U.S. patent application number 11/070084 was filed with the patent office on 2008-03-27 for l-cysteine-producing microorganism and a method for producing l-cysteine.
Invention is credited to Hirotada Mori, Shigeru Nakamori, Hiroshi Takagi, Masaru Wada.
Application Number | 20080076163 11/070084 |
Document ID | / |
Family ID | 34747666 |
Filed Date | 2008-03-27 |
United States Patent
Application |
20080076163 |
Kind Code |
A1 |
Takagi; Hiroshi ; et
al. |
March 27, 2008 |
L-cysteine-producing microorganism and a method for producing
L-cysteine
Abstract
L-cysteine is produced by culturing an Escherichia bacterium
having L-cysteine producing ability and containing a gene encoding
an O-acetylserine sulphydrylase B or MalY regulatory protein that
is modified so that cysteine desulfhydrase activity is reduced or
eliminated. The bacterium is cultured in a medium to produce and
cause accumulation of L-cysteine in the medium, and collecting
L-cysteine from the medium.
Inventors: |
Takagi; Hiroshi; (Fukui-shi,
JP) ; Nakamori; Shigeru; (Fukui, JP) ; Wada;
Masaru; (Fukui, JP) ; Mori; Hirotada;
(Ikoma-shi, JP) |
Correspondence
Address: |
CERMAK & KENEALY LLP;ACS LLC
515 EAST BRADDOCK ROAD, SUITE B
ALEXANDRIA
VA
22314
US
|
Family ID: |
34747666 |
Appl. No.: |
11/070084 |
Filed: |
March 3, 2005 |
Current U.S.
Class: |
435/113 ;
435/252.33 |
Current CPC
Class: |
C12P 13/12 20130101 |
Class at
Publication: |
435/113 ;
435/252.33 |
International
Class: |
C12P 13/12 20060101
C12P013/12; C12N 1/21 20060101 C12N001/21 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 4, 2004 |
JP |
2004-060483 |
Claims
1. An Escherichia bacterium having L-cysteine-producing ability,
wherein said bacterium contains a gene encoding O-acetylserine
sulphydrylase B, and wherein said gene is modified so that cysteine
desulfhydrase activity is reduced or eliminated.
2. An Escherichia bacterium having L-cysteine producing ability,
wherein said bacterium contains a gene encoding MalY regulatory
protein, and wherein said gene is modified so that cysteine
desulfhydrase activity is reduced or eliminated.
3. The Escherichia bacterium according to claim 1, wherein said
gene encoding O-acetylserine sulphydrylase B is disrupted.
4. The Escherichia bacterium according to claim 2, wherein said
gene encoding MalY regulatory protein is disrupted.
5. The Escherichia bacterium according to claim 1, wherein activity
of an L-cysteine biosynthetic enzyme is enhanced.
6. The Escherichia bacterium according to claim 5, wherein said
L-cysteine biosynthetic enzyme is serine acetyltransferase.
7. The Escherichia bacterium according to claim 6, wherein said
serine acetyltransferase is resistant to feedback inhibition by
L-cysteine.
8. The Escherichia bacterium according to claim 1, wherein said
Escherichia bacterium is Escherichia coli.
9. A method of producing L-cysteine comprising culturing the
Escherichia bacterium according to claim 1 in a medium, and
collecting L-cysteine from the medium.
10. The Escherichia bacterium according to claim 2, wherein
activity of an L-cysteine biosynthetic enzyme is enhanced.
11. The Escherichia bacterium according to claim 2, wherein said
Escherichia bacterium is Escherichia coli.
12. A method of producing L-cysteine comprising culturing the
Escherichia bacterium according to claim 2 in a medium, and
collecting L-cysteine from the medium.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Technical field
[0002] 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.
[0003] 2. Background art
[0004] 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.
[0005] Furthermore, production of L-cysteine has also been
attempted by fermentation utilizing a microorganism. A method of
producing L-cysteine using a microorganism has been reported,
wherein said microorganism contains a DNA encoding serine
acetyltransferase (SAT) with a mutation which prevents feedback
inhibition by L-cysteine (WO 97/15673). A method of producing
L-cysteine using a strain of Escherichia coli which contains a gene
encoding SAT isozyme of Arabidopsis thaliana is disclosed in FEMS
Microbiol. Lett., vol. 179 (1999) p 453-459. This SAT isozyme gene
is resistant to feedback inhibition by L-cysteine. Also, a method
of producing L-cysteine using a microorganism which overexpresses a
gene encoding a protein that excretes an antibiotic or a toxic
substance is disclosed in JP11-56381A.
[0006] Furthermore, the inventors of the present invention have
disclosed a method of producing L-cysteine using a strain of
Escherichia coli which contains serine acetyltransferase with
reduced feedback inhibition by L-cysteine, and in which the
L-cysteine-decomposing system is attenuated (JP11-155571A). The
L-cysteine-decomposing system of the bacterium is attenuated by
reduction of the intracellular activity of cysteine desulfhydrase
(hereinafter, also referred to as "CD").
[0007] Enzymes which have been reported to have CD activity in
Escherichia coli include cystathionine-.beta.-lyase (metC gene
product, hereinafter, also referred to as "CBL") (Chandra et. al.,
Biochemistry, vol. 21 (1982) p 3064-3069) and tryptophanase (tnaA
gene product, hereinafter, also referred to as "TNase") (Austin
Newton, et al., J. Biol. Chem. vol. 240 (1965) p 1211-1218). A
method of producing L-cysteine using an Escherichia coli strain
which has reduced activities of cystathionine-.beta.-lyase and
tryptophanase is disclosed in JP2003-169668A (EP1,298,200).
However, no enzymes other than these have been previously reported
to have CD activity.
SUMMARY OF THE INVENTION
[0008] 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.
[0009] In order to attain the above-mentioned object, the inventors
of the present invention made extensive studies and as a result,
have found that the enzymes O-acetylserine sulphydrylase B (OASS-B)
and MalY regulatory protein (MalY) have CD activity in Escherichia
coli. The inventors also found that reducing CD activity by
modifying these genes leads to improvement in the production of
L-cysteine.
[0010] It is an object of the present invention to provide an
Escherichia bacterium having L-cysteine-producing ability, wherein
said bacterium contains a gene encoding O-acetylserine
sulphydrylase B, and wherein said gene is modified so that cysteine
desulfhydrase activity is reduced or eliminated.
[0011] It is a further object of the present invention to provide
an Escherichia bacterium having L-cysteine-producing ability,
wherein said bacterium contains a gene encoding MalY regulatory
protein, and wherein said gene is modified so that cysteine
desulfhydrase activity is reduced or eliminated.
[0012] It is a further object of the present invention to provide
an Escherichia bacterium as described above, wherein said gene
encoding O-acetylserine sulphydrylase B is disrupted.
[0013] It is a further object of the present invention to provide
an Escherichia bacterium as described above, wherein said gene
encoding MalY regulatory protein is disrupted.
[0014] It is a further object of the present invention to provide
an Escherichia bacterium as described above, wherein activity of an
L-cysteine biosynthetic enzyme is enhanced.
[0015] It is a further object of the present invention to provide
an Escherichia bacterium as described above, wherein said
L-cysteine biosynthetic enzyme is serine acetyltransferase.
[0016] It is a further object of the present invention to provide
an Escherichia bacterium as described above, wherein said serine
acetyltransferase is resistant to feedback inhibition by
L-cysteine.
[0017] It is a further object of the present invention to provide
an Escherichia bacterium as described above, wherein said
Escherichia bacterium is Escherichia coli.
[0018] It is a further object of the present invention to provide a
method of producing L-cysteine comprising culturing the Escherichia
bacterium as described above in a medium, and collecting L-cysteine
from the medium.
BRIEF DESCRIPTION OF THE DRAWINGS
[0019] FIG. 1 shows the results of CD activity staining of
Escherichia coli cell extracts on Native-PAGE.
[0020] FIG. 2 shows primers used in gene disruption.
[0021] FIG. 3 shows L-cysteine-producing ability of the control
strain and each CD gene-disrupted strain; JM39 ( ), JM39.DELTA.tnaA
(.box-solid.), JM39.DELTA.metC (.tangle-solidup.), JM39.DELTA.cysm
(*), JM39.DELTA.malY (+), and
JM39.DELTA.tnaA.DELTA.metC.DELTA.malY.DELTA.cysM(.quadrature.).
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0022] Hereinafter, the present invention will be explained in
detail. In the present invention, unless otherwise described,
L-cysteine refers to a reduced-type of L-cysteine, L-cystine, or a
mixture thereof.
[0023] The Escherichia bacterium of the present invention has
L-cysteine-producing ability and contains a gene encoding
O-acetylserine sulphydrylase B (OASS-B) or MalY regulatory protein,
wherein the gene is modified so that the cysteine desulfhydrase
(CD) activity of the bacterium is reduced or eliminated. The
Escherichia bacterium of the present invention may have L-cysteine
producing-ability and may contain both of the genes encoding OASS-B
and MalY regulatory protein which are modified so that the CD
activity of the bacterium is reduced or eliminated. In the
Escherichia bacterium of the present invention, one or both of the
genes encoding tryptophanase (TNase) and cystathionine-.beta.-lyase
(CBL) may also be modified so that the CD activity of the bacterium
is further reduced.
[0024] The term "L-cysteine-producing ability" as used herein
refers to an ability of the Escherichia bacterium of the present
invention to cause accumulation of L-cysteine in a culture medium
to such a degree that L-cysteine can be collected from the medium
when the bacterium is cultured in the medium. The
L-cysteine-producing ability may be imparted to a parent strain of
an Escherichia bacterium by a mutation technique or a recombinant
DNA technique. The recombinant DNA technique includes introduction
of a gene encoding an L-cysteine biosynthetic enzyme.
Alternatively, bacteria having native L-cysteine-producing ability
may also be used. Furthermore, a bacterium imparted with an
L-cysteine-producing ability by modification of a gene encoding
O-acetylserine sulphydrylase B (OASS-B) or MalY regulatory protein
may be used.
[0025] 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).
[0026] 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.
[0027] The method of obtaining the Escherichia bacteria of the
present invention will be explained in detail.
[0028] <1> Modification of a Gene Encoding OASS-B or MalY
Regulatory Protein
[0029] Examples of the methods of modifying a gene encoding OASS-B
or MalY regulatory protein so that the CD activity of the
Escherichia bacteria is reduced or eliminated include a mutation
treatment method and a gene disruption method. Examples of the
mutation treatment method include treating Escherichia bacteria
with ultraviolet ray irradiation or with a mutagen used in ordinary
mutation treatments, such as N-methyl-N'-nitro-N-nitrosoguanidine
(NTG) or nitrous acid, and selecting mutants which contain a
mutation reducing the CD activity in a gene encoding OASS-B or MalY
regulatory protein. To reduce or eliminate the CD activity of
OASS-B or MalY regulatory protein with high accuracy, it is
preferable to disrupt a gene encoding OASS-B or MalY regulatory
protein.
[0030] In Escherichia coli, OASS-B is encoded by the cysM gene, and
MalY regulatory protein is encoded by the malY gene. The nucleotide
sequences of these genes have been already reported (see for cysM;
GenBank accession M32101 (SEQ ID NO: 33), J. Bacteriol. 172 (6),
3351-3357 (1990), and for malY; GenBank accession M60722 (SEQ ID
NO: 35), J. Bacteriol. 173 (15), 4862-4876 (1991)). Accordingly,
DNA fragments which can be used to disrupt the genes can be
obtained by PCR using primers based on the nucleotide sequences
from a chromosomal DNA of Escherichia coli. More specifically, the
cysM gene deletion mutant (deletion-type cysM gene) and the malY
gene deletion mutant (deletion-type malY gene) can be obtained by
PCR using the primers shown in FIG. 2. DNA fragments for gene
disruption are not limited to those derived from Escherichia coli,
and may be DNAs derived from other organisms or synthetic DNAs as
long as they can cause homologous recombination with a chromosomal
DNA of a host bacterium. For example, DNAs having 80% or more,
preferably 90% or more, more preferably 95% or more homology to the
cysM gene or malY gene of Escherichia coli may be used. Homology of
the DNA sequences can be determined using the algorithm BLAST (Pro.
Natl. Acad. Sci. USA, 90, and 5873 (1993)) and FASTA (Methods
Enzymol., 183, and 63 (1990)) by Karlin and Altschul. The BLASTN
and BLASTX programs have been developed based on this algorithm
BLAST. (refer to http://www.ncbi.nlm.nih.gov). Furthermore, DNAs
able to hybridize with the cysM gene or malY gene of Escherichia
coli under stringent conditions may also be used. "Stringent
conditions" as used herein are conditions under which a so-called
specific hybrid is formed, and a non-specific hybrid is not formed.
It is difficult to clearly express this condition by using any
numerical value. However, examples of stringent conditions include,
those under which DNAs having high homology to each other, for
example, DNAs having a homology of not less than 50%, hybridize to
each other, and DNAs having homology lower than 50% do not
hybridize to each other, and those under which DNAs hybridize to
each other at a salt concentration with washing typical of Southern
hybridization, i.e., washing once or preferably 2-3 times under
1.times.SSC, 0.1% SDS at 60.degree. C., preferably 0.1.times.SSC,
0.1% SDS at 60.degree. C., more preferably 0.1.times.SSC, 0.1% SDS
at 68.degree. C.
[0031] Hereinafter, a method of disrupting the gene encoding OASS-B
will be explained. The gene encoding MalY regulatory protein can be
disrupted or mutated in a similar manner.
[0032] 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.
[0033] The gene disruption technique using homologous recombination
has already been established and examples thereof include using a
linear DNA or a plasmid containing a temperature-sensitive
replication origin. Examples of plasmids containing a
temperature-sensitive replication origin for Escherichia coli
include pMAN031 (Yasueda, H. et al., Appl. Microbiol. Biotechnol.,
36, 211 (1991)), pMAN997 (WO 99/03988), and pEL3 (K. A. Armstrong,
et al., J. Mol. Biol. (1984) 175, 331-347).
[0034] 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.
[0035] In the strain in which the recombinant DNA is incorporated
into the chromosomal DNA as described above, the deletion-type cysM
gene is recombined with the native cysM, and the two fusion genes
of the chromosomal cysM gene and the deletion-type cysM gene are
inserted into the chromosome so that the other portions of the
recombinant DNA (vector segment, temperature-sensitive replication
origin and drug resistance marker) are present between the two
fusion genes. Therefore, the transformant strain expresses normal
OASS-B because the normal cysM gene is dominant in this state.
[0036] Then, in order to leave only the deletion-type cysM gene on
the chromosomal DNA, one copy of the cysM gene is eliminated along
with the vector segment (including the temperature-sensitive
replication origin and the drug resistance marker) from the
chromosomal DNA by recombination of two of the cysM genes. In this
case, the normal cysM gene is left on the chromosomal DNA and the
deletion-type cysM gene is excised from the chromosomal DNA, or to
the contrary, the deletion-type cysM gene is left on the
chromosomal DNA and the normal cysM gene is excised from the
chromosomal DNA. In both cases, the excised DNA may be harbored in
the cell as a plasmid when the cell is cultured at a temperature
which allows the temperature-sensitive replication origin to
function. Subsequently, if the cell is cultured at a temperature
which does not allow the temperature-sensitive replication origin
to function, the cysM gene on the plasmid is eliminated with the
plasmid from the cell. Then, a strain having the disrupted cysM
gene left in the chromosome can be selected by PCR, Southern
hybridization, or the like.
[0037] 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.
[0038] The bacteria of the present invention may be strains in
which one or both of the genes encoding tryptophanase (TNase) and
cystathionine-.beta.-lyase (CBL) are modified so that CD activity
of the strain is further reduced. The method of modifying those
genes (tnaA gene or metC gene) is disclosed in detail in JP-A
2003-169668 (EP1,298,200).
[0039] <2> Enhancing L-Cysteine Biosynthetic Enzyme
Activity
[0040] L-cysteine-producing ability may be imparted to a bacterium
by enhancing an activity of an L-cysteine biosynthetic enzyme.
Enhancing an L-cysteine biosynthetic enzyme can be performed by
enhancing, for example, an activity of serine acetyltransferase
(SAT). Enhancing the SAT activity in cells of an Escherichia
bacterium can be attained by increasing a copy number of a SAT
gene. For example, a recombinant DNA can be prepared by ligating a
gene fragment encoding SAT to a vector that functions in
Escherichia bacteria, preferably a multi-copy type vector, and
transforming a host Escherichia bacterium with the vector.
[0041] The SAT gene of the present invention may be derived from
Escherichia bacteria or from any other organism. The cysE SAT gene
has been cloned from a wild-type Escherichia coli strain and an
L-cysteine-secretion mutant strain, and the nucleotide sequence has
been elucidated (Denk, D. and Boeck, A., J. General Microbiol.,
133, 515-525 (1987)). Therefore, a SAT gene can be obtained by PCR
utilizing primers based on the nucleotide sequence (SEQ ID NO: 31)
from a chromosomal DNA of Escherichia bacterium (see JP11-155571A).
Genes encoding SAT derived from other microorganisms can also be
obtained in a similar manner. The SAT gene may be able to hybridize
to a DNA having the nucleotide sequence of SEQ ID NO: 31 under
stringent conditions, and also may encode a protein having SAT
activity, which catalyzes the activation of L-serine by
acetyl-CoA.
[0042] 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).
[0043] In order to introduce the PCR-amplified DNA fragment
containing a SAT gene into an Escherichia bacterium, vectors
typically used for protein expression can be used. Examples of such
vectors include pUC19, pUC18, pHSG299, pHSG399, pHSG398, RSF1010,
pBR322, pACYC184, pMW219, and so forth.
[0044] Introduction of a recombinant vector containing the SAT gene
into Escherichia bacterium can be attained by methods typically
used for transformation of Escherichia bacteria, for example, the
method of D. A. Morrison (Methods in Enzymology, 68, 326 (1979)), a
method of treating recipient cells with calcium chloride so as to
increase the permeability for DNA (Mandel, M. and Higa, A., J. Mol.
Biol., 53, 159 (1970)), and so forth.
[0045] Increasing a copy number of the SAT gene can also be
achieved by introducing multiple copies the gene into the
chromosomal DNA of an Escherichia bacterium. To introduce multiple
copies of the SAT gene into the chromosomal DNA of an Escherichia
bacterium, homologous recombination may be carried out by targeting
a sequence which exists on a chromosomal DNA in multiple copies. As
sequences which exist on a chromosomal DNA in multi-copies,
repetitive DNA or an inverted repeat which exists at the ends of a
transposable element can be used. Furthermore, as disclosed in
J2-109985A, it is also possible to incorporate a SAT gene into a
transposon, and allow it to be transferred so that multiple copies
of the gene are introduced into the chromosomal DNA.
[0046] Besides the aforementioned gene amplification technique,
amplification of the SAT activity can also be attained by replacing
an expression regulatory sequence such as a promoter of the SAT
gene on a chromosomal DNA or on a plasmid with a stronger one
(JP1-215280A). For example, lac promoter, trp promoter, trc
promoter, and so forth are known as strong promoters. Substitution
of an expression regulatory sequence can also be attained by, for
example, gene substitution utilizing a temperature-sensitive
plasmid.
[0047] Furthermore, it is also possible to substitute several
nucleotides in the promoter region of the SAT gene, resulting in
modification of the promoter to make it stronger as disclosed in
WO00/18935. Expression of the SAT gene is enhanced by such
substitution or modification of a promoter, and thereby the SAT
activity is enhanced. These modifications of expression regulatory
sequence may be combined with the increase of a copy number of SAT
gene.
[0048] Furthermore, when a suppression mechanism exists for SAT
gene expression, enhancing the expression can also be enhanced by
modifying an expression regulatory sequence or a gene involved in
the suppression so to eliminate or reduce the suppression.
[0049] The intracellular SAT activity of an Escherichia bacterium
can also be increased by modifying an Escherichia bacterium to
harbor SAT which has reduced or eliminated feedback inhibition by
L-cysteine (henceforth also referred to as "mutant-type SAT").
Examples of the mutant-type SAT include SAT having a mutation
replacing the methionine at a position 256 of wild-type SAT (SEQ ID
32) with an amino acid other than lysine and leucine, or a mutation
deleting a C-terminal region of SAT from the methionine at a
position 256 and thereafter. Examples of the amino acid other than
lysine and leucine include the 17 kinds of amino acid residues
which constitute ordinary proteins with the exceptions of
methionine, lysine, and leucine. Preferably, isoleucine can be
mentioned. A site-specific mutagenesis technique can be used to
introduce a desired mutation into a wild-type SAT gene. As a
mutant-type SAT gene, a mutant-type cysE encoding a mutant-type SAT
of Escherichia coli is known (WO97/15673 and JP11-155571A).
Escherichia coli JM39-8 strain harboring plasmid pCEM256E, which
contains a mutant-type cysE encoding a mutant-type SAT in which the
methionine at a position 256 is replaced with glutamic acid (E.
coli JM39-8(pCEM256E), private number: AJ13391), has been deposited
at the National Institute of Bioscience and Human-Technology,
Agency of Industrial Science and Technology (currently, National
Institute of Advanced Industrial Science and Technology,
International Patent Organism Depositary, Central 6, 1-1, Higashi
1-Chome, Tsukuba-shi, Ibaraki-ken, 305-8566, Japan) on Nov. 20,
1997 under the accession number of FERM P-16527. The original
deposit was converted to an international deposit in accordance
with the Budapest Treaty on Jul. 8, 2002, and given the accession
number of FERM BP-8112.
[0050] Furthermore, an Escherichia bacterium can be modified to
contain a mutant-type SAT by introducing a mutation into a
chromosomal SAT gene which prevents feedback inhibition by
L-cysteine. The mutation can be introduced by ultraviolet
irradiation or a mutagenizing agent used for usual mutagenesis
treatment such as N-methyl-N'-nitro-N-nitrosoguanidine (NTG) or
nitrous acid.
[0051] 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.
[0052] <3> Production of L-Cysteine
[0053] L-Cysteine can be efficiently produced by culturing the
Escherichia bacterium of the present invention obtained as
described above in a suitable medium to cause accumulation of
L-cysteine in the culture medium, and collecting the L-cysteine
from the culture medium. Although L-cysteine produced by the method
of the present invention may contain cystine in addition to
reduced-type cysteine, the target substances produced by the method
of the present invention include cystine and a mixture of
reduced-type cysteine and cystine.
[0054] As culture media, ordinary media containing a carbon source,
nitrogen source, sulfur source, inorganic ions, and other organic
components, if required, can be used. As carbon sources,
saccharides such as glucose, fructose, sucrose, molasses, and
starch hydrolysate, organic acids such as fumaric acid, citric acid
and succinic acid can be used. As nitrogen sources, inorganic
ammonium salts such as ammonium sulfate, ammonium chloride and
ammonium phosphate, organic nitrogen such as soybean hydrolysate,
ammonia gas, aqueous ammonia, and so forth can be used. As sulfur
sources, inorganic sulfur compounds, such as sulfates, sulfites,
sulfides, hyposulfites, and thiosulfates can be used. As organic
trace amount nutrients, it is desirable to add required substances
such as vitamin B1, yeast extract, and so forth in appropriate
amounts. In addition to these components, potassium phosphate,
magnesium sulfate, iron ions, manganese ions, and so forth may be
added in small amounts if required.
[0055] The culture is preferably performed under aerobic conditions
for 30 to 90 hours. The culture temperature is preferably
controlled at 25.degree. C. to 37.degree. C., and pH is preferably
controlled at 5 to 8 during the culture. To adjust the pH,
inorganic or organic, acidic or alkaline substances, ammonia gas,
and so forth can be used. Collecting L-cysteine from the culture
medium can be attained by, for example, an ordinary ion exchange
resin method, precipitation, and other known methods, or
combinations thereof.
EXAMPLES
[0056] Hereinafter, the present invention will be explained in
detail by the following non-limiting examples.
[0057] Strains
[0058] cysE-deficient Escherichia coli JM39 (F+ cysE51 tfr-8)
(Denk, D. and Bock, A., J. Gene. Microbiol., 133, 515-525 (1987))
was used to identify a gene encoding a protein having CD
activity.
[0059] To evaluate L-cysteine productivity of the CD-gene-disrupted
strains, the following strains were used: JM39.DELTA.tnaA,
JM39.DELTA.metC, JM39.DELTA.cysM, JM39.DELTA.malY, and
JM39.DELTA.cysK as a single-CD-gene-disrupted strain;
JM39.DELTA.tnaA.DELTA.metC and JM39.DELTA.cysK.DELTA.cysM as a
double-CD-gene-disrupted strain;
JM39.DELTA.tnaA.DELTA.metC.DELTA.cysM.DELTA.malY as a
quadruple-CD-gene-disrupted strain; and
JM39.DELTA.tnaA.DELTA.metC.DELTA.cysK.DELTA.cysM.DELTA.malY as a
quintuple-CD-gene-disrupted strain. In the production of
L-cysteine, a total of six strains, including JM39,
single-CD-gene-disrupted strains of JM39.DELTA.tnaA,
JM39.DELTA.metC, JM39.DELTA.cysM, and JM39.DELTA.malY, and
quadruple-CD-gene-disrupted strain
JM39.DELTA.tnaA.DELTA.metC.DELTA.cysM.DELTA.malY, all of which
harbors pEAS-m, a plasmid containing SAT gene of Arabidopsis
thaliana (FEMS Microbiol. Lett., 179 (1999) 453-459) were used.
[0060] Plasmids
[0061] A plasmid library containing 4,388 kinds of genes (whole ORF
fragments) of E. coli was used to identify a gene encoding a
protein having CD activity (4,388 kinds of plasmids were
respectively dispensed into the wells of forty eight 96-well
plates). The plasmid library covers all of the 4,388 kinds of ORF
fragments of E. coli located downstream to the lac promoter in the
pCA24N vector and the expression of each ORF is induced by IPTG.
For gene disruption, plasmid pEL3 (K. A. Armstrong et al., J. Mol.
Biol. (1984) 175, 331-347) was used to construct pEL3gdtnaA,
pEL3gdmetC, pEL3gdcysM, pEL3gdcysK, and pEL3gdmalY. The
construction of the plasmids will be described below.
[0062] Culture Media
[0063] For transformation and culture of E. coli, LB medium was
used as a complete medium, and M9 medium (6 g/L Na.sub.2HPO.sub.4,
3 g/L KH.sub.2PO.sub.4, 0.5 g/L NaCl, 0.25 g/L MgSO.sub.4.7H.sub.2,
0.015 mg/L CaCl.sub.2.4H.sub.2O, 4 g/L glucose, and 0.001 g/L
thiamine hydrochloride) was used as a minimum medium. Ampicillin
(Amp) was added if necessary. In some experiments, LB liquid medium
to which 10 to 30 mM cysteine was added was used. Unless otherwise
described, the culture was performed at 37.degree. C. For the
culture of cysteine production (30 g/L glucose, 10 g/L NH.sub.4Cl,
2 g/L KH.sub.2PO.sub.4, 1 g/L MgSO.sub.4.7H.sub.2O, 10 mg/L
FeSO.sub.4.7H.sub.2O, 10 mg/L MnCl.sub.2.4H.sub.2O, and 20 g/L
CaCO.sub.3) sodium thiosulfate was added to the culture. The same
medium was used to determine the quantity of cysteine.
[0064] Preparation of Cell Extract
[0065] 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).
[0066] Composition of Native-PAGE gel and procedure of Native-PAGE
(Polyacrylamide gel electrophoresis under undenatured
conditions)
[0067] Since it was necessary to separate proteins in the cell
extract under an undenatured state, Native-PAGE gel containing no
SDS was prepared for the purpose of identifying and ascertaining a
protein having CD activity, confirming the construction of the
CD-gene-disrupted strains, and so on, by CD activity staining
described hereinbelow. The composition of the Native-PAGE gel for
three gel sheets was 6.4 ml of Acrylamide/Bisacrylamide/amide
(37:5:1), 6.7 ml of 1 M Tris-HCl (pH 8.7), 6.8 ml of dH.sub.2O, 100
.mu.l of 10% APS (Ammonium persulfate), and 10 .mu.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.sub.2O, 100 .mu.l of 10% APS, and 10 .mu.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.sub.2O, 30 .mu.l of 10% APS, and 5 .mu.l of
TEMED. The Native-PAGE was performed using a mini-slab
electrophoretic apparatus (AEV-6500, manufactured by ATTO), and a
mixture of 30 .mu.g to 50 .mu.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.
[0068] CD Activity Staining
[0069] 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.sub.3 (bismuth
chloride), and 200 .mu.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.sub.2S at
the site where a protein having CD activity separated with
Native-PAGE exists on the gel. The generated H.sub.2S reacts with
bismuth chloride (BiCl.sub.3) contained in the CD activity staining
solution to form bismuth sulfide (Bi.sub.2S.sub.3), which exhibits
a black color band.
[0070] Identification of a Gene Encoding a Protein Having CD
Activity using a Plasmid Library Containing E. Coli Whole Genes
[0071] The forty-eight 96-well plates on which respective plasmids
were dispensed were grouped into 5 plates such as 1 to 5, 6 to 10 .
. . , and nine kinds of mixed plasmid solutions obtained from five
plates (each containing 480 kinds of plasmids) were prepared. The
mixed plasmid solutions were used to transform JM39 strains and
about 10,000 colonies of transformants were stocked in glycerol.
The nine kinds of glycerol-stock solutions were inoculated into LB
medium containing chloramphenicol (Cm) and 0.01 mM IPTG and
cultured. Then, cell extract was prepared and subjected to
Native-PAGE. CD activity staining was performed to detect which
mixed plasmid solution contained a candidate gene encoding a
protein having CD activity. The population containing a candidate
gene presumed to encode a protein having CD activity was downsized
to a population of 480 kinds of plasmids, and then, further
downsizing of the population to that of 96 kinds of plasmids was
performed. 480 kinds of the plasmids were divided into five groups
of 96 to prepare five kinds of mixed plasmid solutions. JM39
strains were transformed with the mixed plasmid solutions and about
6,000 colonies of the transformants were stocked in glycerol.
Thereafter, the transformants were cultured and CD activity
staining was performed to confirm if the mixed plasmid solution
contains a candidate gene encoding a protein having CD activity.
After the population containing a candidate gene presumed to encode
a protein having CD activity was downsized to 96 kinds of plasmids,
the population was further reduced to 8 kinds of plasmids. Finally,
eight proteins were each expressed from the 8 kinds of plasmids and
CD activity staining was performed to confirm if they are the
target protein having CD activity.
[0072] Construction of Plasmids for CD Gene Disruption
[0073] To disrupt each CD gene, five kinds of plasmids for gene
disruption, i.e., pEL3gdtnaA, pEL3gdmetC, pEL3gdcysM, pEL3gdcysK,
and pEL3dgmalY were constructed using plasmid pEL3 having a
temperature-sensitive replication origin. The preparation methods
for these plasmids are described below. That is, using the genome
of E. coli JM39 as a template, two kinds of 300 to 700 bp DNA
fragments each covering a part of the respective CD gene was
amplified by PCR. The DNA fragments were designated homologous
region DNA fragments-A and -B, respectively. The primers used are
described in FIG. 2. For the amplification of the homologous region
DNA fragment-A, CD gene disruption primers-1 and -2 were used, and
for the amplification of the homologous region DNA fragment-B, CD
gene disruption primers-3 and -4 were used. These primers had a
restriction enzyme recognition site at the 5'-side so that the
amplified homologous region DNA fragments contain restriction
enzyme recognition sites at both ends. After treatment with
appropriate restriction enzymes of both fragments A and B (KpnI,
HindIII, or EcoRI), both the enzyme-treated fragments were ligated
to each other to form a template for preparing CD gene disruption
fragments. The CD gene disruption fragments were prepared in large
amounts by PCR using the CD gene disruption primers-1 and -4. The
disruption fragments and pEL3 were treated with the restriction
enzyme BamHI and ligated to each other to construct the CD gene
disruption plasmids. The construction was confirmed by DNA
sequencing.
[0074] Disruption of CD Gene
[0075] A CD gene-disrupted strain was constructed from E. coli JM39
strain with the disruption plasmid as described in section 1-9.
First, disruption plasmids were introduced into JM39 to obtain
transformants. The limiting temperature for temperature-sensitive
plasmid pEL3 is 42.degree. C. Alternatively, the non-limiting
temperature, a temperature not higher than the limiting
temperature, for the plasmid is generally 37.degree. C., which is
an ordinary culture temperature for E. coli. However, the culture
was performed at 30.degree. C. in this experiment to ensure the
temperature sensitivity of the plasmid. Then, after each
transformant was cultured overnight at 30.degree. C. in an LB+Amp
medium, the culture broth was diluted to 10.sup.3-fold, and 200
.mu.l of the diluted solution was spread on the LB+Amp plate.
Culture was performed at 42.degree. C., which is the temperature at
which the plasmid becomes unreplicable and the growth of the
transformants is inhibited by Amp, and therefore no colonies form.
Thereby, homologous recombination occurred between each disrupted
fragment on the plasmid with suppressed replication and a
homologous region on the chromosome of the JM39 strain. This
allowed the whole length of the disruption plasmid to be
incorporated into the chromosome. Then, the recombinant strain was
selected which was able to form an Amp resistant colony by
incorporation of the disruption plasmid. The incorporation of the
disruption plasmid into the chromosome was confirmed by PCR using
FW and RV of each CD gene disruption primer as described in FIG. 2.
The colony having a confirmed disruption plasmid incorporated into
the chromosome was cultured in an LB liquid medium to cause further
homologous recombination. This was done so that the disrupted
fragment remains on the chromosome and the fragment containing a
plasmid sequence and a chromosomal gene is removed. The
transformants were subcultured several times in an LB liquid
medium. Then, the culture broth was spread on an LB agar medium
after dilution to a concentration that would cause 200 to 300
colonies to form on the LB agar medium. The colonies were
replicated on an LB plate and an LB+Amp agar plate to select for
Amp-sensitive colonies. By performing colony PCR using FW and RV
primers, CD-gene-disrupted strains having only the disrupted
fragment on the chromosome were selected.
[0076] The CD-gene-disrupted strains were subjected to CD activity
staining and disappearance of the CD activity due to gene
disruption was confirmed. A multiple CD-gene-disrupted strain was
constructed by repeating the operation of disrupting the target CD
genes.
[0077] Measurement of Total CD Activity (sulfide/H.sub.2S
Quantification)
[0078] The total CD activity in the cell extract was measured by
determining the amount of hydrogen sulfide (H.sub.2S) generated by
degradation of cysteine by CD. A strain was cultured in 5 ml of LB
medium and 5 ml of LB+10 mM cysteine medium at 37.degree. C.
overnight, and then the cell extract was prepared as in the section
2-2-4. The composition of the buffer used for measuring the CD
activity was 100 mM Tris-HCl (pH 8.6), 100 .mu.M DTT, 10 mM PLP, 2
.mu.M L-cysteine. 10 ml of the cell extract was added to 1 ml of
the buffer and the reaction was carried out at 30.degree. C. for 10
minutes. A standard curve was prepared by adding 10 .mu.l aliquots
of water, or 10 .mu.l of 0.1 mM, 0.2 mM, or 2 mM of Na.sub.2S 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.sub.3 (in 1.2 N HCl) were added, vigorously
mixed, and left to stand in the dark for 15 minutes. Iron chloride
acts as an oxidizing agent under acidic conditions adjusted by
hydrochloric acid, and the N,N-dimethyl-p-phenyldiamine sulfate
reacts with a sulfide in the sample to form a thiazine dye. As a
result, Methylene Blue exhibits a greenish blue or blue color. The
mixture was left to stand for 15 minutes, then, OD650 of the
reaction mixture was measured and the activity was calculated by
defining an amount of enzyme giving 1 .mu.mol H.sub.2S as 1U.
[0079] Cysteine Production Culture
[0080] 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.degree.
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)).
[0081] 2. Results
[0082] 2-1. Confirmation of Existence of a Protein having CD
Activity in E. coli
[0083] To confirm the existence of a protein having CD activity in
E. coli, a cell extract of JM39 strain was prepared and subjected
to Native-PAGE, and electrophoresis was performed for about 2 hours
to separate proteins, which was then subjected to activity
staining. FIG. 1 shows the results. Five bands exhibiting CD
activity were detected. This experiment indicates that at least
five kinds of proteins having CD activity are present in E. coli.
Of those, two were identified as tryptophanase (TNase) and
cystathionine-.beta.-lyase (CBL) by amino acid sequencing analysis
(JP 2003-169668A). To identify the remaining three, the following
experiments were performed.
[0084] 2-3. Identification of the Unidentified CD Proteins using E.
coli Total Gene Plasmid Library
[0085] The genome of E. coli is presumed to have a total of 4,388
genes (ORF). Using the E. coli whole ORF library in which all ORFs
were inserted into each plasmid, the operation for identification
of a protein having a CD activity was repeated by the procedure
described in the section 1-7. By detecting the band of the
unidentified CD protein by CD activity staining, the population of
plasmids containing a gene encoding an unidentified CD protein was
reduced from 4,388 kinds to 480 kinds, 96 kinds, and 8 kinds,
sequentially. Finally, the selected 8 kinds of plasmids were
analyzed and the proteins encoded by the cysM gene, cysK gene, and
malY gene were found to be the unidentified CD proteins. The cysM
gene of E. coli has been reported to encode O-acetyl L-serine
sulphydrylase-B (OASS-B) (see J. Bacteriol. 172 (6), 3351-3357
(19890)). The cysK has been reported to encode O-acetyl L-serine
sulphydrylase (OASS-A) (Mol. Microbiol. 2 (6), 777-783 (1988)).
Furthermore, it has been reported that the malY gene encodes a MalY
protein which is a regulatory factor for maltose metabolism pathway
gene group and has a conformation close to that of CBL and
catalyzes the C-S lyase reaction (EMBO J. 2000, March;
19(5):831-842).
[0086] 2-4. Confirmation of CD Activity
[0087] The OASS-B, OASS-A, and MalY identified in section 2-3 were
confirmed to have the CD activity by overexpressing the genes in
the JM39 strain. That is, when the respective genes were
overexpressed and protein bands of cell extract were analyzed by CD
activity staining, the stained band was denser than the band of the
control JM strain, indicating that each gene encodes a protein
having CD activity.
[0088] 2-5. Construction of CD-Gene-Disrupted Strain
[0089] Then, each CD-gene-disrupted strain was constructed. Methods
of preparing JM39.DELTA.tnaA and JM39.DELTA.metC strains are
disclosed in JP 2003-169668A. First, disruption plasmids
pEL3gdtnaA, pEL3gdmetC, pEL3gdcysM, pEL3gdcysK, and pEL3gdmalY for
disrupting tnaA, metC, cysM, cysK, and malY, respectively, were
constructed and introduced into the JM39 strain to construct
single-disrupted strains by homologous recombination. Furthermore,
the gene disruption step was repeated to prepare multiple-disrupted
strains, such as a quadruple disrupted strain
JM39.DELTA.tnaA.DELTA.metC.DELTA.cysM.DELTA.malY in which tnaA,
metC, cysM, and malY were disrupted. After the operation of gene
disruption, gene disruption was confirmed based on the length of
the DNA fragment amplified by colony PCR. Furthermore, it was
confirmed by CD activity staining that the CD activity of a protein
encoded by each gene was eliminated due to gene disruption.
[0090] 2-6. Measurement of Total CD Activity
[0091] According to the method in the section 1-11, the total CD
activities of all the CD-gene-disrupted strains used in this
experiment were measured. The results are shown in Table 1. As a
result, comparison of the total CD activity of each strain cultured
in LB medium with that of the parent strain JM39 indicated a
decrease in the CD activity for all the disrupted strains.
Comparison of the activity of the multiple-disrupted strain with
the activity of JM39 indicated a considerable decrease in the CD
activity except for JM39.DELTA.cysK.DELTA.cysM. The decrease in the
CD activity in multi-disrupted strains was significant as compared
with the decrease in the activity in each single-disrupted strain.
The activity of JM39.DELTA.cysK.DELTA.cysM decreased as compared
with the activities of single-disrupted strains. Then, the total CD
activity of the CD-gene-disrupted strain cultured in a medium to
which cysteine was added was analyzed. In the strains other than
JM39.DELTA.tnaA, the CD activity of the strain cultured in a
cysteine-containing medium increased considerably as compared with
the CD activity of the same strain cultured in an LB medium.
TABLE-US-00001 TABLE 1 total CD activity (mU/mg) medium LB + 10 mM
Strain LB L-cysteine JM39 20.6 .+-. 0 27.6 .+-. 0 JM39.DELTA.tnaA
15.7 .+-. 0 14.1 .+-. 0 JM39.DELTA.metC 15.0 .+-. 0 27.6 .+-. 0.46
JM39.DELTA.cysK 18.2 .+-. 0.52 29.9 .+-. 0 JM39.DELTA.cysM 17.9
.+-. 0.46 27.8 .+-. 0 JM39.DELTA.malY 15.3 .+-. 0 27.1 .+-. 0.46
JM39.DELTA.tnaADmetC 9.6 .+-. 0 16.2 .+-. 0
JM39.DELTA.cysK.DELTA.cysM 17.2 .+-. 0.58 27.0 .+-. 0
JM39.DELTA.tnaADmetC.DELTA.cysM.DELTA.malY 9.1 .+-. 0.46 19.6 .+-.
0 JM39.DELTA.tnaADmetC.DELTA.cysK.DELTA.cysM.DELTA.malY 8.7 .+-.
0.46 11.5 .+-. 0
[0092] 2-7. Cysteine Production using CD-Gene-Disrupted Strains
[0093] pEAS-m, a plasmid containing SAT-m gene of A. thaliana, was
introduced into a total of six strains, i.e., a control JM39
strain, four single-CD-gene-disrupted strains of JM39.DELTA.tnaA,
JM39.DELTA.metC, JM39.DELTA.cysM, and JM39.DELTA.malY, and a
quadruple-CD-gene-disrupted strain of
JM39.DELTA.tnaA.DELTA.metC.DELTA.malY.DELTA.cysM, and the
transformants were used for the production of cysteine. Cysteine
production culture was performed according to the method in the
section 1-12 and the amount of produced cysteine was quantified.
Time courses of the amounts of produced cysteine of the control
strain and each of the CD-gene-disrupted strain per growth (growth:
value of OD.sub.562) are shown in Table 2 and FIG. 3. The growth
decreased slightly in the case of JM39.DELTA.tnaA but the growth of
other disrupted strains was substantially the same as that of the
control strain JM39.
TABLE-US-00002 TABLE 2 L-Cys (mg/L) hr Strain 24 48 72 96 JM39 416
.+-. 96 720 .+-. 161 587 .+-. 164 415 .+-. 111 JM39.DELTA.tnaA 1206
.+-. 26 1195 .+-. 95 1287 .+-. 74 1077 .+-. 124 JM39.DELTA.metC
1408 .+-. 98 930 .+-. 47 1243 .+-. 101 853 .+-. 13 JM39.DELTA.malY
1291 .+-. 95 1213 .+-. 93 1359 .+-. 87 1256 .+-. 75 JM39.DELTA.cysM
1369 .+-. 67 1123 .+-. 148 1100 .+-. 66 840 .+-. 56
JM39.DELTA.tnaA.DELTA.metC.DELTA.malY.DELTA.cysM 1291 .+-. 21 1117
.+-. 21 1080 .+-. 13 847 .+-. 69
[0094] The L-cysteine production of the respective gene-disrupted
strains exceeded the value of the control strain JM39. Therefore,
the disruption of the CD genes to inhibit the CD activity is
effective for increasing the production of cysteine. When cysK
gene-disrupted strains were used, almost no cysteine could be
obtained when a cysteine production C1 medium containing sodium
thiosulfate was used.
INDUSTRIAL APPLICABILITY
[0095] By using the bacteria of the present invention, L-cysteine
can be produced efficiently. L-cysteine and its derivatives are
useful in the fields of medicine, cosmetics, foods, and the
like.
[0096] While the invention has been described in detail with
reference to preferred embodiments thereof, it will be apparent to
one skilled in the art that various changes can be made, and
equivalents employed, without departing from the scope of the
invention. Each of the aforementioned documents, including the
foreign priority documents, Japanese Patent No. 2004-060483 filed
on Mar. 4, 2004, is incorporated by reference herein in its
entirety.
Sequence CWU 1
1
36126DNAArtificialprimer 1cgcggatcca agccgcattc tgactg
26227DNAArtificialprimer 2cccaagcttc tgactcgggc taacgca
27326DNAArtificialprimer 3cccaagcttg ccggtttcac tggcaa
26427DNAArtificialprimer 4ctatggatcc ttatagccac tctgtag
27527DNAArtificialprimer 5ctatggatcc ttatagccac tctgtag
27622DNAArtificialprimer 6caccggggaa tttacttcag ac
22729DNAArtificialprimer 7cgcggatcca acagagcttc tgcgatacc
29829DNAArtificialprimer 8cggggtacca ctagcatgaa tattcgcgg
29931DNAArtificialprimer 9cggggtacct accgcctata tataaccagc c
311019DNAArtificialprimer 10aatatgagga tccgccagc
191121DNAArtificialprimer 11gttatagata acgaccgcag g
211220DNAArtificialprimer 12cgcccctgaa tataacttag
201337DNAArtificialprimer 13gcggcgggat cctaggttga gtgaatgtta
aacgccc 371437DNAArtificialprimer 14ggggggaagc ttggtgttac
cactggtggc ttcgatt 371537DNAArtificialprimer 15ggggggaagc
ttaatattct gtggcgtcag ctccggc 371636DNAArtificialprimer
16gcggcgggat ccatactgca tttgtcggca gcaaca 361725DNAArtificialprimer
17aacccgcgat gaggaacttg ctctc 251825DNAArtificialprimer
18ttcaatgacc ttacggcgtt tcctc 251937DNAArtificialprimer
19cgccgcggat cccaatctac cggttatttt gataacc
372037DNAArtificialprimer 20cggcggggta ccttttcggc atcccaaatc
atgttgg 372137DNAArtificialprimer 21gccgccggta ccattaaacc
tggcccgcat aaaattc 372237DNAArtificialprimer 22cgccgcggat
cccaagctgg cattactgtt gcaattc 372324DNAArtificialprimer
23ctatcgcgat aaacacgcga tgtg 242423DNAArtificialprimer 24ggcgaaagtt
tgaagcaggc cac 232528DNAArtificialprimer 25atccagtcga tgatcgatac
cgggatcc 282626DNAArtificialprimer 26ggcgctacga acaggaacag gaattc
262735DNAArtificialprimer 27ggccgaattc cgtcatggtg tgcgggttat ttccg
352835DNAArtificialprimer 28cgcgggatcc ttaacgaaca gcgcggatgg cgtta
352925DNAArtificialprimer 29ttctgaaagc caataacatc cagag
253025DNAArtificialprimer 30ggtaaaaatc cacgattgcg caacg
25311134DNAEscherichia coliCDS(223)..(1044) 31tccgcgaact ggcgcatcgc
ttcggcgttg aaatgccaat aaccgaggaa atttatcaag 60tattatattg cggaaaaaac
gcgcgcgagg cagcattgac tttactaggt cgtgcacgca 120aggacgagcg
cagcagccac taaccccagg gaacctttgt taccgctatg acccggcccg
180cgcagaacgg gccggtcatt atctcatcgt gtggagtaag ca atg tcg tgt gaa
234 Met Ser Cys Glu 1gaa ctg gaa att gtc tgg aac aat att aaa gcc
gaa gcc aga acg ctg 282Glu Leu Glu Ile Val Trp Asn Asn Ile Lys Ala
Glu Ala Arg Thr Leu5 10 15 20gcg gac tgt gag cca atg ctg gcc agt
ttt tac cac gcg acg cta ctc 330Ala Asp Cys Glu Pro Met Leu Ala Ser
Phe Tyr His Ala Thr Leu Leu 25 30 35aag cac gaa aac ctt ggc agt gca
ctg agc tac atg ctg gcg aac aag 378Lys His Glu Asn Leu Gly Ser Ala
Leu Ser Tyr Met Leu Ala Asn Lys 40 45 50ctg tca tcg cca att atg cct
gct att gct atc cgt gaa gtg gtg gaa 426Leu Ser Ser Pro Ile Met Pro
Ala Ile Ala Ile Arg Glu Val Val Glu 55 60 65gaa gcc tac gcc gct gac
ccg gaa atg atc gcc tct gcg gcc tgt gat 474Glu Ala Tyr Ala Ala Asp
Pro Glu Met Ile Ala Ser Ala Ala Cys Asp 70 75 80att cag gcg gtg cgt
acc cgc gac ccg gca gtc gat aaa tac tca acc 522Ile Gln Ala Val Arg
Thr Arg Asp Pro Ala Val Asp Lys Tyr Ser Thr85 90 95 100ccg ttg tta
tac ctg aag ggt ttt cat gcc ttg cag gcc tat cgc atc 570Pro Leu Leu
Tyr Leu Lys Gly Phe His Ala Leu Gln Ala Tyr Arg Ile 105 110 115ggt
cac tgg ttg tgg aat cag ggg cgt cgc gca ctg gca atc ttt ctg 618Gly
His Trp Leu Trp Asn Gln Gly Arg Arg Ala Leu Ala Ile Phe Leu 120 125
130caa aac cag gtt tct gtg acg ttc cag gtc gat att cac ccg gca gca
666Gln Asn Gln Val Ser Val Thr Phe Gln Val Asp Ile His Pro Ala Ala
135 140 145aaa att ggt cgc ggt atc atg ctt gac cac gcg aca ggc atc
gtc gtt 714Lys Ile Gly Arg Gly Ile Met Leu Asp His Ala Thr Gly Ile
Val Val 150 155 160ggt gaa acg gcg gtg att gaa aac gac gta tcg att
ctg caa tct gtg 762Gly Glu Thr Ala Val Ile Glu Asn Asp Val Ser Ile
Leu Gln Ser Val165 170 175 180acg ctt ggc ggt acg ggt aaa tct ggt
ggt gac cgt cac ccg aaa att 810Thr Leu Gly Gly Thr Gly Lys Ser Gly
Gly Asp Arg His Pro Lys Ile 185 190 195cgt gaa ggt gtg atg att ggc
gcg ggc gcg aaa atc ctc ggc aat att 858Arg Glu Gly Val Met Ile Gly
Ala Gly Ala Lys Ile Leu Gly Asn Ile 200 205 210gaa gtt ggg cgc ggc
gcg aag att ggc gca ggt tcc gtg gtg ctg caa 906Glu Val Gly Arg Gly
Ala Lys Ile Gly Ala Gly Ser Val Val Leu Gln 215 220 225ccg gtg ccg
ccg cat acc acc gcc gct ggc gtt ccg gct cgt att gtc 954Pro Val Pro
Pro His Thr Thr Ala Ala Gly Val Pro Ala Arg Ile Val 230 235 240ggt
aaa cca gac agc gat aag cca tca atg gat atg gac cag cat ttc 1002Gly
Lys Pro Asp Ser Asp Lys Pro Ser Met Asp Met Asp Gln His Phe245 250
255 260aac ggt att aac cat aca ttt gag tat ggg gat ggg atc taa
1044Asn Gly Ile Asn His Thr Phe Glu Tyr Gly Asp Gly Ile 265
270tgtcctgtga tcgtgccgga tgcgatgtaa tcatctatcc ggcctacagt
aactaatctc 1104tcaataccgc tcccgatacc ccaactgtcg
113432273PRTEscherichia coli 32Met Ser Cys Glu Glu Leu Glu Ile Val
Trp Asn Asn Ile Lys Ala Glu1 5 10 15Ala Arg Thr Leu Ala Asp Cys Glu
Pro Met Leu Ala Ser Phe Tyr His 20 25 30Ala Thr Leu Leu Lys His Glu
Asn Leu Gly Ser Ala Leu Ser Tyr Met 35 40 45Leu Ala Asn Lys Leu Ser
Ser Pro Ile Met Pro Ala Ile Ala Ile Arg 50 55 60Glu Val Val Glu Glu
Ala Tyr Ala Ala Asp Pro Glu Met Ile Ala Ser65 70 75 80Ala Ala Cys
Asp Ile Gln Ala Val Arg Thr Arg Asp Pro Ala Val Asp 85 90 95Lys Tyr
Ser Thr Pro Leu Leu Tyr Leu Lys Gly Phe His Ala Leu Gln 100 105
110Ala Tyr Arg Ile Gly His Trp Leu Trp Asn Gln Gly Arg Arg Ala Leu
115 120 125Ala Ile Phe Leu Gln Asn Gln Val Ser Val Thr Phe Gln Val
Asp Ile 130 135 140His Pro Ala Ala Lys Ile Gly Arg Gly Ile Met Leu
Asp His Ala Thr145 150 155 160Gly Ile Val Val Gly Glu Thr Ala Val
Ile Glu Asn Asp Val Ser Ile 165 170 175Leu Gln Ser Val Thr Leu Gly
Gly Thr Gly Lys Ser Gly Gly Asp Arg 180 185 190His Pro Lys Ile Arg
Glu Gly Val Met Ile Gly Ala Gly Ala Lys Ile 195 200 205Leu Gly Asn
Ile Glu Val Gly Arg Gly Ala Lys Ile Gly Ala Gly Ser 210 215 220Val
Val Leu Gln Pro Val Pro Pro His Thr Thr Ala Ala Gly Val Pro225 230
235 240Ala Arg Ile Val Gly Lys Pro Asp Ser Asp Lys Pro Ser Met Asp
Met 245 250 255Asp Gln His Phe Asn Gly Ile Asn His Thr Phe Glu Tyr
Gly Asp Gly 260 265 270Ile33912DNAEscherichia coliCDS(1)..(912)
33gtg agt aca tta gaa caa aca ata ggc aat acg cct ctg gtg aag ttg
48Val Ser Thr Leu Glu Gln Thr Ile Gly Asn Thr Pro Leu Val Lys Leu1
5 10 15cag cga atg ggg ccg gat aac ggc agt gaa gtg tgg tta aaa ctg
gaa 96Gln Arg Met Gly Pro Asp Asn Gly Ser Glu Val Trp Leu Lys Leu
Glu 20 25 30ggc aat aac ccg gca ggt tcg gtg aaa gat cgt gcg gca ctt
tcg atg 144Gly Asn Asn Pro Ala Gly Ser Val Lys Asp Arg Ala Ala Leu
Ser Met 35 40 45atc gtc gag gcg gaa aag cgc ggg gaa att aaa ccg ggt
gat gtc tta 192Ile Val Glu Ala Glu Lys Arg Gly Glu Ile Lys Pro Gly
Asp Val Leu 50 55 60atc gaa gcc acc agt ggt aac acc ggc att gcg ctg
gca atg att gcc 240Ile Glu Ala Thr Ser Gly Asn Thr Gly Ile Ala Leu
Ala Met Ile Ala65 70 75 80gcg ctg aaa ggc tat cgc atg aaa ttg ctg
atg ccc gac aac atg agc 288Ala Leu Lys Gly Tyr Arg Met Lys Leu Leu
Met Pro Asp Asn Met Ser 85 90 95cag gaa cgc cgt gcg gcg atg cgt gct
tat ggt gcg gaa ctg att ctt 336Gln Glu Arg Arg Ala Ala Met Arg Ala
Tyr Gly Ala Glu Leu Ile Leu 100 105 110gtc acc aaa gag cag ggc atg
gaa ggt gcg cgc gat ctg gcg ctg gag 384Val Thr Lys Glu Gln Gly Met
Glu Gly Ala Arg Asp Leu Ala Leu Glu 115 120 125atg gcg aat cgt ggc
gaa gga aag ctg ctc gat cag ttc aat aat ccc 432Met Ala Asn Arg Gly
Glu Gly Lys Leu Leu Asp Gln Phe Asn Asn Pro 130 135 140gat aac cct
tat gcg cat tac acc acc act ggg ccg gaa atc tgg cag 480Asp Asn Pro
Tyr Ala His Tyr Thr Thr Thr Gly Pro Glu Ile Trp Gln145 150 155
160caa acc ggc ggg cgc atc act cat ttt gtc tcc agc atg ggg acg acc
528Gln Thr Gly Gly Arg Ile Thr His Phe Val Ser Ser Met Gly Thr Thr
165 170 175ggc act atc acc ggc gtc tca cgc ttt atg cgc gaa caa tcc
aaa ccg 576Gly Thr Ile Thr Gly Val Ser Arg Phe Met Arg Glu Gln Ser
Lys Pro 180 185 190gtg acc att gtc ggc ctg caa ccg gaa gag ggc agc
agc att ccc ggc 624Val Thr Ile Val Gly Leu Gln Pro Glu Glu Gly Ser
Ser Ile Pro Gly 195 200 205att cgc cgc tgg cct acg gaa tat ctg ccg
ggg att ttc aac gct tct 672Ile Arg Arg Trp Pro Thr Glu Tyr Leu Pro
Gly Ile Phe Asn Ala Ser 210 215 220ctg gtg gat gag gtg ctg gat att
cat cag cgc gat gcg gaa aac acc 720Leu Val Asp Glu Val Leu Asp Ile
His Gln Arg Asp Ala Glu Asn Thr225 230 235 240atg cgc gaa ctg gcg
gtg cgg gaa gga ata ttc tgt ggc gtc agc tcc 768Met Arg Glu Leu Ala
Val Arg Glu Gly Ile Phe Cys Gly Val Ser Ser 245 250 255ggc ggc gcg
gtt gcc gga gca ctg cgg gtg gca aaa gct aac cct gac 816Gly Gly Ala
Val Ala Gly Ala Leu Arg Val Ala Lys Ala Asn Pro Asp 260 265 270gcg
gtg gtg gtg gcg atc atc tgc gat cgt ggc gat cgc tac ctt tct 864Ala
Val Val Val Ala Ile Ile Cys Asp Arg Gly Asp Arg Tyr Leu Ser 275 280
285acc ggg gtg ttt ggg gaa gag cat ttt agc cag ggg gcg ggg att taa
912Thr Gly Val Phe Gly Glu Glu His Phe Ser Gln Gly Ala Gly Ile 290
295 30034303PRTEscherichia coli 34Val Ser Thr Leu Glu Gln Thr Ile
Gly Asn Thr Pro Leu Val Lys Leu1 5 10 15Gln Arg Met Gly Pro Asp Asn
Gly Ser Glu Val Trp Leu Lys Leu Glu 20 25 30Gly Asn Asn Pro Ala Gly
Ser Val Lys Asp Arg Ala Ala Leu Ser Met 35 40 45Ile Val Glu Ala Glu
Lys Arg Gly Glu Ile Lys Pro Gly Asp Val Leu 50 55 60Ile Glu Ala Thr
Ser Gly Asn Thr Gly Ile Ala Leu Ala Met Ile Ala65 70 75 80Ala Leu
Lys Gly Tyr Arg Met Lys Leu Leu Met Pro Asp Asn Met Ser 85 90 95Gln
Glu Arg Arg Ala Ala Met Arg Ala Tyr Gly Ala Glu Leu Ile Leu 100 105
110Val Thr Lys Glu Gln Gly Met Glu Gly Ala Arg Asp Leu Ala Leu Glu
115 120 125Met Ala Asn Arg Gly Glu Gly Lys Leu Leu Asp Gln Phe Asn
Asn Pro 130 135 140Asp Asn Pro Tyr Ala His Tyr Thr Thr Thr Gly Pro
Glu Ile Trp Gln145 150 155 160Gln Thr Gly Gly Arg Ile Thr His Phe
Val Ser Ser Met Gly Thr Thr 165 170 175Gly Thr Ile Thr Gly Val Ser
Arg Phe Met Arg Glu Gln Ser Lys Pro 180 185 190Val Thr Ile Val Gly
Leu Gln Pro Glu Glu Gly Ser Ser Ile Pro Gly 195 200 205Ile Arg Arg
Trp Pro Thr Glu Tyr Leu Pro Gly Ile Phe Asn Ala Ser 210 215 220Leu
Val Asp Glu Val Leu Asp Ile His Gln Arg Asp Ala Glu Asn Thr225 230
235 240Met Arg Glu Leu Ala Val Arg Glu Gly Ile Phe Cys Gly Val Ser
Ser 245 250 255Gly Gly Ala Val Ala Gly Ala Leu Arg Val Ala Lys Ala
Asn Pro Asp 260 265 270Ala Val Val Val Ala Ile Ile Cys Asp Arg Gly
Asp Arg Tyr Leu Ser 275 280 285Thr Gly Val Phe Gly Glu Glu His Phe
Ser Gln Gly Ala Gly Ile 290 295 300351173DNAEscherichia
coliCDS(1)..(1173) 35atg ttc gat ttt tca aag gtc gtg gat cgt cat
ggc aca tgg tgt aca 48Met Phe Asp Phe Ser Lys Val Val Asp Arg His
Gly Thr Trp Cys Thr1 5 10 15cag tgg gat tat gtc gct gac cgt ttc ggc
act gct gac ctg tta ccg 96Gln Trp Asp Tyr Val Ala Asp Arg Phe Gly
Thr Ala Asp Leu Leu Pro 20 25 30ttc acg att tca gac atg gat ttt gcc
act gcc ccc tgc att atc gag 144Phe Thr Ile Ser Asp Met Asp Phe Ala
Thr Ala Pro Cys Ile Ile Glu 35 40 45gcg ctg aat cag cgc ctg atg cac
ggc gta ttt ggc tac agc cgc tgg 192Ala Leu Asn Gln Arg Leu Met His
Gly Val Phe Gly Tyr Ser Arg Trp 50 55 60aaa aac gat gag ttt ctc gcg
gct att gcc cac tgg ttt tcc acc cag 240Lys Asn Asp Glu Phe Leu Ala
Ala Ile Ala His Trp Phe Ser Thr Gln65 70 75 80cat tac acc gcc atc
gat tct cag acg gtg gtg tat ggc cct tct gtc 288His Tyr Thr Ala Ile
Asp Ser Gln Thr Val Val Tyr Gly Pro Ser Val 85 90 95atc tat atg gtt
tca gaa ctg att cgt cag tgg tct gaa aca ggt gaa 336Ile Tyr Met Val
Ser Glu Leu Ile Arg Gln Trp Ser Glu Thr Gly Glu 100 105 110ggc gtg
gtg atc cac aca ccc gcc tat gac gca ttt tac aag gcc att 384Gly Val
Val Ile His Thr Pro Ala Tyr Asp Ala Phe Tyr Lys Ala Ile 115 120
125gaa ggt aac cag cgc aca gta atg ccc gtt gct tta gag aag cag gct
432Glu Gly Asn Gln Arg Thr Val Met Pro Val Ala Leu Glu Lys Gln Ala
130 135 140gat ggt tgg ttt tgc gat atg ggc aag ttg gaa gcc gtg ttg
gcg aaa 480Asp Gly Trp Phe Cys Asp Met Gly Lys Leu Glu Ala Val Leu
Ala Lys145 150 155 160cca gaa tgt aaa att atg ctc ctg tgt agc cca
cag aat cct acc ggg 528Pro Glu Cys Lys Ile Met Leu Leu Cys Ser Pro
Gln Asn Pro Thr Gly 165 170 175aaa gtg tgg acg tgc gat gag ctg gag
atc atg gct gac ctg tgc gag 576Lys Val Trp Thr Cys Asp Glu Leu Glu
Ile Met Ala Asp Leu Cys Glu 180 185 190cgt cat ggt gtg cgg gtt att
tcc gat gaa atc cat atg gat atg gtt 624Arg His Gly Val Arg Val Ile
Ser Asp Glu Ile His Met Asp Met Val 195 200 205tgg ggc gag cag ccg
cat att ccc tgg agt aat gtg gct cgc gga gac 672Trp Gly Glu Gln Pro
His Ile Pro Trp Ser Asn Val Ala Arg Gly Asp 210 215 220tgg gcg ttg
cta acg tcg ggc tcg aaa agt ttc aat att ccc gcc ctg 720Trp Ala Leu
Leu Thr Ser Gly Ser Lys Ser Phe Asn Ile Pro Ala Leu225 230 235
240acc ggt gct tac ggg att ata gaa aat agc agt agc cgc gat gcc tat
768Thr Gly Ala Tyr Gly Ile Ile Glu Asn Ser Ser Ser Arg Asp Ala Tyr
245 250 255tta tcg gca ctg aaa ggc cgt gat ggg ctt tct tcc cct tcg
gta ctg 816Leu Ser Ala Leu Lys Gly Arg Asp Gly Leu Ser Ser Pro Ser
Val Leu 260 265 270gcg tta act gcc cat atc gcc gcc tat cag caa ggc
gcg ccg tgg ctg 864Ala Leu Thr Ala His Ile Ala Ala Tyr Gln Gln Gly
Ala Pro Trp Leu 275 280 285gat gcc tta cgc atc tat ctg aaa gat aac
ctg acg tat atc gca gat 912Asp Ala Leu Arg Ile Tyr Leu Lys Asp Asn
Leu Thr Tyr Ile Ala Asp 290
295 300aaa atg aac gcc gcg ttt cct gaa ctc aac tgg cag atc cca caa
tcc 960Lys Met Asn Ala Ala Phe Pro Glu Leu Asn Trp Gln Ile Pro Gln
Ser305 310 315 320act tat ctg gca tgg ctt gat tta cgt ccg ttg aat
att gac gac aac 1008Thr Tyr Leu Ala Trp Leu Asp Leu Arg Pro Leu Asn
Ile Asp Asp Asn 325 330 335gcg ttg caa aaa gca ctt atc gaa caa gaa
aaa gtc gcg atc atg ccg 1056Ala Leu Gln Lys Ala Leu Ile Glu Gln Glu
Lys Val Ala Ile Met Pro 340 345 350ggg tat acc tac ggt gaa gaa ggt
cgt ggt ttt gtc cgt ctc aat gcc 1104Gly Tyr Thr Tyr Gly Glu Glu Gly
Arg Gly Phe Val Arg Leu Asn Ala 355 360 365ggc tgc cca cgt tcg aaa
ctg gaa aaa ggt gtg gct gga tta att aac 1152Gly Cys Pro Arg Ser Lys
Leu Glu Lys Gly Val Ala Gly Leu Ile Asn 370 375 380gcc atc cgc gct
gtt cgt taa 1173Ala Ile Arg Ala Val Arg385 39036390PRTEscherichia
coli 36Met Phe Asp Phe Ser Lys Val Val Asp Arg His Gly Thr Trp Cys
Thr1 5 10 15Gln Trp Asp Tyr Val Ala Asp Arg Phe Gly Thr Ala Asp Leu
Leu Pro 20 25 30Phe Thr Ile Ser Asp Met Asp Phe Ala Thr Ala Pro Cys
Ile Ile Glu 35 40 45Ala Leu Asn Gln Arg Leu Met His Gly Val Phe Gly
Tyr Ser Arg Trp 50 55 60Lys Asn Asp Glu Phe Leu Ala Ala Ile Ala His
Trp Phe Ser Thr Gln65 70 75 80His Tyr Thr Ala Ile Asp Ser Gln Thr
Val Val Tyr Gly Pro Ser Val 85 90 95Ile Tyr Met Val Ser Glu Leu Ile
Arg Gln Trp Ser Glu Thr Gly Glu 100 105 110Gly Val Val Ile His Thr
Pro Ala Tyr Asp Ala Phe Tyr Lys Ala Ile 115 120 125Glu Gly Asn Gln
Arg Thr Val Met Pro Val Ala Leu Glu Lys Gln Ala 130 135 140Asp Gly
Trp Phe Cys Asp Met Gly Lys Leu Glu Ala Val Leu Ala Lys145 150 155
160Pro Glu Cys Lys Ile Met Leu Leu Cys Ser Pro Gln Asn Pro Thr Gly
165 170 175Lys Val Trp Thr Cys Asp Glu Leu Glu Ile Met Ala Asp Leu
Cys Glu 180 185 190Arg His Gly Val Arg Val Ile Ser Asp Glu Ile His
Met Asp Met Val 195 200 205Trp Gly Glu Gln Pro His Ile Pro Trp Ser
Asn Val Ala Arg Gly Asp 210 215 220Trp Ala Leu Leu Thr Ser Gly Ser
Lys Ser Phe Asn Ile Pro Ala Leu225 230 235 240Thr Gly Ala Tyr Gly
Ile Ile Glu Asn Ser Ser Ser Arg Asp Ala Tyr 245 250 255Leu Ser Ala
Leu Lys Gly Arg Asp Gly Leu Ser Ser Pro Ser Val Leu 260 265 270Ala
Leu Thr Ala His Ile Ala Ala Tyr Gln Gln Gly Ala Pro Trp Leu 275 280
285Asp Ala Leu Arg Ile Tyr Leu Lys Asp Asn Leu Thr Tyr Ile Ala Asp
290 295 300Lys Met Asn Ala Ala Phe Pro Glu Leu Asn Trp Gln Ile Pro
Gln Ser305 310 315 320Thr Tyr Leu Ala Trp Leu Asp Leu Arg Pro Leu
Asn Ile Asp Asp Asn 325 330 335Ala Leu Gln Lys Ala Leu Ile Glu Gln
Glu Lys Val Ala Ile Met Pro 340 345 350Gly Tyr Thr Tyr Gly Glu Glu
Gly Arg Gly Phe Val Arg Leu Asn Ala 355 360 365Gly Cys Pro Arg Ser
Lys Leu Glu Lys Gly Val Ala Gly Leu Ile Asn 370 375 380Ala Ile Arg
Ala Val Arg385 390
* * * * *
References