US20080076163A1 - L-cysteine-producing microorganism and a method for producing L-cysteine - Google Patents

L-cysteine-producing microorganism and a method for producing L-cysteine Download PDF

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US20080076163A1
US20080076163A1 US11/070,084 US7008405A US2008076163A1 US 20080076163 A1 US20080076163 A1 US 20080076163A1 US 7008405 A US7008405 A US 7008405A US 2008076163 A1 US2008076163 A1 US 2008076163A1
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cysteine
gene
activity
escherichia
strain
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Hiroshi Takagi
Shigeru Nakamori
Masaru Wada
Hirotada Mori
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Ajinomoto Co Inc
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Assigned to AJINOMOTO CO., INC. reassignment AJINOMOTO CO., INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: MORI, HIROTADA, NAKAMORI, SHIGERU, TAKAGI, HIROSHI, WADA, MASARU
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/12—Methionine; Cysteine; Cystine

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  • 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.
  • 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.
  • 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.
  • 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.
  • 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- ⁇ -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).
  • CBL cystathionine- ⁇ -lyase
  • TNase tryptophanase
  • 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.
  • 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 .
  • OASS-B O-acetylserine sulphydrylase B
  • MalY MalY regulatory protein
  • the inventors also found that reducing CD activity by modifying these genes leads to improvement in the production of L-cysteine.
  • 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.
  • 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 ( ⁇ ), JM39 ⁇ tnaA ( ⁇ ), JM39 ⁇ metC ( ⁇ ), JM39 ⁇ cysm (*), JM39 ⁇ malY (+), and JM39 ⁇ tnaA ⁇ metC ⁇ malY ⁇ cysM( ⁇ ).
  • 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.
  • OASS-B O-acetylserine sulphydrylase B
  • CD cysteine desulfhydrase
  • 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.
  • one or both of the genes encoding tryptophanase (TNase) and cystathionine- ⁇ -lyase (CBL) may also be modified so that the CD activity of the bacterium is further reduced.
  • L-cysteine-producing ability 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.
  • bacteria having native L-cysteine-producing ability may also be used.
  • 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.
  • 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.
  • NTG N-methyl-N′-nitro-N-nitrosoguanidine
  • nitrous acid nitrous acid
  • OASS-B is encoded by the cysM gene
  • 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 .
  • 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.
  • 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.
  • BLAST Pro. Natl. Acad. Sci. USA, 90, and 5873 (1993)
  • FASTA Methods Enzymol., 183, and 63 (1990)
  • BLASTN and BLASTX programs have been developed based on this algorithm BLAST. (refer to http://www.ncbi.nlm.nih.gov).
  • 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.
  • 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.
  • 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 cysMgene 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.
  • 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.
  • 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.
  • the deletion-type 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.
  • 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.
  • 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- ⁇ -lyase (CBL) are modified so that CD activity of the strain is further reduced.
  • TNase tryptophanase
  • CBL cystathionine- ⁇ -lyase
  • the method of modifying those genes is disclosed in detail in JP-A 2003-169668 (EP1,298,200).
  • 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).
  • SAT serine acetyltransferase
  • Enhancing the SAT activity in cells of an Escherichia bacterium can be attained by increasing a copy number of a SAT gene.
  • 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).
  • 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).
  • 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.
  • 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.
  • homologous recombination may be carried out by targeting a sequence which exists on a chromosomal DNA in multiple copies.
  • 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.
  • 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.
  • 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).
  • 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).
  • JP1-215280A 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.
  • SAT gene 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.
  • 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”).
  • 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.
  • amino acid other than lysine and leucine examples include the 17 kinds of amino acid residues which constitute ordinary proteins with the exceptions of methionine, lysine, and leucine.
  • isoleucine can be mentioned.
  • a site-specific mutagenesis technique can be used to introduce a desired mutation into a wild-type SAT gene.
  • 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
  • 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.
  • 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.
  • 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.
  • 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.
  • culture media ordinary media containing a carbon source, nitrogen source, sulfur source, inorganic ions, and other organic components, if required, can be used.
  • 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.
  • 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.
  • inorganic sulfur compounds such as sulfates, sulfites, sulfides, hyposulfites, and thiosulfates can be used.
  • organic trace amount nutrients it is desirable to add required substances such as vitamin B1, yeast extract, and so forth in appropriate amounts.
  • 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.
  • pH is preferably controlled at 5 to 8 during the culture.
  • 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.
  • JM39 ⁇ tnaA, JM39 ⁇ metC, JM39 ⁇ cysM, JM39 ⁇ malY, and JM39 ⁇ cysK as a single-CD-gene-disrupted strain
  • JM39 ⁇ tnaA ⁇ metC and JM39 ⁇ cysK ⁇ cysM as a double-CD-gene-disrupted strain
  • JM39 ⁇ tnaA ⁇ metC ⁇ cysM ⁇ malY as a quadruple-CD-gene-disrupted strain
  • JM39 ⁇ tnaA ⁇ metC ⁇ cysK ⁇ cysM ⁇ malY as a quintuple-CD-gene-disrupted strain.
  • 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.
  • plasmid pEL3 K. A. Armstrong et al., J. Mol. Biol.
  • 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).
  • 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 dH 2 O, 100 ⁇ l of 10% APS (Ammonium persulfate), and 10 ⁇ l 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 dH 2 O, 100 ⁇ l of 10% APS, and 10 ⁇ l 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 dH 2 O, 30 ⁇ l of 10% APS, and 5 ⁇ l of TEMED.
  • the Native-PAGE was performed using a mini-slab electrophoretic apparatus (AEV-6500, manufactured by ATTO), and a mixture of 30 ⁇ g to 50 ⁇ g 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.
  • 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 BiCl 3 (bismuth chloride), and 200 ⁇ l 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 H 2 S at the site where a protein having CD activity separated with Native-PAGE exists on the gel.
  • the generated H 2 S reacts with bismuth chloride (BiCl 3 ) contained in the CD activity staining solution to form bismuth sulfide (Bi 2 S 3 ), which exhibits a black color band.
  • 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.
  • 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.
  • 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.
  • a CD gene-disrupted strain was constructed from E. coli JM39 strain with the disruption plasmid as described in section 1-9.
  • disruption plasmids were introduced into JM39 to obtain transformants.
  • the limiting temperature for temperature-sensitive plasmid pEL3 is 42° C.
  • 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 .
  • 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.
  • the culture broth was diluted to 10 3 -fold, and 200 ⁇ l 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.
  • a standard curve was prepared by adding 10 ⁇ l aliquots of water, or 10 ⁇ l of 0.1 mM, 0.2 mM, or 2 mM of Na 2 S 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 FeCl 3 (in 1.2 N HCl) were added, vigorously mixed, and left to stand in the dark for 15 minutes.
  • 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)).
  • 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- ⁇ -lyase (CBL) by amino acid sequencing analysis (JP 2003-169668A). To identify the remaining three, the following experiments were performed.
  • TNase tryptophanase
  • CBL cystathionine- ⁇ -lyase
  • 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)).
  • OASS-A O-acetyl L-serine sulphydrylase
  • 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).
  • the gene disruption step was repeated to prepare multiple-disrupted strains, such as a quadruple disrupted strain JM39 ⁇ tnaA ⁇ metC ⁇ cysM ⁇ malY in which tnaA, metC, cysM, and malY were disrupted.
  • 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.

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US8647847B2 (en) 2009-11-30 2014-02-11 Ajinomoto Co., Inc. L-cysteine-producing bacterium and a method for producing L-cysteine
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US20110033902A1 (en) * 2008-02-21 2011-02-10 Gen Nonaka l-cysteine-producing bacterium and a method for producing l-cysteine
US8278075B2 (en) 2008-02-21 2012-10-02 Ajinomoto Co., Inc. L-cysteine-producing bacterium and a method for producing L-cysteine
US8383372B2 (en) 2008-03-06 2013-02-26 Ajinomoto Co., Inc. L-cysteine producing bacterium and a method for producing L-cysteine
US8008048B2 (en) 2008-03-06 2011-08-30 Ajinomoto Co., Inc. L-cysteine-producing bacterium and a method for producing L-cysteine
US20090226983A1 (en) * 2008-03-06 2009-09-10 Gen Nonaka L-cysteine-producing bacterium and a method for producing l-cysteine
US20090226984A1 (en) * 2008-03-06 2009-09-10 Gen Nonaka L-cysteine producing bacterium and a method for producing l-cysteine
US20100209977A1 (en) * 2009-02-16 2010-08-19 Kazuhiro Takumi L-amino acid-producing bacterium and a method for producing an l-amino acid
US9458206B2 (en) 2009-02-16 2016-10-04 Ajinomoto Co., Inc. L-amino acid-producing bacterium and a method for producing an L-amino acid
US20100216196A1 (en) * 2009-02-25 2010-08-26 Gen Nonaka L-cysteine-producing bacterium and a method for producing l-cysteine
US8293506B2 (en) 2009-02-25 2012-10-23 Ajinomoto Co., Inc. L-cysteine-producing bacterium and a method for producing L-cysteine
US20100233765A1 (en) * 2009-03-12 2010-09-16 Gen Nonaka L-cysteine-producing bacterium and a method for producing l-cysteine
US8647847B2 (en) 2009-11-30 2014-02-11 Ajinomoto Co., Inc. L-cysteine-producing bacterium and a method for producing L-cysteine
US8962284B2 (en) 2010-09-14 2015-02-24 Ajinomoto Co., Inc. Sulfur-containing amino acid-producing bacterium and method for producing sulfur-containing amino acid
US9234223B2 (en) 2011-04-01 2016-01-12 Ajinomoto Co., Inc. Method for producing L-cysteine

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