(19)
(11)EP 2 138 585 A1

(12)EUROPEAN PATENT APPLICATION

(43)Date of publication:
30.12.2009 Bulletin 2009/53

(21)Application number: 09003107.1

(22)Date of filing:  04.03.2009
(51)Int. Cl.: 
C12P 13/12  (2006.01)
C12R 1/19  (2006.01)
C12N 1/20  (2006.01)
(84)Designated Contracting States:
AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO SE SI SK TR
Designated Extension States:
AL BA RS

(30)Priority: 06.03.2008 JP 2008056371

(71)Applicant: Ajinomoto Co., Inc.
Tokyo 104-8315 (JP)

(72)Inventors:
  • Nonaka, Gen
    Kawasaki-shi Kanagawa 210-8681 (JP)
  • Takumi, Kazuhiro
    Kawasaki-shi Kanagawa 210-8681 (JP)

(74)Representative: Strehl Schübel-Hopf & Partner 
Maximilianstrasse 54
80538 München
80538 München (DE)

  


(54)An L-cysteine producing bacterium and a method for producing L-cysteine


(57) A bacterium belonging to the family Enterobacteriaceae, which has L-cysteine-producing ability and has been modified to decrease the activity of a protein encoded by the d0191 gene is cultured in a medium, and L-cysteine, L-cystine, a derivative thereof or a mixture thereof is collected from the medium to produce these compounds.


Description

BACKGROUND OF THE INVENTION


Field of the Invention



[0001] The present invention relates to a method for producing L-cysteine or related substances. More precisely, the present invention relates to a bacterium suitable for the production of L-cysteine or related substances and a method for producing L-cysteine or related substances utilizing such a bacterium. L-cysteine and L-cysteine-related substances are used in the fields of drugs, cosmetics and foods.

Brief Description of the Related Art



[0002] Microorganisms which are able to produce L-cysteine are known. For example, a coryneform bacterium with increased intracellular serine acetyltransferase activity produces L-cysteine (Japanese Patent Laid-open (Kokai) No. 2002-233384). L-cysteine-producing ability can also be increased by incorporating serine acetyltransferase which has been mutated to attenuate feedback inhibition by L-cysteine (Japanese Patent Laid-open No. 11-155571, U.S. Patent Published Application No. 20050112731, U.S. Patent No. 6,218,168).

[0003] Furthermore, L-cysteine-producing ability in a microorganism can be enhanced by suppressing the L-cysteine decomposition system. Examples of such microorganisms include coryneform bacteria or Escherichia bacteria in which the activity of cystathionine-β-lyase (U.S. Patent Published Application No. 20050112731), tryptophanase (Japanese Patent Laid-open No. 2003-169668), or O-acetylserine sulfhydrylase B (Japanese Patent Laid-open No. 2005-245311) is attenuated or deleted.

[0004] Furthermore, it is known that the ydeD gene which encodes the YdeD protein participates in secretion of the metabolic products of the cysteine pathway (Dabler et al., Mol. Microbiol., 36, 1101-1112 (2000)). Furthermore, techniques of enhancing L-cysteine-producing ability by increasing expression of the mar-locus, emr-locus, acr-locus, cmr-locus, mex-gene, bmr-gene or qacA-gene, are also known. These loci and/or genes encode proteins which cause secretion of toxic substances from cells (U.S. Patent No. 5,972,663). emrAB, emrKY, yojIH, acrEF, bcr and cusA are further examples (Japanese Patent Laid-open No. 2005-287333).

[0005] An Escherichia coli has been reported which produces L-cysteine and which has increased activity of the positive transcriptional control factor of the cysteine regulon encoded by the cysB gene (International Patent Publication WO01/27307).

[0006] The d0191 gene is a novel gene of Pantoea ananatis found by the inventor of the present invention. Although genes presumed to be homologue genes of this gene have been found in various bacteria by homology search, functions of all of them are unknown, and relation thereof with L-cysteine production is not known.

Summary of the Invention



[0007] The present invention provides a novel technique for improving bacterial L-cysteine-producing ability, and thereby provide an L-cysteine-producing bacterium, as well as a method for producing L-cysteine, L-cystine, their derivatives, or a mixture of these by using such a bacterium.

[0008] A novel gene encoding a protein having cysteine desulfhydrase activity in Pantoea ananatis was isolated. L-cysteine-producing ability of a bacterium is enhanced by modifying the bacterium so that the activity of that protein is decreased.

[0009] It is an aspect of the present invention to provide a bacterium belonging to the family Enterobacteriaceae, which has L-cysteine-producing ability and has been modified to decrease the activity of the protein selected from the group consisting of:
  1. (A) a protein comprising the amino acid sequence of SEQ ID NO: 2, and
  2. (B) a protein comprising the amino acid sequence of SEQ ID NO: 2, but which includes substitutions, deletions, insertions or additions of one or several amino acid residues, and having cysteine desulfhydrase activity.


[0010] It is a further aspect of the present invention to provide the aforementioned bacterium, wherein the activity of the protein is decreased by decreasing expression of a gene encoding the protein or by disrupting the gene.

[0011] It is a further aspect of the present invention to provide the aforementioned bacterium, wherein the gene is selected from the group consisting of:
  1. (a) a DNA comprising the nucleotide sequence of SEQ ID NO: 1, and
  2. (b) a DNA which is able to hybridize with a sequence complementary to the nucleotide sequence of SEQ ID NO: 1 or a probe prepared from the nucleotide sequence under stringent conditions, and encodes a protein having cysteine desulfhydrase activity.


[0012] It is a further aspect of the present invention to provide the aforementioned bacterium, in which serine acetyltransferase has been mutated so that feedback inhibition by L-cysteine is attenuated.

[0013] It is a further aspect of the present invention to provide the aforementioned bacterium, which is a Pantoea bacterium.

[0014] It is a further aspect of the present invention to provide the aforementioned bacterium, which is Pantoea ananatis.

[0015] It is a further aspect of the present invention to provide a method for producing L-cysteine, L-cystine, a derivative thereof or a mixture thereof, which comprises culturing the aforementioned bacterium in a medium and collecting L-cysteine, L-cystine, a derivative thereof or a mixture thereof from the medium.

[0016] It is a further aspect of the present invention to provide the aforementioned method, wherein the derivative of L-cysteine is a thiazolidine derivative.

[0017] It is a further aspect of the present invention to provide a DNA encoding the protein selected from the group consisting of:
  1. (A) a protein comprising the amino acid sequence of SEQ ID NO: 2, and
  2. (B) a protein comprising the amino acid sequence of SEQ ID NO: 2, but which includes substitutions, deletions, insertions or additions of one or several amino acid residues, and having cysteine desulfhydrase activity.


[0018] It is a further aspect of the present invention to provide the aforementioned DNA, which is a DNA selected from the group consisting of:
  1. (a) a DNA comprising the nucleotide sequence of SEQ ID NO: 1, and
  2. (b) a DNA which is able to hybridize with a sequence complementary to the nucleotide sequence of SEQ ID NO: 1 or a probe prepared from the nucleotide sequence under stringent conditions, and encodes a protein having cysteine desulfhydrase activity.


[0019] According to the present invention, L-cysteine-producing ability of bacteria can be improved. Furthermore, according to the present invention, L-cysteine, L-cystine, their derivatives, or a combinations thereof can be efficiently produced.

[0020] Moreover, the present invention provides a novel gene encoding a protein having cysteine desulfhydrase activity.

Brief Description of the Drawings



[0021] 

Fig. 1 shows the structure of the helper plasmid RSF-Red-TER.

Fig. 2 shows the construction of the helper plasmid RSF-Red-TER.

Fig. 3 shows the scheme of deletion of the d0191 gene.

Fig. 4 shows the construction of the pMIV-5JS plasmid.

Fig. 5 shows the construction of the pM12 plasmid.

Fig. 6 shows the construction of the pM12-ter(thr) plasmid. The sequences in the drawing are shown as SEQ ID NOS: 24 and 25.

Fig. 7 shows the construction of the IntJS cassette.

Fig. 8 shows growth of a d0191-deficient strain and a d0191-enhanced strain of P. ananatis in the presence of L-cysteine.

Fig. 9 shows growth of a d0191-enhanced strain and a d0191-non-enhanced strain of E. coli in the presence of L-cysteine.

Fig. 10 shows results of cysteine desulfhydrase activity staining of d0191 product.


Detailed Description of the Preferred Embodiments


<1> Bacterium



[0022] The bacterium belongs to the family Enterobacteriaceae, and is able to produce L-cysteineFurthermore, the bacterium has been modified to decrease the activity of the protein encoded by the d0191 gene should be decreased. The d0191 gene will be explained later.

[0023] The "ability to produce L-cysteine" or the "L-cysteine-producing ability" means an ability of the bacterium to produce and cause accumulation of L-cysteine in a medium or the bacterial cells in such an amount that the L-cysteine can be collected from the medium or cells when the bacterium is cultured in the medium. A bacterium having L-cysteine-producing ability means a bacterium which can produce and cause accumulation of a larger amount of L-cysteine as compared to a wild-type, parent, or unmodified strain, and preferably a bacterium which can produce and cause accumulation of L-cysteine in a medium in an amount of 0.3 g/L or more, more preferably 0.4 g/L or more, particularly preferably 0.5 g/L or more.

[0024] The L-cysteine produced by the bacterium may change into L-cystine in the medium by the formation of a disulfide bond. Furthermore, as described later, S-sulfocysteine may be generated by the reaction of L-cysteine and thiosulfuric acid in the medium (Szczepkowski T.W., Nature, vol. 182 (1958)). Furthermore, the L-cysteine generated in bacterial cells may be condensed with a ketone, aldehyde, or, for example, pyruvic acid, which is present in the cells, to produce a thiazolidine derivative via a hemithioketal intermediate (refer to Japanese Patent No. 2992010). This thiazolidine derivative and hemithioketal may be present as an equilibrated mixture. Therefore, the L-cysteine-producing ability is not limited to the ability to accumulate only L-cysteine in the medium or cells, but also includes the ability to accumulate, in addition to L-cysteine, L-cystine or derivatives thereof such as S-sulfocysteine, a thiazolidine derivative, or a hemithioketal or a mixture thereof in the medium.

[0025] The bacterium having L-cysteine-producing ability may have the ability to produce L-cysteine, or it may be imparted by modifying a microorganism such as those described below by mutagenesis or a recombinant DNA techniqueUunless specially mentioned, the term L-cysteine refers to the reduced-type L-cysteine, L-cystine, derivative such as those mentioned above, or a mixture thereof.

[0026] The bacterium is not particularly limited so long as the bacterium belongs to the family Enterobacteriaceae and has L- cysteine-producing ability. Such bacteria include those of the genera Escherichia, Enterobacter, Pantoea, Klebsiella, Serratia, Erwinia, Salmonella and Morganella. Specifically, those classified into the family Enterobacteriaceae according to the taxonomy used in the NCBI (National Center for Biotechnology Information) database (http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?id=91347) can be used. As the parent strain of the family Enterobacteriaceae which can be used to perform the modification, it is desirable to use, especially, a bacterium of the genus Escherichia, Enterobacter, Pantoea, Erwinia, or Klebsiella.

[0027] Although the Escherichia bacteria are not particularly limited, specifically, those described in the work of Neidhardt et al. (Backmann B.J., 1996, Derivations and Genotypes of some mutant derivatives of Escherichia coli K-12, p.2460-2488, Table 1, In F.D. Neidhardt (ed.), Escherichia coli and Salmonella Cellular and Molecular Biology/Second Edition, American Society for Microbiology Press, Washington, D.C.) can be used. Escherichia coli is preferable. Examples of Escherichia coli include Escherichia coli W3110 (ATCC 27325), Escherichia coli MG1655 (ATCC 47076), and so forth, and include those derived from the prototype wild-type strain, K12 strain.

[0028] These strains are available from, for example, American Type Culture Collection (Address: 12301 Parklawn Drive, Rockville, Maryland 20852, P.O. Box 1549, Manassas, VA 20108, United States of America). That is, registration numbers are given to each of the strains, and the strains can be ordered by using these registration numbers (refer to http://www.atcc.org/). The registration numbers of the strains are listed in the catalogue of the American Type Culture Collection.

[0029] Examples of the Enterobacter bacteria include Enterobacter agglomerans, Enterobacter aerogenes and so forth, and examples of the Pantoea bacteria include Pantoea ananatis. Some strains of Enterobacter agglomerans were recently reclassified into Pantoea agglomerans, Pantoea ananatis, or Pantoea stewartii on the basis of the nucleotide sequence analysis of 16S rRNA etc.. A bacterium belonging to either Enterobacter or Pantoea may be used so long as it is classified as the family Enterobacteriaceae.

[0030] In particular, Pantoea bacteria, Erwinia bacteria, and Enterobacter bacteria are classified as γ-proteobacteria, and they are taxonomically very close to one another (J. Gen. Appl. Microbiol., 1997, 43, 355-361; Int. J. Syst. Bacteriol., 1997, 43, 1061-1067). In recent years, some bacteria belonging to the genus Enterobacter were reclassified as Pantoea agglomerans, Pantoea dispersa, or the like, on the basis of DNA-DNA hybridization experiments etc. (International Journal of Systematic Bacteriology, July 1989, 39:337-345). Furthermore, some bacteria belonging to the genus Erwinia were reclassified as Pantoea ananas or Pantoea stewartii (refer to Int. J. Syst. Bacteriol., 1993, 43:162-173).

[0031] Examples of the Enterobacter bacteria include, but are not limited to, Enterobacter agglomerans, Enterobacter aerogenes, and so forth. Specifically, the strains exemplified in European Patent Publication No. 952221 can be used.

[0032] A typical strain of the genus Enterobacter is the Enterobacter agglomeranses ATCC 12287 strain.

[0033] Typical strains of the Pantoea bacteria include, but are not limited to, Pantoea ananatis, Pantoea stewartii, Pantoea agglomerans, and Pantoea citrea.

[0034] Specific examples of Pantoea ananatis include the Pantoea ananatis AJ13355 strain, SC 17 strain and SC 17(0) strain. The SC 17 strain was selected as a low phlegm-producing mutant strain from the AJ13355 strain (FERM BP-6614), which was isolated from soil in Iwata-shi, Shizuoka-ken, Japan for it's ability to proliferate in a low pH medium containing L-glutamic acid and a carbon source (U.S. Patent No. 6,596,517). The SC 17(0) strain was constructed as a strain resistant to the λ Red gene product for performing gene disruption in Pantoea ananatis (refer to Reference Example 1).

[0035] The Pantoea ananatis AJ13355 strain was deposited at the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry (currently, the National Institute of Advanced Industrial Science and Technology, International Patent Organism Depositary, Address: Tsukuba Central 6, 1-1, Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, 305-8566, Japan) on February 19, 1998 and assigned an accession number of FERM P-16644. It was then converted to an international deposit under the provisions of Budapest Treaty on January 11, 1999 and assigned an accession number of FERM BP-6614. This strain was identified as Enterobacter agglomerans when it was isolated and deposited as the Enterobacter agglomerans AJ13355 strain. However, it was recently reclassified as Pantoea ananatis on the basis of nucleotide sequencing of 16S rRNA and so forth.

[0036] Examples of the Erwinia bacteria include, but are not limited to, Erwinia amylovora and Erwinia carotovora, and examples of the Klebsiella bacteria include Klebsiella planticola.

[0037] Hereinafter, methods for imparting L-cysteine-producing ability to bacteria belonging to Enterobacteriaceae, or methods for enhancing L-cysteine-producing ability of such bacteria, are described.

[0038] To impart the ability to produce L-cysteine, methods conventionally employed in the breeding of coryneform bacteria or bacteria of the genus Escherichia (see "Amino Acid Fermentation", Gakkai Shuppan Center (Ltd.), 1st Edition, published May 30, 1986, pp. 77-100) can be used. Such methods include by acquiring the properties of an auxotrophic mutant, an analogue-resistant strain, or a metabolic regulation mutant, or by constructing a recombinant strain so that it overexpresses an L-cysteine biosynthesis enzyme. Here, in the breeding of an L-cysteine-producing bacteria, one or more of the above described properties may be imparted. The expression of L-cysteine biosynthesis enzyme(s) can be enhanced alone or in combinations of two or more. Furthermore, imparting properties such as an auxotrophic mutation, analogue resistance, or metabolic regulation mutation may be combined with the methods of enhancing the biosynthesis enzymes.

[0039] An auxotrophic mutant strain, L-cysteine analogue-resistant strain, or metabolic regulation mutant strain with the ability to produce L-cysteine can be obtained by subjecting a parent, wild-type, or unmodified strain to conventional mutatagenesis, such as exposure to X-rays or UV irradiation, or by treating with a mutagen such as N-methyl-N'-nitro-N-nitrosoguanidine, etc., then selecting those which exhibit autotrophy, analogue resistance, or a metabolic regulation mutation and which also have the ability to produce L-cysteine.

[0040] Specific examples of L-cysteine-producing bacteria include, but are not limited to, E. coli JM 15 transformed with multiple kinds of cysE gene alleles encoding serine acetyltransferase resistant to feedback inhibition (US Patent 6,218,168), E. coli W3110 in which a gene encoding a protein responsible for excretion of cytotoxic substances is overexpressed (U.S. Patent No. 5,972,663), an E. coli strain having decreased cysteine desulfhydrase activity (Japanese Patent Laid-open No. 11-155571), and E. coli W3110 with increased activity of the positive transcriptional control factor of the cysteine regulon encoded by the cysB gene is increased (WO01/27307).

[0041] The bacterium of the present invention has been modified to decrase the activity of the protein encoded by the d0191 gene. As described later, the protein has cysteine desulfhydrase activity. The following proteins are known to have the cysteine desulfhydrase activity of E. coli, cystathionine-β-lyase (metC product, Japanese Patent Laid-open No. 11-155571, Chandra et al., Biochemistry, 21 (1982) 3064-3069), tryptophanase (tnaA product, Japanese Patent Laid-open No. 2003-169668, Austin Newton et al., J. Biol. Chem., 240 (1965) 1211-1218)), O-acetylserine sulfhydrylase B (cysM gene product, Japanese Patent Laid-open No. 2005-245311) and the malY gene product (Japanese Patent Laid-open No. 2005-245311) are known. In addition to the activity of the protein encoded by the d0191 gene, the activities of these proteins may be decreased.

[0042] The L-cysteine-producing bacterium preferably has a SAT which has been mutated to be resistant to feedback inhibition. The following mutation in SAT are known to induce resistance to feedback inhibition and are derived from Escherichia coli: when the methionine residue at position 256 is replaced with a glutamate residue (Japanese Patent Laid-open No. 11-155571), when the methionine residue at position 256 is replaced with an isoleucine residue (Denk, D. and Boeck, A., J. General Microbiol., 133, 515-525 (1987)), a mutation in the region from the amino acid residue at position 97 to the amino acid residue at position 273 or a deletion of the C-terminus region from the amino acid residue at position 227 (International Patent Publication WO97/15673, U.S. Patent No. 6,218,168), when the amino acid sequence corresponding to positions 89 to 96 of wild-type SAT contains one or more mutations (U.S. Patent Published Application No. 20050112731(A1)) and so forth. In the cysE5 gene which encodes the mutant SAT described in the examples, the Val residue and the Asp residue at positions 95 and 96 of the wild-type SAT are replaced with an Arg residue and Pro residue, respectively.

[0043] The SAT gene is not limited to the gene of Escherichia coli, but can be any gene encoding a protein having the SAT activity. An SAT isozyme of Arabidopsis thaliana and desensitized to feedback inhibition by L-cysteine is known, and the gene encoding this SAT can also be used (FEMS Microbiol. Lett., 179 (1999) 453-459).

[0044] If a gene encoding a mutant SAT is introduced into a bacterium, L-cysteine-producing ability is imparted to the bacterium. To introduce a mutant SAT gene into a bacterium, various vectors which are 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.

[0045] In order to introduce a recombinant vector containing a SAT gene into a bacterium, methods which are typically used to transform bacteria can be used, such as the method of D.A. Morrison (Methods in Enzymology, 68, 326 (1979)), treating recipient cells with calcium chloride to increase permeability of the cells for DNA (Mandel, M. and Higa, A., J. Mol. Biol., 53, 159 (1970)), and a method based on electroporation.

[0046] Furthermore, the SAT activity can also be enhanced by increasing the copy number of the SAT gene. The copy number of the SAT gene can be increased by introducing the SAT gene into a bacterium by using a vector such as those described above, or by introducing multiple copies of the SAT gene onto the chromosomal DNA of a bacterium. Multiple copies of the SAT gene are introduced by homologous recombination which targets a sequence present on the chromosomal DNA in multiple copies. A repetitive DNA or inverted repeat present at the end of a transposable element can be used as a sequence which is present on the chromosomal DNA in multiple copies. Alternatively, as disclosed in Japanese Patent Laid-open No. 2-109985, multiple copies of the SAT gene can be introduced into the chromosomal DNA by incorporating them into a transposon and transferring it.

[0047] The bacterium can be obtained by modifying a bacterium which belongs to the family Enterobacteriaceae and which has L-cysteine-producing ability and the d0191 gene such as those described above, so that the activity of the protein encoded by the d0191 gene (henceforth also referred to as "D0191 ") is decreased. Alternatively, after such modification that the activity of the D0191 protein is decreased, L-cysteine-producing ability may be imparted.

[0048] The inventor of the present invention found a novel gene encoding a protein having cysteine desulfhydrase activity from the chromosomal DNA of Pantoea ananatis, and designated it as d0191 gene. The nucleotide sequence of this gene and the amino acid sequence encoded by the gene are shown in SEQ ID NOS: 1 and 2, respectively.

[0049] When databases were searched for the sequence of the d0191 gene of Pantoea ananatis, genes considered to be homologue genes of the d0191 gene of which functions were unknown were found in the following bacteria. The nucleotide sequences of these genes and the amino acid sequences encoded by those nucleotide sequences are shown in SEQ ID NOS: 34 to 52. Accession numbers in the NCBI (National Center for Biotechnology Information) database are shown in the parentheses (GenBank Identifier(gi)|RefSeq accession (ref)).

[0050] Citrobacter koseri ATCC BAA-895 (accession:

gi|157146936|ref|YP_001454255.1|)



[0051] Klebsiella pneumoniae subsp. pneumoniae MGH 78578 (accession:

gi|152968982|ref|YP_001334091.1|)



[0052] Enterobacter sp. 638 (accession: gi|146310575|ref|YP_001175649.1|)

[0053] Salmonella typhimurium LT2 (accession: gi|16763839|ref|NP_459454.1|)

[0054] Serratia proteamaculans 568 (accession: gi|157369348|ref|YP_001477337.1|)

[0055] Erwinia carotovora subsp. atroseptica SCRI1043 (accession:

gi|50120095|ref|YP_049262.1|)



[0056] Vibrio cholerae O1 biovar eltor str. N16961 (accession:

gi|15641074|ref|NP_230706.1|)



[0057] Pseudomonas fluorescens PfO-1 (accession: gi|77457462|ref|YP_346967.1|)

[0058] Streptomyces coelicolor A3(2) (accession: gi|21219544|ref|NP_625323.1|)

[0059] Mycobacterium avium 104 (accession: gi|118467280|ref|YP_79726.1|)

[0060] In the present invention, the d0191 gene of Pantoea ananatis and homologue genes of this gene possessed by other bacteria may be called d0191 gene.

[0061] The phrase "decrease the activity of the protein encoded by the d0191 gene" means that the activity of the D0191 protein encoded by the d0191 gene is decreased as compared to a non-modified strain such as a wild-type strain or parent strain, and also means the complete disappearance of the activity.

[0062] As described in the examples, it was demonstrated that the protein encoded by the d0191 gene, i.e., the D0191 protein, had the cysteine desulfhydrase activity by activity staining. Therefore, the activity of the D0191 protein specifically means the cysteine desulfhydrase activity. The cysteine desulfhydrase activity can be measured by, for example, the method described in Japanese Patent Laid-open No. 2002-233384.

[0063] Modifications to decrease the activity of the D0191 proteinare attained by, for example, reducing the expression of the d0191 gene. Specifically, for example, intracellular activity of the protein can be reduced by deleting a part or all of the coding region of the d0191 gene on the chromosome. Furthermore, the activity of the D0191 protein can also be decreased by reducing the expression, for example, by modifying an expression control sequence of the gene such as a promoter or the Shine-Dalgamo (SD) sequence. Furthermore, the expression amount of the gene can also be reduced by modifying a non-translated region other than the expression control sequence. Furthermore, the whole gene as well as the sequences on both sides of the gene on the chromosome may be deleted. Furthermore, mutations which cause an amino acid substitution (missense mutation), a stop codon (nonsense mutation), or a frame shift mutation which adds or deletes one or two nucleotides into the coding region of the d0191 gene on the chromosome can be introduced (Journal of Biological Chemistry, 272:8611-8617 (1997); Proceedings of the National Academy of Sciences, USA, 95 5511-5515 (1998); Journal of Biological Chemistry, 266, 20833-20839 (1991)).

[0064] Furthermore, a transcriptional regulator, ybaO gene (synonym: c0263), is present upstream of the d0191 gene. The activity of the D0191 protein can be decreased by inactivating the ybaO gene. The nucleotide sequence of the ybaO gene from Pantoea ananatis and the amino acid sequence encoded by the gene are shown in SEQ ID NOS: 66 and 67, respectively.

[0065] Furthermore, modification can be caused by a conventional mutagenesis based on X-ray or ultraviolet irradiation or the use of a mutagen such as N-methyl-N'-nitro-N-nitrosoguanidine, so long as the activity of the D0191 protein is decreased.

[0066] An expression control sequence can be modified for preferably one or more nucleotides, more preferably two or more nucleotides, particularly preferably three or more nucleotides. When a coding region is deleted, the region to be deleted may be an N-terminus region, an internal region or a C-terminus region, or even the entire coding region, so long as the function of the D0191 protein is decreased or deleted. Deletion of a longer region can usually more surely inactivate a gene. Further, it is preferred that the deleted reading frames upstream and downstream of the region are not the same.

[0067] When another sequence is inserted into the coding region of the d0191 gene, the sequence may be inserted into any region of the gene, and insertion of a longer sequence can usually more surely inactivate the gene. It is preferred that the reading frames upstream and downstream of the insertion site are not the same. The other sequence is not particularly limited so long as a sequence which decreases or deletes function of the encoded D0191 protein is chosen, and examples include a transposon carrying an antibiotic resistance gene, a gene useful for L-cysteine production and so forth.

[0068] The d0191 gene on the chromosome can be modified as described above by, for example, preparing a deletion-type version of the gene in which a partial sequence of the gene is deleted so that the deletion-type gene does not produce normally-functioning D0191 proteins, and transforming a bacterium with a DNA containing the deletion-type gene to cause homologous recombination between the deletion-type gene and the native gene on the chromosome, which results in the substitution of the deletion-type gene for the gene on the genome. The D0191 protein encoded by the deletion-type gene has a conformation different from that of the wild-type enzyme protein, if it is even produced, and thus the function is reduced or deleted. Such gene disruption based on gene substitution utilizing homologous recombination is known, and examples includes Red-driven integration (Datsenko, K.A, and Wanner, B.L., Proc. Natl. Acad. Sci. USA, 97:6640-6645 (2000)), using a linear DNA in Red driven integration in combination with an excisive system derived from λ phage (Cho, E.H., Gumport, R.I., Gardner, J.F., J. Bacteriol., 184:5200-5203 (2002)), using a plasmid containing a temperature sensitive replication origin or a plasmid capable of conjugative transfer, utilizing a suicide vector without a replication origin in a host (U.S. Patent No. 6,303,383, Japanese Patent Laid-open No. 05-007491), and so forth.

[0069] Decrease of the expression of the d0191 gene can be confirmed by comparing the amount of mRNA transcribed from the gene with that in a wild-type strain or unmodified strain. The expression amount can be confirmed by Northern hybridization, RT-PCR (Sambrook, J., Fritsch, E. F., and Maniatis, T. 1989, Molecular Cloning A Laboratory Manual/Second Edition, Cold Spring Harbor Laboratory Press, New York.), and the like.

[0070] Decrease of the amount of D0191 protein can be confirmed by Western blotting using antibodies (Molecular cloning, Cold spring Harbor Laboratory Press, Cold spring Harbor, USA, 2001).

[0071] Decrease of the amount of the D0191 protein can also be confirmed by measuring the cysteine desulfhydrase activity of cell.

[0072] Since the nucleotide sequence of the d0191 gene may different depending on species or strain to which a bacterium belongs, the d0191 gene to be modified may be a variant of the nucleotide sequence of SEQ ID NO: 1. Moreover, the d0191 gene to be modified may be any of the aforementioned d0191 gene homologues, for example, a variant of a gene having the nucleotide sequence shown as SEQ ID NOS: 33, 35, 37, 39, 41, 43, 45, 47, 49 or 51.

[0073] Variant of the d0191 gene can be searched for with BLAST (http://blast.genome.jp/) or the like by referring to the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 33, 35, 37, 39, 41, 43, 45, 47, 49 or 51. Moreover, variants of the d0191 gene include genes which can be amplified by PCR using a chromosome of bacterium belonging to the family Enterobacteriaceae or the like as a template, and synthetic oligonucleotides prepared on the basis of the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 33, 35, 37, 39, 41, 43, 45, 47, 49 or 51.

[0074] Moreover, the d0191 gene may also be a gene encoding a protein having a sequence corresponding to the amino acid sequence of the D0191 protein, such as a sequence of SEQ ID NO: 2 or SEQ ID NO: 34, 36, 38, 40, 42, 44, 46, 48, 50 or 52, including substitutions, deletions, insertions, additions or the like of one or several amino acid residues at one or several positions. Although the number of the "one or several" amino acid residues may differ depending on their position in the three-dimensional structure or the types of amino acid residues of the proteins, it is preferably 1 to 20, more preferably 1 to 10, particularly preferably 1 to 5. These substitutions, deletions, insertions, or additions of one or several amino acids are preferably conservative mutations so that the function of the protein is maintained. A conservative mutation is a mutation wherein substitution takes place mutually among Phe, Trp and Tyr, if the substitution site is an aromatic amino acid; among Leu, Ile and Val, if the substitution site is a hydrophobic amino acid; between Gln and Asn, if it is a polar amino acid; among Lys, Arg and His, if it is a basic amino acid; between Asp and Glu, if it is an acidic amino acid; and between Ser and Thr, if it is an amino acid having a hydroxyl group. Typical examples of conservative mutations are conservative substitutions. Specific examples of conservative substitutions include: substitution of Ser or Thr for Ala; substitution of Gln, His or Lys for Arg; substitution of Glu, Gln, Lys, His or Asp for Asn; substitution of Asn, Glu or Gln for Asp; substitution of Ser or Ala for Cys; substitution of Asn, Glu, Lys, His, Asp or Arg for Gln; substitution of Gly, Asn, Gln, Lys or Asp for Glu; substitution of Pro for Gly; substitution of Asn, Lys, Gln, Arg or Tyr for His; substitution of Leu, Met, Val or Phe for Ile; substitution of Ile, Met, Val or Phe for Leu; substitution of Asn, Glu, Gln, His or Arg for Lys; substitution of Ile, Leu, Val or Phe for Met; substitution of Trp, Tyr, Met, Ile or Leu for Phe; substitution of Thr or Ala for Ser; substitution of Ser or Ala for Thr; substitution of Phe or Tyr for Trp; substitution of His, Phe or Trp for Tyr; and substitution of Met, Ile or Leu for Val. The above-mentioned amino acid substitution, deletion, insertion, addition, inversion, etc. may be the result of a naturally-occurring mutation or variation due to an individual difference, or a difference of species of a bacterium.

[0075] The d0191 gene provided by the present invention as a novel gene encodes a protein having the amino acid sequence of SEQ ID NO: 2, or a protein having an amino acid sequence of SEQ ID NO: 2 including substitutions, deletions, insertions or additions of 1 to 50, preferably 1 to 40, more preferably 1 to 30, still more preferably 1 to 20, further still more preferably 1 to 10, particularly preferably 1 to 5, of amino acid residues, and having the cysteine desulfhydrase activity.

[0076] Furthermore, the gene having such a conservative mutation as mentioned above may be a gene encoding a protein showing a homology of 80% or more, preferably 90% or more, more preferably 95% or more, still more preferably 97% or more, further still more preferably 98% or more, particularly preferably 99% or more, to the entire encoded amino acid sequence, and which has a function equivalent to that of a wild-type D0191 protein. In the present specification, the term "homology" may mean "identity".

[0077] The d0191 gene may be a DNA which hybridizes with a probe prepared from known gene sequences, for example, the above described gene sequences or sequences complementary to the sequences under stringent conditions and which encodes a protein which is a functional equivalent to the D0191 protein. The term "stringent conditions" refers to conditions where a so-called specific hybrid is formed and a non-specific hybrid is not formed. Examples thereof include conditions where DNAs having high homology, for example, at least 80%, preferably 90%, more preferably 95%, more preferably 97%, more preferably 98%, further preferably 99% homology, hybridize with each other and DNAs having homology less than the value do not hybridize with each other; and specifically include conditions corresponding to a salt concentration and temperature of washing which are typical of Southern hybridization, e.g., washing at 60°C, 1×SSC, 0.1% SDS, preferably 60°C, 0.1×SSC, 0.1% SDS, more preferably 68°C, 0.1×SSC, 0.1% SDS, once or preferably twice or three times.

[0078] The probe may be a partial sequence of the gene. Such a probe can be prepared by PCR using oligonucleotides prepared based on the known nucleotide sequences of genes as primers, and a DNA fragment containing these sequences as the template. When a DNA fragment of a length of about 300 bp is used as the probe, the conditions of washing after hybridization can be, for example, 50°C, 2×SSC, and 0.1% SDS.

[0079] The above descriptions about variants of genes and proteins are similarly applied to enzymes such as serine acetyltransferase and genes coding for them.

<2> Method for producing L-cysteine, L-cystine, derivative thereof or mixture thereof



[0080] These compounds can be produced by culturing the bacterium obtained as described above in a medium, and collecting L-cysteine, L-cystine, a derivative thereof or a mixture thereof from the medium. Examples of a derivative of L-cysteine include S-sulfocysteine, a thiazolidine derivative, a hemithioketal corresponding the thiazolidine derivative mentioned above and so forth.

[0081] Examples of the medium used for the culture include ordinary media containing a carbon source, nitrogen source, sulfur source, inorganic ions, and other organic components as required.

[0082] 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.

[0083] 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 so forth can be used.

[0084] As the sulfur source, inorganic sulfur compounds, such as sulfates, sulfites, sulfides, hyposulfites and thiosulfates can be used.

[0085] As organic trace amount nutrients, it is desirable to add required substances such as vitamin B1, yeast extract, and so forth in appropriate amounts. Other than these, potassium phosphate, magnesium sulfate, iron ions, manganese ions and so forth are added in small amounts.

[0086] The culture is preferably performed under aerobic conditions for 30 to 90 hours. The culture temperature is preferably controlled to be 25°C to 37°C, and the pH is preferably controlled to be 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. Collection of L-cysteine from the culture can be attained by, for example,any combination of known ion exchange resin method, precipitation and other known methods.

[0087] L-cysteine obtained as described above can be used toproduce L-cysteine derivatives. The cysteine derivatives include methylcysteine, ethylcysteine, carbocysteine, sulfocysteine, acetylcysteine, and so forth.

[0088] Furthermore, when a thiazolidine derivative of L-cysteine is produced in the medium, L-cysteine can be produced by collecting the thiazolidine derivative from the medium to break the reaction equilibrium between the thiazolidine derivative and L-cysteine so that L-cysteine should be excessively produced.

[0089] Furthermore, when S-sulfocysteine is produced in the medium, it can be converted into L-cysteine by reduction with a reducing agent such as dithiothreitol.

Examples



[0090] Hereinafter, the present invention will be explained more specifically with reference to the following non-limiting examples.

Reference Example 1: Construction of Pantoea ananatis strain resistant to λ Red gene product



[0091] In order to perform gene disruption in Pantoea ananatis, a recipient strain for efficiently carrying out the method called "Red-driven integration" or "Red-mediated integration" (Proc. Natl. Acad. Sci. USA., 97, 6640-6645 (2000)) was constructed.

[0092] First, a novel helper plasmid RSF-Red-TER which expresses the gam, bet and exo genes of λ (henceforth referred to as "λ Red gene") was constructed (Fig. 1). The details are described in Reference Example 2.

[0093] This plasmid can be used for a wide range of hosts of different genetic backgrounds. This is because 1) this plasmid has the replicon of the RSF1010 wide host spectrum plasmid (Scholz, et al., 1989; Buchanan-Wollaston et al., 1987), which may be stably maintained by many gram negative and gram positive bacteria, and even plants, 2) the λ Red gene, gam, bet and exo genes, are under the control of the PlacUV5 promoter, which is recognized by RNA polymerases of many bacteria (for example, Brunschwig, E. and Darzins, A., Gene, 111, 1, 35-41 (1992); Dehio, M. et al, Gene, 215, 2, 223-229 (1998)), and 3) the autoregulation factor PlacUV5-lacI and the p-non-dependent transcription terminator (TrrnB) of the rrnB operon of Escherichia coli lower the basal expression level of the λ Red gene (Skorokhodova, A. Yu et al, Biotekhnologiya (Rus), 5, 3-21 (2004)). Furthermore, the RSF-Red-TER plasmid contains the levansucrase gene (sacB), and by using this gene, the plasmid can be collected from cells in a medium containing sucrose.

[0094] In Escherichia coli, frequency of integration of a DNA fragment generated by PCR together with the short flanking region provided by the RSF-Red-TER plasmid is high to the same extent as that in the case where the pKD46 helper plasmid (Datsenko, KA., Wanner, B.L., Proc. Natl. Acad. Sci. USA, 97, 6640-6645 (2000)) is used. However, expression of the λ Red gene is toxic for Pantoea ananatis. The cells transformed with the RSF-Red-TER helper plasmid extremely slowly grew in the LB medium containing IPTG (isopropyl-β-D-thiogalactopyranoside, 1 mM) and an appropriate antibiotic (25 µg/ml of chloramphenicol or 40 µg/ml of kanamycin), and the efficiency of λ Red-mediated recombination is extremely low (10-8), even if it is observed.

[0095] A variant strain of Pantoea ananatis resistant to expression of all the three genes of λ Red gene was selected. For this purpose, the RSF-Red-TER plasmid was introduced into the Pantoea ananatis SC17 strain (U.S. Patent No. 6,596,517) by electroporation. After culture of 18 hours, about 106 of transformants were obtained, 10 clones among them formed colonies of large size, and all the remainder formed extremely small colonies. After culture of 18 hours, the large colonies had a size of about 2 mm, and the small colonies had a size of about 0.2 mm. Whereas the small colonies did not grow any more even if the culture was extended until 24 hours, the large colonies continued to grow. One of the Pantoea ananatis variant strains forming large colonies, which was resistant to expression of all the three genes of λ Red gene (gam, bet, and exo), was used for the further analysis.

[0096] The RSF-Red-TER plasmid DNA was isolated from one clone among those of the large colonies and several clones among those of small colonies, and transformed again into Escherichia coli MG1655 to examine the ability of the plasmid to synthesize an active Red gene product. By a control experiment for Red-dependent integration in the obtained transformants, it was demonstrated that only the plasmid isolated from the clone of the large colony induced expression of the λ Red gene required for the Red-dependent integration. In order to investigate whether the Red-mediated integration occurs in the selected clone of the large colony, electroporation was performed by using a linear DNA fragment produced by PCR, which was designed so that it contains a KmR marker and a flanking region of 40 bp homologous to the hisD gene, and it is integrated in the hisD gene of Pantoea ananatis at the SmaI recognition site. Two clones of small colonies were used as control. The nucleotide sequence of the hisD gene of Pantoea ananatis is shown in SEQ ID NO: 3. For PCR, the oligonucleotides of SEQ ID NOS: 4 and 5 were used as primers, and a pMW 118-(λatt-Kmr-λatt) plasmid was used as a template. The two clones of small colonies, which were not resistant to the λ Red gene, were used as control. Construction of the pMW118-(λattL-Kmr-λattR) plasmid will be explained in detail in Reference Example 3.

[0097] The RSF-Red-TER plasmid can induce expression of the Red gene with the lacI gene carried on the plasmid. Two kinds of induction conditions were investigated. In the first group, IPTG (1 mM) was added 1 hour before the electroporation, and in the second group, IPTG was added at the start of the culture for preparation of cells of which electroporation is possible. The growth rate of the progeny of the cells harboring RSF-Red-TER derived from the clone of large colony was not significantly lower than that of a strain not harboring the SC17 plasmid. The addition of IPTG only slightly decreased the growth rate of these cultures. On the other hand, the progeny of the clones of small colonies extremely slowly grew even without addition of IPTG, and after induction, growth was substantially arrested. After electroporation of the cells of the progeny of the clone of large colony, many KmR clones grew (18 clones after a short induction time, and about 100 clones after extended induction time). All the 100 clones investigated had a His- phenotype, and about 20 clones were confirmed by PCR to have the expected structure of chromosome in the cells thereof. On the other hand, even when electroporation was performed for the cells of the progeny of the clones of small colonies, any integrated strain could not be obtained.

[0098] The obtained clone of large colony was grown on a plate containing 7% sucrose to eliminate the plasmid, and transformed again with RSF-Red-TER. A strain not harboring the plasmid was designated SC 17(0). This strain was deposited at the Russian National Collection of Industrial Microorganisms (VKPM, GNII Genetica, address: Russia, 117545 Moscow, 1 Dorozhny proezd. 1) on September 21, 2005, and assigned an accession number of VKPM B-9246.

[0099] All the clones grown after the aforementioned re-transformation showed large colony sizes like the parent strain clone SC17(0). The Red-mediated integration experiment was performed in the SC 17(0) strain re-transformed with the RSF-Red-TER plasmid. Three of the obtained independent transformants were investigated by using the same DNA fragment as that used for the previous experiment. The short induction time (1 hour before electroporation) was employed. KmR Clones exceeding ten clones grew in each experiment. All the examined clones had the His- phenotype. In this way, a variant strain designated SC 17(0) resistant to the expression of the λ Red gene was selected. This strain can be used as a recipient strain suitable for the Red-dependent integration into the Pantoea ananatis chromosome.

Reference Example 2: Construction of helper plasmid RSF-Red-TER



[0100] The construction scheme of the helper plasmid RSF-Red-TER is shown in Fig. 2.

[0101] As a first step of the construction, a RSFsacBPlacMCS vector was designed. For this purpose, DNA fragments containing the cat gene of pACYC184 plasmid and the structural gene region of the sacB gene of Bacillus subtilis were amplified by PCR using oligonucleotide of SEQ ID NOS: 6 and 7, and 8 and 9, respectively. These oligonucleotides contained convenient BglII, SacI, XbaI and BamHI restriction enzyme sites required for further cloning in the 5' end regions, respectively. The obtained sacB fragment of 1.5 kb was cloned into the previously obtained pMW119-PlaclacI vector at the XbaI-BamHI site. This vector was constructed in the same manner as that described for the pMW118-PlaclacI vector (Skorokhodova, A. Yu et al, Biotekhnologiya (Rus), 5, 3-21 (2004)). However, this vector contained a polylinker moiety derived from pMW219 instead of the pMW218 plasmid.

[0102] Then, the aforementioned cat fragment of 1.0 kb was treated with BglII and SacI, and cloned into the RSF-PlaclacIsacB plasmid obtained in the previous step at the BamHI-SacI site. The obtained plasmid pMW-PlaclacIsacBcat contained the PlacUV5-lacI-sacB-cat fragment. In order to subclone this fragment into the RSF1010 vector, pMW-PlaclacIsacBcat was digested with BglII, blunt-ended by a treatment with DNA polymerase I Klenow fragment, and successively digested with SacI. A 3.8 kb BglII-SacI fragment of the pMWPlaclacIsacBcat plasmid was eluted from 1% agarose gel, and ligated with the RSF1010 vector treated with PstI and SacI. Escherichia coli TG1 was transformed with the ligation mixture, and plated on the LB medium containing chloramphenicol (50 mg/L). The plasmids isolated from the grown clones were analyzed with restriction enzymes to obtain an RSFsac B plasmid. In order to construct a RSFsacBPlacMCS vector, a DNA fragment containing the PlacUV5 promoter was amplified by PCR using oligonucleotides of SEQ ID NOS: 10 and 11 as primers and the pMW119-PlaclacI plasmid as a template. The obtained fragment of 146 bp was digested with SacI and NotI, and ligated with the SacI-NotI large fragment of the RSFsacB plasmid. Then, by PCR using the oligonucleotides of SEQ ID NOS: 12 and 13 as primers, and the pKD46 plasmid (Datsenko, KA., Wanner, B.L., Proc. Natl. Acad. Sci. USA, 97, 6640-6645 (2000)) as a template, a DNA fragment of 2.3 kb containing the λRedαβγ gene and the transcription terminator tL3 was amplified. The obtained fragment was cloned into the RSFsacBPlacMCS vector at the PvuI-NotI site. In this way, the RSFRed plasmid was designed.

[0103] In order to eliminate the read through transcription of the Red gene, a p-dependent transcription terminator of the rrnB operon of Escherichia coli was inserted at a position between the cat gene and the PlacUV5 promoter. For this purpose, a DNA fragment containing the PlacUV5 promoter and the TrrnB terminator was amplified by PCR using the oligonucleotides of SEQ ID NOS: 14 and 11 as primers and the chromosome of Escherichia coli BW3350 as a template. These obtained fragments were treated with KpnI and ligated. Then, the 0.5 kb fragment containing both PlacUV5 and TrrnB was amplified by PCR using the oligonucleotides of SEQ ID NOS: 11 and 15 as primers. The obtained DNA fragment was digested with EcoRI, blunt-ended by a treatment with DNA polymerase I Klenow fragment, digested with BamHI, and ligated with the Ecl136II-BamHI large fragment of the RSFsacBPlacMCS vector. The obtained plasmid was designated RSF-Red-TER.

Reference Example 3: Construction of the pMW 118-(λattL-KmrattR) plasmid.



[0104] The pMW118-(λattL-KmrattR) plasmid was constructed based on the pMW118-attL-Tc-attR (W02005/010175) plasmid by substitution of the tetracycline resistance marker gene with the kanamycin resistance marker gene from the pUC4K plasmid (Vieira, J. and Messing, J., Gene, 19(3): 259-68 (1982)). For that purpose, the large EcoRI-HindIII fragment from pMW118-attL-Tc-attR plasmid was ligated to two fragments from the pUC4K plasmid: HindIII - PstI fragment (676 bp) and EcoRI-HindIII fragment (585 bp). Basic pMW1118-attL-Tc-attR was obtained by ligation of the following four DNA fragments:
  1. 1) the BglII-EcoRI fragment (114 bp) carrying attL (SEQ ID NO: 26) which was obtained by PCR amplification of the corresponding region of the E. coli W3350 (contained λ prophage) chromosome using oligonucleotides P1 and P2 (SEQ ID NOS: 53 and 54) as primers (these primers contained the subsidiary recognition sites for BglII and EcoRI endonucleases);
  2. 2) the PstI-HindIII fragment (182 bp) carrying attR (SEQ ID NO: 58) which was obtained by PCR amplification of the corresponding region of the E. coli W3350 (contained λ prophage) chromosome using the oligonucleotides P3 and P4 (SEQ ID NOS: 56 and 57) as primers (these primers contained the subsidiary recognition sites for PstI and HindIII endonucleases);
  3. 3) the large BglII-HindIII fragment (3916 bp) of pMW118-ter_rrnB. The plasmid pMW118-ter_rrnB was obtained by ligation of the following three DNA fragments:
    1. a) the large DNA fragment (2359 bp) carrying the AatII-EcoRI fragment of pMW118 that was obtained by the following way: pMW118 was digested with EcoRI restriction endonuclease, treated with Klenow fragment of DNA polymerase I, and then digested with AatII restriction endonuclease;
    2. b) the small AatII-BglII fragment (1194 bp) of pUC19 carrying the bla gene for ampicillin resistance (ApR) was obtained by PCR amplification of the corresponding region of the pUC 19 plasmid using oligonucleotides P5 and P6 (SEQ ID NOS: 59 and 60) as primers (these primers contained the subsidiary recognition sites for AatII and BglII endonucleases);
    3. c) the small BglII-PstIpol fragment (363 bp) of the transcription terminator ter_rrnB was obtained by PCR amplification of the corresponding region of the E. coli MG1655 chromosome using oligonucleotides P7 and P8 (SEQ ID NOS: 61 and 62) as primers (these primers contained the subsidiary recognition sites for BglII and PstI endonucleases);
  4. 4) the small EcoRI-PstI fragment (1388 bp) (SEQ ID NO: 63) of pML-Tc-ter_thrL bearing the tetracycline resistance gene and the ter_thrL transcription terminator; the pML-Tc-ter_thrL plasmid was obtained in two steps:
    • the pML-ter_thrL plasmid was obtained by digesting the pML-MCS plasmid (Mashko, S.V. et al., Biotekhnologiya (in Russian), 2001, no. 5, 3-20) with the XbaI and BamHI restriction endonucleases, followed by ligation of the large fragment (3342 bp) with the XbaI-BamHI fragment (68 bp) carrying terminator ter_thrL obtained by PCR amplification of the corresponding region of the E. coli MG1655 chromosome using oligonucleotides P9 and P10 (SEQ ID NOS: 64 and 65) as primers (these primers contained the subsidiary recognition sites for the XbaI and BamHI endonucleases);
    • the pML-Tc-ter_thrL plasmid was obtained by digesting the pML-ter_thrL plasmid with the KpnI and XbaI restriction endonucleases followed by treatment with Klenow fragment of DNA polymerase I and ligation with the small EcoRI-Van91I fragment (1317 bp) of pBR322 bearing the tetracycline resistance gene (pBR322 was digested with EcoRI and Van91I restriction endonucleases and then treated with Klenow fragment of DNA polymerase I).

Example 1


(1) Cloning of d0191 gene from P. ananatis SC17 strain



[0105] By PCR using the genomic DNA of P. ananatis SC17 strain (U.S. Patent No. 6,596,517) as a template, primers d0191(Pa)-FW (CGCGGATCCAAGCTTTTCATTATCCAGCAGAGCG, SEQ ID NO: 16), and d0191(Pa)-RV (CGCGGATCCTAATGCTGTAGGGCCTGAACCAG, SEQ ID NO: 17), a d0191 gene fragment containing 300 bp upstream and 200 bp downstream from the d0191 gene was obtained. Restriction enzyme BamHI sites were designed at the 5' ends of these primers. For PCR, Pyrobest polymerase (Takara) was used, and after a reaction at 94°C for 5 minutes, a cycle of 98°C for 5 seconds, 55°C for 5 seconds and 72°C for 1 minute and 30 seconds was repeated 30 times in the standard reaction composition described in the protocol of the polymerase to amplify the target fragment of about 1.6 kb. The obtained fragment was treated with BamHI and inserted into pSTV29 (Takara) at the BamHI site (inserted in the same direction as the direction of the lacZ gene on the vector) to obtain a plasmid pSTV-d0191F (having a chloramphenicol resistance marker) in which d0191 was cloned. The same PCR fragment was also inserted into pACYC177 (Nippon Gene) at the BamHI site (inserted in the same direction as the direction of the kanamycin resistance gene on the vector) to obtain a plasmid pACYC-d0191F (having kanamycin resistant marker). It was confirmed by sequencing that there was no PCR error. In this way, the plasmids having two kinds of different antibiotic resistance markers and cloned with the same d0191 region (both had the same p15A origin) were prepared. Furthermore, pHSG-d0191F was prepared, which corresponded to the high copy vector pHSG299 (Takara) inserted with the same DNA fragment of about 1.6 kb as mentioned above at the BamHI site (having kanamycin resistant marker, inserted in the same direction as that of the lacZ gene on the vector).

(2) Construction of d0191-enhanced strains from P. ananatis SC17 strain (SC17/pSTV-d0191F, SC17/pHSG-d0191F)



[0106] P. ananatis SC 17 was transformed with the plasmid pSTV-d0191F constructed as described above to prepare a d0191-enhanced strain, SC17/pSTV-d0191F strain. As a control strain, a strain introduced with the empty vector, SC17/pSTV29 strain, was also prepared. Furthermore, a d0191-enhanced strain SC 17/pHSG-d0191 F strain was also prepared by transforming P. ananatis SC 17 with the high copy pHSG-d0191F. As a control strain, a strain introduced with the empty vector, SC17/pHSG299 strain, was also prepared. The transformation of P. ananatis was performed by a conventional method based on electroporation, and selection of the transformants was performed on the LB agar medium (5 g/L of yeast extract, 10 g/L of tryptone, 10 g/L of sodium chloride, 15 g/L of agar) containing an antibiotic corresponding to the antibiotic resistance marker of the plasmid (25 mg/L in the case of chloramphenicol, 20 mg/L in the case of kanamycin).

(3) Construction of d0191-enhanced strain from E. coli MG1655 strain (MG1655/pSTV-d0191F)



[0107] E. coli MG1655 was transformed with the plasmid pSTV-d0191F constructed as described above to prepare a d0191-enhanced strain, MG1655/pSTV-d0191F strain. As a control strain, a strain introduced with the empty vector, MG1655/pSTV29 strain, was also prepared. The transformation of E. coli was performed by a conventional method based on electroporation, and selection of the transformants was performed on the LB agar medium (5 g/L of yeast extract, 10 g/L of tryptone, 10 g/L of sodium chloride, 15 g/L of agar) containing an antibiotic corresponding to the antibiotic resistance marker of the plasmid (25 mg/L in the case of chloramphenicol, 20 mg/L in the case of kanamycin).

(4) Construction of d0191-deficient strains from P. ananatis SC17 strain (SC17 d0191::Kmr strain, SC17 Dd0191 strain)



[0108] Deletion of the d0191 gene was performed by the method called "Red-driven integration", first developed by Datsenko and Wanner (Proc. Natl. Acad. Sci. USA, 2000, vol. 97, No. 12, pp.6640-6645). According to the "Red-driven integration" method, using a PCR product obtained by using synthetic oligonucleotides in which a part of a target gene is designed on the 5' side, and a part of antibiotic resistance gene is designed on the 3' side, respectively, as primers, a gene-disrupted strain can be constructed in one step.

[0109] A DNA fragment comprising homologous sequences of the both ends of the d0191 gene and an antibiotic resistance gene (kanamycin resistance gene) between them was obtained by PCR. As primers, Dd0191-FW (CCGTGTCTGAAGCCTATTTTGCCCGCCTGCTGGGCTTGCCTTTTATTGCCTGA AGCCTGCTTTTTTATACTAAGTTGGCA, SEQ ID NO: 18), and Dd0191-RV (CTAGCCCAGTTCGCGCTGCCAGGGCGTAATATCGCCAATGTGCTCGGCAACG CTCAAGTTAGTATAAAAAAGCTGAACGA, SEQ ID NO: 19) were used, and as a template, pME118-( λ attL-Kmr - λ attR) (WO2006/093322A2, SEQ ID NO: 18) was used.

[0110] For PCR, Pyrobest polymerase (Takara) was used, and after a reaction at 94°C for 5 minutes, a cycle of 98°C for 5 seconds, 55°C for 5 seconds and 72°C for 2 minute and 30 seconds was repeated 30 times in the standard reaction composition described in the protocol of the polymerase to amplify the target DNA fragment. The obtained DNA fragment (DNA fragment comprising the kanamycin resistant marker and homologous sequences of d0191 on both sides of the marker, Fig. 3) was introduced into P. ananatis SC 17(0)/RSF-Red-TER strain by electroporation to obtain a kanamycin-resistant strain. It was confirmed that the obtained kanamycin-resistant strain was a d0191 gene-disrupted strain in which the kanamycin resistant cassette was inserted into the d0191 gene on the chromosome with the homologous sequences of the d0191 gene region existing at the both ends of the DNA fragment (SC17(0) d0191::Kmr strain, Fig. 3).

[0111] Then, by introducing the chromosomal DNA prepared from this SC 17(0) d0191::Kmr strain into the SC17 strain by electroporation, a SC17 d0191::Kmr strain, which was a d0191 gene-disrupted strain derived from the SC 17 strain, was finally obtained.

(5) Construction of plasmid carrying inhibition-desensitized type SAT (serine acetyltransferase) gene (pMIV-CysE5)



[0112] It is known that the pMIV-CysE5 plasmid carries the cysE5 gene encoding a mutant SAT which is desensitized to feedback inhibition (U.S. Patent Published Application No. 20050112731(A1)). A cysteine-producing bacterium which produces a marked amount of cysteine can be prepared by introducing this plasmid into the bacterium (U.S. Patent Published Application No. 20050112731(A1), U.S. Patent No. 5,972,663 etc.). The construction method of pMIV-CysE5 is described below.

[0113] The mutant allele E. coli cysE gene, cysE5 (U.S. Patent Published Application No. 20050112731(A1)) was obtained by PCR using primers cysEplF (5'-agc-tga-gtc-gac-atg-tcg-tgt-gaa-gaa-ctg-gaa-3', SEQ ID NO: 20), cysER (5'-agc-tga-tct-aga-ata-gat-gat-tac-atc-gca-tcc-3', SEQ ID NO: 21) and the template pMW-PompC-cysE5 (EP1650296A1). A cycle of 94°C for 0.5 minute, 57°C for 0.5 minute was conducted, then cycles of and 72°C for 1 minute were repeated 27 times, and then the reaction was maintained at 72°C for 7 minutes. The cysEplF primer was designed so as to bind with the start codon ATG of the E. coli cysE gene and a downstream sequence, and has a 6-mer SalI site at the 5' end. The cysER primer was designed so as to bind with the stop codon of the E. coli cysE gene and an upstream sequence, and has a 6-mer XbaI site at the 5' end. A DNA fragment of about 0.7 kb obtained by PCR was digested with SalI and XbaI, the digestion product was inserted into the pMIV-PompC plasmid which had been similarly digested with SalI and XbaI to construct pMIV-CysE5.

[0114] The pMIV-PompC plasmid described above was constructed as follows. By conducting PCR using the genomic DNA of the E. coli MG1655 strain as the template, primers PrOMPCF (5'-agc-tga-gtc-gac-aac-cct-ctg-tta-tat-gcc-ttt-a-3', SEQ ID NO: 22) and PrOMPCR (5'-agc-tga-gca-tgc-gag-tga-agg-ttt-tgt-ttt-gac-3', SEQ ID NO: 23), a DNA fragment containing about 0.3 kb of a promoter region of the ompC gene was obtained, and this fragment was inserted into the pMIV-5JS plasmid at the PaeI and SalI sites to construct pMIV-PompC. The plasmid pMIV-5JS was constructed by ligating the BamHI and HindIII sites designed beforehand at both ends of the intJS cassette (described later) with the BglII and HindIII sites of pM12-ter(thr) (described later) (Fig. 4).

[0115] The pM12-ter(thr) plasmid was constructed (Fig. 6) by inserting a double stranded DNA fragment (thrL terminator, designed to have HindIII and PstI sites at both ends) produced by annealing a synthetic oligonucleotide (aagcttaaca cagaaaaaag cccgcacctg acagtgcggg cttttttttt cgaccactgc ag, SEQ ID NO: 24) and a complementary synthetic oligonucleotide (ttcgaattgt gtcttttttc gggcgtggac tgtcacgccc gaaaaaaaaa gctggtgacg tc, SEQ ID NO: 25) into the pM 12 plasmid which contains the integration cassette derived from Mu phage (EP1486570(A1), Fig. 5) at the HindIII and Mph1103I sites. The IntJS cassette was constructed by the following procedures (a) to (g) (Fig. 7).
  1. (a) By conducting PCR using an upstream primer (ccagatcttg aagcctgctt ttttatacta agttggc, SEQ ID NO: 26, designed to have a BglII site), a downstream primer (gaaatcaaat aatgatttta ttttg, SEQ ID NO: 27, phosphorylated) and the pMW118-attL-tet-attR-ter_rrnB plasmid (W02005/010175) as the template, a LattL fragment of 0.12 kbp was obtained.
  2. (b) By conducting PCR using an upstream primer (ttacgccccg ccctgccact catcgc, SEQ ID NO: 28, phosphorylated), a downstream primer (gtcactgcag ctgatgtccg gcggtgcttt tgcc, SEQ ID NO: 29, designed to have a PstI site) and the pACYC184 plasmid (New England Biolabs) as the template, a CmR fragment of 1.03 kbp was obtained.
  3. (c) By conducting PCR using an upstream primer (cagctgcagt ctgttacagg tcactaatac c, SEQ ID NO: 30, designed to have a PstI site), a downstream primer (ccgagctccg ctcaagttag tataaaaaag ctgaacg, SEQ ID NO: 31, designed to have SacI site), and the pMW118-attL-tet-attR-ter_rrnB plasmid (W02005/010175) as the template, a LattR fragment of 0.16 kbp was obtained.
  4. (d) By ligation of the LattL and CmR fragments, a LattL-CmR fragment of 1.15 kbp was obtained.
  5. (e) By ligation of the LattL-CmR and LattR fragments digested with PstI, a LattL-CmR-LattR fragment of 1.31 kbp was obtained.
  6. (f) By annealing a synthetic oligonucleotide (cccgagctcg gtacctcgcg aatgcatcta gatgggcccg tcgactgcag aggcctgcat gcaagcttcc, SEQ ID NO: 32) and with its complementary strand, a double stranded DNA fragment of 70 bp containing a multi-cloning site (MCS) was obtained.
  7. (g) By ligation of the LattL-CmR-LattR fragment and the double stranded DNA fragment containing a multi-cloning site (MCS), and digested both with SacI, an IntJS fragment of 1.38 kbp was obtained.

(6) Construction of a P. ananatis strain having cysteine-producing ability and enhanced d0191 (SC17/pMIV-CysE5/pACYC-d0191F strain)



[0116] The pACYC-d0191F plasmid containing the d0191 gene (kanamycin resistant) constructed as described above was introduced into the SC 17 strain (SC17/pACYC-d0191F), along with the inhibition-desensitized type SAT gene-carrying plasmid pMIV-CysE5 (chloramphenicol resistant) to construct a P. ananatis strain having cysteine-producing ability and enhanced d0191 (SC17/pMIV-CysE5/pACYC-d0191F strain). Furthermore, as a control strain, SC 17/pMIV-CysE5/pACYC 177 strain was prepared, which was transformed with the empty vector pACYC177 instead of pACYC-d0191F.

(7) Construction of a P. ananatis strain with cysteine-producing ability and deficient in d0191 (SC17 d0191::Kmr/pMIV-CysE5 strain).



[0117] The d0191-deficient strain (SC 17 d0191::Kmr), constructed as described above was transformed with the pMIV-CysE5 plasmid to prepare a d0191-deficient cysteine-producing bacterium, SC 17 d0191::Kmr/pMIV-CysE5. The SC17/pMIV-CysE5 strain was prepared as a control, which corresponded to the SC 17 strain transformed with pMIV-CysE5.

(8) Effect of deletion and enhancement of d0191 on cysteine resistance in the P. ananatis SC 17 strain



[0118] In order to investigate the influence of the d0191 gene on cysteine resistance, the d0191-deficient strain SC17 d0191::Kmr, the d0191-amplified strain SC17/pSTV-d0191F, as well as their control strains (SC 17 and SC17/pSTV29) were each cultured in M9 minimal medium (Sambrook et al., Molecular Cloning, 3rd edition, 2001, Cold Spring Harbor Laboratory Press) which contained cysteine at different concentrations. The difference in cysteine resistance was evaluated by determining change in of the growth. As the resistance to cysteine increased, the OD of the medium more quickly increased, and as the resistance to cysteine decreased, the OD of the mediumincreased more slowly. The procedure of the experiment was as follows. Each strain was precultured overnight in M9 minimal medium containing 0.4% of glucose, but no cysteine (3-ml test tube, 34°C, shaking culture), and then inoculated into the main culture medium. At the time of the inoculation, the OD of the preculture was measured, and the amount inoculated was adjusted so that the main culture begun with the same amount. The OD at the time of the start of the main culture was about 0.007.

[0119] The main culture was performed in 4 ml of M9 minimal medium containing cysteine at the following concentrations: 0 mM, 1 mM, and 5 mM, and 0.4% of glucose using an automatically OD measuring culture apparatus, BIO-PHOTORECORDER TN-1506 (ADVANTEC) and the L-shaped test tube for the apparatus. To the medium for the strains harboring the plasmid, 25 mg/L of chloramphenicol was added. The progress of the culture (growth curves) is shown in Fig. 8. It was observed that as the concentration of cysteine increased, the OD increased more slowly. However, the d0191-enhanced strain, SC17/pSTV-d0191F grew more quickly as compared to the control SC17/pSTV29 strain, and it was found to be resistant to cysteine. Furthermore, since the growth of the d0191-deficient strain, SC 17 d0191::Kmr was completely inhibited by addition of cysteine, it was found that the cysteine resistance thereof had decreased.

(9) Effect of enhancing d0191 on cysteine production in E. coli MG1655 strain



[0120] In order to investigate the effect of enhancing the d0191 gene on cysteine resistance, the cysteine-producing abilities of the d0191-amplified strain MG1655/pSTV-d0191F, the control strain thereof MG1655/pSTV29, were each cultured in the M9 minimal medium containing cysteine of different concentrations, and difference in cysteine resistance was evaluated by determining rising of the growth. The culture was performed basically according to the method described in (8), provided that the method was different from the method of (8) in that OD at the start of the main culture was about 0.006, the culture temperature was 37°C, and the cysteine concentrations were 0 mM, 0.1 mM, 0.2 mM. Progress of the culture (growth curves) are shown in Fig. 9. There was observed a tendency that as the concentration of cysteine increased, the rise of OD became later. However, the d0191-enhanced strain, MG1655/pSTV-d0191F strain grew earlier compared with the MG1655/pSTV29 strain as the control, and it was found that it was imparted with cysteine resistance. From the above results, it was found that when d0191 was enhanced, cysteine resistance was enhanced regardless of whether the host is P. ananatis or E. coli.

(10) Detection of cysteine desulfhydrase activity of d0191 product by activity staining



[0121] The phenotype concerning cysteine resistance suggested a possibility of involvement of the d0191 product in cysteine decomposition. Therefore, in order to examine whether the d0191 gene product had the cysteine desulfhydrase activity (henceforth abbreviated as the "CD activity") known as a cysteine decomposition activity, a d0191-deficient strain, a d0191-enhanced strain and control strains of them were cultured, proteins in cell extracts of them were separated by native PAGE, and the CD activity of them was detected by activity staining. The methods of native PAGE and activity staining were according to the descriptions ofN. Awano et al. (Effect of cysteine desulfhydrase gene disruption on L-cysteine overproduction in Escherichia coli, Appl. Microbiol. Biotechnol., 2003 Aug; 62(2-3):239-43).

[0122] First, the four strains, the d0191-deficient strain SC17 d0191::Kmr strain constructed as described above and the control strain thereof, the SC 17 strain, as well as the d0191-enhanced strain SC 17/pHSG-d0191F strain and the control strain thereof, SC17/pHSG299, were cultured by the method described below, and cell extracts of them were prepared. Overnight culture in LB medium (5 g/L of yeast extract, 10 g/L of tryptone, 10 g/L of sodium chloride) of each strain was inoculated into 50 ml of LB medium contained in a Sakaguchi flask so as to be diluted 100 times, and culture was performed with shaking. After 3 hours, cells in the logarithmic phase were collected from the medium (OD was around 0.5), washed with a washing buffer (10 mM Tris-HCl (pH 8.6), 0.1 mM DTT (dithiothreitol), 0.01 mM PLP (pyridoxal-5'-phosphate)), then suspended in 1 ml of the washing buffer, and disrupted by ultrasonication, and the supernatant (cell extract) was obtained by centrifugation. The culture was performed at 34°C for all the strains, and 20 mg/L of kanamycin was added to the medium for the strains harboring a plasmid. Protein concentration of each cell extract prepared as described above was quantified, and the extract was diluted to a protein concentration of 2.5 mg/ml with the washing buffer, and added 5 x loading buffer (10 mM Tris-HCl (pH 8.6), 30% glycerol, 0.005% BPB (Bromophenol Blue)) to prepare a sample of final concentration of 2 mg/ml.

[0123] One microliter (2 µg of protein) of each sample was loaded on 10% native PAGE gel (TEFCO), and native PAGE was performed at 4°C and 20 mA for 1.5 hours. The composition of the gel-running buffer used in the native PAGE consisted of 25 mM Tris-HCl (pH 8.3) and 192 mM glycine. After the electrophoresis, the gel was immersed in a staining solution (100 mM Tris-HCl (pH 8.6), 10 mM EDTA (ethylenediaminetetraacetic acid), 50 mM L-cysteine, 0.02 mM PLP, 1.6 mM bismuth(III) chloride), and activity staining was performed at room temperature by slowly shaking the gel until appropriate coloring was observed. The detected bands of the d0191 product presented by the CD activity are shown in Fig. 10. A band not seen with the d0191-deficient strain was observed with the control strain, and increased intensity of this band was observed with the d0191-enhanced strain. From these results, it was considered a band of the d0191 product stained by the CD activity. From the above results, it was revealed that d0191 coded for a novel cysteine desulfhydrase, and was involved in cysteine resistance.

(11) Effect of d0191 enhancement on cysteine production in cysteine-producing bacterium



[0124] In order to investigate effect of enhancement of the d0191 gene on cysteine production, cysteine-producing abilities of the cysteine-producing bacterium, P. ananatis SC17/pMIV-CysE5/pACYC-d0191F strain, of which d0191 was enhanced, and the SC17/pMIV-CysE5/pACYC177 strain as a control strain thereof were compared. For the culture, a cysteine production medium (composition: 15 g/L of ammonium sulfate, 1.5 g/L of potassium dihydrogenphosphate, 1 g/L of magnesium sulfate heptahydrate, 0.1 mg/L of thiamine hydrochloride, 1.7 mg/L of ferrous sulfate heptahydrate, 0.15 mg/L of sodium molybdate dihydrate, 0.7 mg/L of cobalt chloride hexahydrate, 1.6 mg/L of manganese chloride tetrahydrate, 0.3 mg/L of zinc sulfate heptahydrate, 0.25 mg/L of copper sulfate pentahydrate, 0.6 g/L of tryptone, 0.3 g/L of yeast extract, 0.6 g/L of sodium chloride, 20 g/L of calcium carbonate, 135 mg/L of L-histidine monohydrochloride monohydrate, 4 g/L of sodium thiosulfate, 2 mg/L of pyridoxine hydrochloride, 60 g/L of glucose, 25 mg/L of chloramphenicol and 20 mg/L of kanamycin) was used.

[0125] The culture was performed according to the following procedures. The SC 17/pMIV-CysE5/pACYC-d0191F and the SC 17/pMIV-CysE5/pACYC 177 strains were each applied and spread on LB agar medium containing chloramphenicol and kanamycin, and precultured overnight at 34°C. The cellson about 7 cm on the plate were scraped with an inoculating loop of 10 µl (NUNC Blue Loop), and inoculated into 2 ml of the cysteine production medium in a large test tube (internal diameter: 23 mm, length: 20 cm). The amounts of the inoculated cells were adjusted so that the cell amounts at the time of the start of the culture are substantially the same. The culture was performed at 32°C with shaking. For the both strains, when glucose was completely consumed, the culture was ended (about 22 to 25 hours), and the amount of cysteine which had accumulated in the medium was quantified. The quantification of cysteine was performed by the method described by Gaitonde, M.K. (Biochem. J., 1967 Aug., 104(2):627-33). The experiment was performed in tetraplicate for the both strains, and averages and standard deviations of the accumulated cysteine amounts are shown in Table 1. As shown in Table 1, it was revealed that enhancing d0191 caused decrease in the accumulation of cysteine.
Table 1
StrainGene typeCys (mg/L)
SC17/pMIV-CysE5/pACYC177 Vector 169.5 ± 3.7
SC17/pMIV-CysE5/pACYC-d0191F d0191 (plasmid) 71.3 ± 4.6

(9) Effect of a deficiency of d0191 on cysteine-producing ability in cysteine-producing bacterium



[0126] Since it became clear that d0191 participated in decomposition of cysteine, and enhancement of the gene reduced cysteine production, whether suppression of the activity of d0191 (specifically, deficiency of the gene) had a positive effect on the cysteine production was then examined. In order to examine whether the d0191-deficient cysteine-producing strain SC17 d0191::Kmr/pMIV-CysE5, prepared as described above had superior cysteine production ability as compared to the d0191-non-disrupted strain SC17/pMIV-CysE5, they were cultured for cysteine production, and the abilities thereof were compared. The methods for the cysteine production culture and quantification of cysteine were the same as those described in (11), except that, as for the antibiotics, only chloramphenicol was added to the medium. The experiment was performed in tetraplicate for the control strain, and in octaplicate for the d0191-deficient strain. Averages and standard deviations of the accumulated cysteine amounts are shown in Table 2. As shown in Table 2, it was revealed that suppression of the activity of d0191 had an effect of increasing accumulation amount of cysteine.
Table 2
StrainGene typeCys (mg/L)
SC17/pMIV-CysE5 Vector 202 ± 14
SC 17 d0191::Kmr/pMIV-CysE5 Δd0191 516 ± 46

[Explanation of Sequence Listing]



[0127] 

SEQ ID NO: 1: Nucleotide sequence of P. ananatis d0191 gene

SEQ ID NO: 2: Amino acid sequence of P. ananatis D0191

SEQ ID NO: 3: Nucleotide sequence of P. ananatis hisD gene

SEQ ID NOS: 4 to 32: PCR primers

SEQ ID NO: 33: Nucleotide sequence of Citrobacter koseri d0191 gene

SEQ ID NO: 34: Amino acid sequence of Citrobacter koseri D0191

SEQ ID NO: 35: Nucleotide sequence of Klebsiella pneumoniae d0191 gene

SEQ ID NO: 36: Amino acid sequence of Klebsiella pneumoniae D0191

SEQ ID NO: 37: Nucleotide sequence of Enterobacter sp. 638 d0191 gene

SEQ ID NO: 38: Amino acid sequence of Enterobacter sp. 638 D0191

SEQ ID NO: 39: Nucleotide sequence of Salmonella typhimurium d0191 gene

SEQ ID NO: 40: Amino acid sequence of Salmonella typhimurium D0191

SEQ ID NO: 41: Nucleotide sequence of Serratia proteamaculans d0191 gene

SEQ ID NO: 42: Amino acid sequence of Serratia proteamaculans D0191

SEQ ID NO: 43: Nucleotide sequence of Erwinia carotovora d0191 gene

SEQ ID NO: 44: Amino acid sequence of Erwinia carotovora D0191

SEQ ID NO: 45: Nucleotide sequence of Vibrio cholerae d0191 gene

SEQ ID NO: 46: Amino acid sequence of Vibrio cholerae D0191

SEQ ID NO: 47: Nucleotide sequence of Pseudomonas fluorescens d0191 gene

SEQ ID NO: 48: Amino acid sequence of Pseudomonas fluorescens D0191

SEQ ID NO: 49: Nucleotide sequence of Streptomyces coelicolor d0191 gene

SEQ ID NO: 50: Amino acid sequence of Streptomyces coelicolor D0191

SEQ ID NO: 51: Nucleotide sequence of Mycobacterium avium d0191 gene

SEQ ID NO: 52: Amino acid sequence of Mycobacterium avium D0191

SEQ ID NO: 53: Primer for attL amplification

SEQ ID NO: 54: Primer for attL amplification

SEQ ID NO: 55: Nucleotide sequence of attL

SEQ ID NO: 56: Primer for attR amplification

SEQ ID NO: 57: Primer for attR amplification

SEQ ID NO: 58: Nucleotide sequence of attR

SEQ ID NO: 59: Primer for amplification of DNA fragment containing bla gene

SEQ ID NO: 60: Primer for amplification of DNA fragment containing bla gene

SEQ ID NO: 61: Primer for amplification of DNA fragment containing ter_rrnB

SEQ ID NO: 62: Primer for amplification of DNA fragment containing ter_rrnB

SEQ ID NO: 63: Nucleotide sequence of the DNA fragment containing ter_thrL terminator

SEQ ID NO: 64: Primer for amplification of DNA fragment containing ter_thrL terminator

SEQ ID NO: 65: Primer for amplification of DNA fragment containing ter_thrL terminator

SEQ ID NO: 66: Nucleotide sequence of P. ananatis ybaO gene

SEQ ID NO: 67: Amino acid sequence of P. ananatis YbaO
































































Claims

1. A bacterium belonging to the family Enterobacteriaceae, which has L-cysteine-producing ability and has been modified to decrease the activity of the the protein selected from the group consisting of:

(A) a protein comprising the amino acid sequence of SEQ ID NO: 2, and

(B) a protein comprising the amino acid sequence of SEQ ID NO: 2, but which includes substitutions, deletions, insertions or additions of one or several amino acid residues, and having cysteine desulfhydrase activity.


 
2. The bacterium according to claim 1, wherein the activity of the protein is decreased by decreasing expression of a gene encoding the protein or by disrupting the gene.
 
3. The bacterium according to claim 1 or 2, wherein the gene is selected from the group consisting of:

(a) a DNA comprising the nucleotide sequence of SEQ ID NO: 1, and

(b) a DNA which is able to hybridize with a sequence complementary to the nucleotide sequence of SEQ ID NO: 1 or a probe prepared from the nucleotide sequence under stringent conditions, and encodes a protein having cysteine desulfhydrase activity.


 
4. The bacterium according to any one of claims 1 to 3, in which serine acetyltransferase has been mutated so that feedback inhibition by L-cysteine is attenuated.
 
5. The bacterium according to any one of claims 1 to 4, which is a Pantoea bacterium.
 
6. The bacterium according to claim 5, which is Pantoea ananatis.
 
7. A method for producing L-cysteine, L-cystine, a derivative thereof or a mixture thereof, which comprises culturing the bacterium according to any one of claims 1 to 6 in a medium and collecting L-cysteine, L-cystine, a derivative thereof or a mixture thereof from the medium.
 
8. The method according to claim 7, wherein the derivative of L-cysteine is a thiazolidine derivative.
 
9. A DNA encoding the protein selected from the group consisting of:

(A) a protein comprising the amino acid sequence of SEQ ID NO: 2, and

(B) a protein comprising the amino acid sequence of SEQ ID NO: 2, but which includes substitutions, deletions, insertions or additions of 1 or several amino acid residues, and having cysteine desulfhydrase activity.


 
10. The DNA according to claim 9, which is a DNA selected from the group consisting of:

(a) a DNA comprising the nucleotide sequence of SEQ ID NO: 1, and

(b) a DNA which is able to hybridize with a sequence complementary to the nucleotide sequence of SEQ ID NO: 1 or a probe prepared from the nucleotide sequence under stringent conditions, and encodes a protein having cysteine desulfhydrase activity.


 




Drawing











































REFERENCES CITED IN THE DESCRIPTION



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Patent documents cited in the description




Non-patent literature cited in the description