruption primers-3 and -4 were used. These primers had a restriction enzyme recognition site at the 5′-side so that the amplified homologous region DNA fragments contain restriction enzyme recognition sites at both ends. After treatment with appropriate restriction enzymes of both fragments A and B (KpnI, HindIII, or EcoRI), both the enzyme-treated fragments were ligated to each other to form a template for preparing CD gene disruption fragments. The CD gene disruption fragments were prepared in large amounts by PCR using the CD gene disruption primers-1 and -4. The disruption fragments and pEL3 were treated with the restriction enzyme BamHI and ligated to each other to construct the CD gene disruption plasmids. The construction was confirmed by DNA sequencing.
Disruption of CD Gene
A CD gene-disrupted strain was constructed from E. coli JM39 strain with the disruption plasmid as described in section 1-9. First, disruption plasmids were introduced into JM39 to obtain transformants. The limiting temperature for temperature-sensitive plasmid pEL3 is 42° C. Alternatively, the non-limiting temperature, a temperature not higher than the limiting temperature, for the plasmid is generally 37° C., which is an ordinary culture temperature for E. coli. However, the culture was performed at 30° C. in this experiment to ensure the temperature sensitivity of the plasmid. Then, after each transformant was cultured overnight at 30° C. in an LB+Amp medium, the culture broth was diluted to 103-fold, and 200 μl of the diluted solution was spread on the LB+Amp plate. Culture was performed at 42° C., which is the temperature at which the plasmid becomes unreplicable and the growth of the transformants is inhibited by Amp, and therefore no colonies form. Thereby, homologous recombination occurred between each disrupted fragment on the plasmid with suppressed replication and a homologous region on the chromosome of the JM39 strain. This allowed the whole length of the disruption plasmid to be incorporated into the chromosome. Then, the recombinant strain was selected which was able to form an Amp resistant colony by incorporation of the disruption plasmid. The incorporation of the disruption plasmid into the chromosome was confirmed by PCR using FW and RV of each CD gene disruption primer as described in FIG. 2. The colony having a confirmed disruption plasmid incorporated into the chromosome was cultured in an LB liquid medium to cause further homologous recombination. This was done so that the disrupted fragment remains on the chromosome and the fragment containing a plasmid sequence and a chromosomal gene is removed. The transformants were subcultured several times in an LB liquid medium. Then, the culture broth was spread on an LB agar medium after dilution to a concentration that would cause 200 to 300 colonies to form on the LB agar medium. The colonies were replicated on an LB plate and an LB+Amp agar plate to select for Amp-sensitive colonies. By performing colony PCR using FW and RV primers, CD-gene-disrupted strains having only the disrupted fragment on the chromosome were selected.
The CD-gene-disrupted strains were subjected to CD activity staining and disappearance of the CD activity due to gene disruption was confirmed. A multiple CD-gene-disrupted strain was constructed by repeating the operation of disrupting the target CD genes.
Measurement of Total CD Activity (Sulfide/H2S Quantification)
The total CD activity in the cell extract was measured by determining the amount of hydrogen sulfide (H2S) generated by degradation of cysteine by CD. A strain was cultured in 5 ml of LB medium and 5 ml of LB+10 mM cysteine medium at 37° C. overnight, and then the cell extract was prepared as in the section 2-2-4. The composition of the buffer used for measuring the CD activity was 100 mM Tris-HCl (pH 8.6), 100 μM DTT, 10 mM PLP, 2 μM L-cysteine. 10 ml of the cell extract was added to 1 ml of the buffer and the reaction was carried out at 30° C. for 10 minutes. A standard curve was prepared by adding 10 μl aliquots of water, or 10 μl of 0.1 mM, 0.2 mM, or 2 mM of Na2S to the buffer and the mixture was incubated in the same way. After completion of the reaction, 100 ml of 20 mM N,N-dimethyl-p-phenyldiamine sulfate (in 7.2 N HCl) and the same amount of 30 mM FeCl3 (in 1.2 N HCl) were added, vigorously mixed, and left to stand in the dark for 15 minutes. Iron chloride acts as an oxidizing agent under acidic conditions adjusted by hydrochloric acid, and the N,N-dimethyl-p-phenyldiamine sulfate reacts with a sulfide in the sample to form a thiazine dye. As a result, Methylene Blue exhibits a greenish blue or blue color. The mixture was left to stand for 15 minutes, then, OD650 of the reaction mixture was measured and the activity was calculated by defining an amount of enzyme giving 1 μmol H2S as 1 U.
Cysteine Production Culture
Each of the obtained transformants was inoculated in a Sakaguchi flask containing 20 ml of C1 medium with sodium thiosulfate (15 g/L thiosulfuric acid), and cultured at 37° C. The amount of L-cysteine in the supernatant after 24, 48, 72, and 96 hours was quantified. The amount of L-cysteine was measured as a total amount of reduced cysteine and cystine by the bioassay using Leuconostoc mesenteroides (Tsunoda, T. et al., Amino acids, 3, 7-13 (1961)).
2. Results
2-1. Confirmation of Existence of a Protein Having CD activity in E. coli 
To confirm the existence of a protein having CD activity in E. coli, a cell extract of JM39 strain was prepared and subjected to Native-PAGE, and electrophoresis was performed for about 2 hours to separate proteins, which was then subjected to activity staining. FIG. 1 shows the results. Five bands exhibiting CD activity were detected. This experiment indicates that at least five kinds of proteins having CD activity are present in E. coli. Of those, two were identified as tryptophanase (TNase) and cystathionine-β-lyase (CBL) by amino acid sequencing analysis (JP 2003-169668A). To identify the remaining three, the following experiments were performed.
2-3. Identification of the Unidentified CD Proteins Using E. coli Total Gene Plasmid Library
The genome of E. coli is presumed to have a total of 4,388 genes (ORF). Using the E. coli whole ORF library in which all ORFs were inserted into each plasmid, the operation for identification of a protein having a CD activity was repeated by the procedure described in the section 1-7. By detecting the band of the unidentified CD protein by CD activity staining, the population of plasmids containing a gene encoding an unidentified CD protein was reduced from 4,388 kinds to 480 kinds, 96 kinds, and 8 kinds, sequentially. Finally, the selected 8 kinds of plasmids were analyzed and the proteins encoded by the cysM gene, cysK gene, and malY gene were found to be the unidentified CD proteins. The cysM gene of E. coli has been reported to encode O-acetyl L-serine sulphydrylase-B (OASS-B) (see J. Bacteriol. 172 (6), 3351-3357 (19890)). The cysK has been reported to encode O-acetyl L-serine sulphydrylase (OASS-A) (Mol. Microbiol. 2 (6), 777-783 (1988)). Furthermore, it has been reported that the malY gene encodes a MalY protein which is a regulatory factor for maltose metabolism pathway gene group and has a conformation close to that of CBL and catalyzes the C—S lyase reaction (EMBO J. 2000, March; 19(5):831-842).
2-4. Confirmation of CD Activity
The OASS-B, OASS-A, and MalY identified in section 2-3 were confirmed to have the CD activity by overexpressing the genes in the JM39 strain. That is, when the respective genes were overexpressed and protein bands of cell extract were analyzed by CD activity staining, the stained band was denser than the band of the control JM strain, indicating that each gene encodes a protein having CD activity.
2-5. Construction of CD-Gene-Disrupted Strain
Then, each CD-gene-disrupted strain was constructed. Methods of preparing JM39ΔtnaA and JM39ΔmetC strains are disclosed in JP 2003-169668A. First, disruption plasmids pEL3gdtnaA, pEL3gdmetC, pEL3gdcysM, pEL3gdcysK, and pEL3gdmalY for disrupting tnaA, metC, cysM, cysK, and malY, respectively, were constructed and introduced into the JM39 strain to construct single-disrupted strains by homologous recombination. Furthermore, the gene disruption step was repeated to prepare multiple-disrupted strains, such as a quadruple disrupted strain JM39ΔtnaAΔmetCΔcysMΔmalY in which tnaA, metC, cysM, and malY were disrupted. After the operation of gene disruption, gene disruption was confirmed based on the length of the DNA fragment amplified by colony PCR. Furthermore, it was confirmed by CD activity staining that the CD activity of a protein encoded by each gene was eliminated due to gene disruption.
2-6. Measurement of Total CD Activity
According to the method in the section 1-11, the total CD activities of all the CD-gene-disrupted strains used in this experiment were measured. The results are shown in Table 1. As a result, comparison of the total CD activity of each strain cultured in LB medium with that of the parent strain JM39 indicated a decrease in the CD activity for all the disrupted strains. Comparison of the activity of the multiple-disrupted strain with the activity of JM39 indicated a considerable decrease in the CD activity except for JM39ΔcysKΔcysM. The decrease in the CD activity in multi-disrupted strains was significant as compared with the decrease in the activity in each single-disrupted strain. The activity of JM39ΔcysKΔcysM decreased as compared with the activities of single-disrupted strains. Then, the total CD activity of the CD-gene-disrupted strain cultured in a medium to which cysteine was added was analyzed. In the strains other than JM39ΔtnaA, the CD activity of the strain cultured in a cysteine-containing medium increased considerably as compared with the CD activity of the same strain cultured in an LB medium.









TABLE 1









medium



total CD activity (mU/mg)











LB + 10 mM


Strain
LB
L-cysteine





JM39
20.6 ± 0
27.6 ± 0


JM39ΔtnaA
15.7 ± 0
14.1 ± 0


JM39ΔmetC
15.0 ± 0
27.6 ± 0.46


JM39ΔcysK
18.2 ± 0.52
29.9 ± 0


JM39ΔcysM
17.9 ± 0.46
27.8 ± 0


JM39ΔmalY
15.3 ± 0
27.1 ± 0.46


JM39ΔtnaADmetC
 9.6 ± 0
16.2 ± 0


JM39ΔcysKΔcysM
17.2 ± 0.58
27.0 ± 0


JM39ΔtnaADmetCΔcysMΔmalY
 9.1 ± 0.46
19.6 ± 0


JM39ΔtnaADmetCΔcysKΔcysMΔmalY
 8.7 ± 0.46
11.5 ± 0









2-7. Cysteine Production Using CD-Gene-Disrupted Strains
pEAS-m, a plasmid containing SAT-m gene of A. thaliana, was introduced into a total of six strains, i.e., a control JM39 strain, four single-CD-gene-disrupted strains of JM39ΔtnaA, JM39ΔmetC, JM39ΔcysM, and JM39ΔmalY, and a quadruple-CD-gene-disrupted strain of JM39ΔtnaAΔmetCΔmalYΔcysM, and the transformants were used for the production of cysteine. Cysteine production culture was performed according to the method in the section 1-12 and the amount of produced cysteine was quantified. Time courses of the amounts of produced cysteine of the control strain and each of the CD-gene-disrupted strain per growth (growth: value of OD562) are shown in Table 2 and FIG. 3. The growth decreased slightly in the case of JM39ΔtnaA but the growth of other disrupted strains was substantially the same as that of the control strain JM39.









TABLE 2









L-Cys (mg/L)



hr











Strain
24
48
72
96





JM39
 416 ± 96
 720 ± 161
 587 ± 164
 415 ± 111


JM39ΔtnaA
1206 ± 26
1195 ± 95
1287 ± 74
1077 ± 124


JM39ΔmetC
1408 ± 98
 930 ± 47
1243 ± 101
 853 ± 13


JM39ΔmalY
1291 ± 95
1213 ± 93
1359 ± 87
1256 ± 75


JM39ΔcysM
1369 ± 67
1123 ± 148
1100 ± 66
 840 ± 56


JM39ΔtnaAΔmetCΔmalYΔcysM
1291 ± 21
1117 ± 21
1080 ± 13
 847 ± 69









The L-cysteine production of the respective gene-disrupted strains exceeded the value of the control strain JM39. Therefore, the disruption of the CD genes to inhibit the CD activity is effective for increasing the production of cysteine. When cysK gene-disrupted strains were used, almost no cysteine could be obtained when a cysteine production C1 medium containing sodium thiosulfate was used.
INDUSTRIAL APPLICABILITY
By using the bacteria of the present invention, L-cysteine can be produced efficiently. L-cysteine and its derivatives are useful in the fields of medicine, cosmetics, foods, and the like.
While the invention has been described in detail with reference to preferred embodiments thereof, it will be apparent to one skilled in the art that various changes can be made, and equivalents employed, without departing from the scope of the invention. Each of the aforementioned documents, including the foreign priority documents, Japanese Patent No. 2004-060483 filed on Mar. 4, 2004, is incorporated by reference herein in its entirety.

## Claims

1. A method of producing L-cysteine comprising:
A) culturing an Escherichia bacterium in a medium, and
B) collecting L-cysteine from the medium or the bacterium,

wherein said bacterium contains a gene encoding O-acetylserine sulphydrylase B, and wherein said gene is modified so that cysteine desulfhydrase activity is reduced or eliminated as compared to the cysteine desulfhydrase activity in a bacterium containing a non-modified gene.

2. The method according to claim 1, wherein said gene encoding O-acetylserine sulphydrylase B is disrupted.

3. The method according to claim 1, wherein activity of an L-cysteine biosynthetic enzyme is enhanced.

4. The method according to claim 3, wherein said L-cysteine biosynthetic enzyme is serine acetyltransferase.

5. The method according to claim 4, wherein said serine acetyltransferase is resistant to feedback inhibition by L-cysteine.

6. The method according to claim 1, wherein said Escherichia bacterium is Escherichia coli.