U.S. patent application number 10/557306 was filed with the patent office on 2007-05-10 for dna encoding a protein having d-lactate dehydrogenase activity and uses thereof.
Invention is credited to Masana Hirai, Nobuhiro Ishida, Eiji Nagamori, Tohru Ohnishi, Satoshi Saitoh, Haruo Takahashi, Kenro Tokuhiro.
Application Number | 20070105202 10/557306 |
Document ID | / |
Family ID | 33475225 |
Filed Date | 2007-05-10 |
United States Patent
Application |
20070105202 |
Kind Code |
A1 |
Ishida; Nobuhiro ; et
al. |
May 10, 2007 |
Dna encoding a protein having d-lactate dehydrogenase activity and
uses thereof
Abstract
This invention provides a polynucleotide that encodes a protein
having lactate dehydrogenase activity and such protein that can be
used for producing D-lactic acid. This polynucleotide has the
nucleotide sequence as shown in SEQ ID NO: 1 (a), and it hybridizes
under stringent conditions with a probe comprising all or part of
the nucleotide sequence as shown in SEQ ID NO: 1 or a complementary
strand thereof and encodes a protein having D-lactate dehydrogenase
activity (b).
Inventors: |
Ishida; Nobuhiro; (Aichi,
JP) ; Tokuhiro; Kenro; (Aichi, JP) ;
Takahashi; Haruo; (Gifu, JP) ; Nagamori; Eiji;
(Aichi, JP) ; Hirai; Masana; (Aichi, JP) ;
Saitoh; Satoshi; (Aichi, JP) ; Ohnishi; Tohru;
(Aichi, JP) |
Correspondence
Address: |
FINNEGAN, HENDERSON, FARABOW, GARRETT & DUNNER;LLP
901 NEW YORK AVENUE, NW
WASHINGTON
DC
20001-4413
US
|
Family ID: |
33475225 |
Appl. No.: |
10/557306 |
Filed: |
May 21, 2004 |
PCT Filed: |
May 21, 2004 |
PCT NO: |
PCT/JP04/07317 |
371 Date: |
November 18, 2005 |
Current U.S.
Class: |
435/135 ;
435/139; 435/189; 435/254.21; 435/483; 525/437; 536/23.2 |
Current CPC
Class: |
C12P 7/56 20130101; C12N
9/0006 20130101; C12P 7/625 20130101 |
Class at
Publication: |
435/135 ;
435/189; 435/254.21; 435/483; 536/023.2; 435/139; 525/437 |
International
Class: |
C12P 7/62 20060101
C12P007/62; C12P 7/56 20060101 C12P007/56; C07H 21/04 20060101
C07H021/04; C12N 9/02 20060101 C12N009/02; C12N 15/74 20060101
C12N015/74 |
Foreign Application Data
Date |
Code |
Application Number |
May 22, 2003 |
JP |
2003-145085 |
Claims
1. A polynucleotide as described in any of the following (a) to
(g): (a) a polynucleotide comprising the nucleotide sequence as
shown in SEQ ID NO: 1; (b) a polynucleotide that hybridizes under
stringent conditions with a probe comprising all or part of the
nucleotide sequence as shown in SEQ ID NO: 1 or a complementary
strand thereof and that encodes a protein having D-lactate
dehydrogenase activity; (c) a polynucleotide that encodes a protein
consisting of the amino acid sequence as shown in SEQ ID NO: 2; (d)
a polynucleotide that encodes a protein consisting of an amino acid
sequence derived from the amino acid sequence as shown in SEQ ID
NO: 2 by substitution, deletion, insertion, or addition of one or
several amino acid residues and having D-lactate dehydrogenase
activity; (e) a polynucleotide that encodes a protein having 70% or
higher homology to the amino acid sequence as shown in SEQ ID NO: 2
and having D-lactate dehydrogenase activity; (f) a polynucleotide
that encodes a protein having an amino acid sequence derived from
the amino acid sequence as shown in SEQ ID NO: 4 by substitution of
one or more amino acid residues selected from the list of amino
acid residue substitutions shown in Table 1 and having D-lactate
dehydrogenase activity: TABLE-US-00005 TABLE 1 List of amino acid
residue substitutions Substitution type Position of substitution
Amino acid substituent 1 40 Valine (Val) 2 112 Isoleucine (Ile) 3
131 Histidine (His) 4 139 Isoleucine (Ile) 5 181 Glutamic acid
(Glu) 6 266 Glycine (Gly) 7 267 Leucine (Leu) 8 268 Phenylalanine
(Phe) 9 269 Asparagine (Asn) 10 270 Glutamic acid (Glu) 11 271
Aspartic acid (Asp) 12 272 Tryptophan (Trp) 13 273 Serine (Ser) 14
274 Glycine (Gly) 15 276 Glutamic acid (Glu) 16 277 Phenylalanine
(Phe) 17 287 Serine (Ser) 18 292 Leucine (Leu) 19 293 Valine
(Val)
wherein positions of substitution are indicated as the positions
from methionine, which corresponds to the initiation codon; or (g)
a polynucleotide that encodes a protein having an amino acid
sequence containing at least amino acid residues 78 and 79, 152 to
175, 235, and 296 of the amino acid sequence as shown in SEQ ID NO:
2 and having D-lactate dehydrogenase activity.
2. A protein as described in any of the following (h) to (1): (h) a
protein that consists of the amino acid sequence as shown in SEQ ID
NO: 2 of the Sequence Listing; (i) a protein that consists of an
amino acid sequence derived from the amino acid sequence as shown
in SEQ ID NO: 2 of the Sequence Listing by substitution, deletion,
insertion, or addition of one or several amino acid residues and
that has D-lactate dehydrogenase activity; (j) a protein that has
70% or higher homology to the amino acid sequence as shown in SEQ
ID NO: 2 and that has D-lactate dehydrogenase activity; (k) a
protein that has an amino acid sequence derived from the amino acid
sequence as shown in SEQ ID NO: 4 by substitution of one or more
amino acid residues selected from the list of amino acid residue
substitutions shown in Table 1 and that has D-lactate dehydrogenase
activity; or (l) a protein that has an amino acid sequence
containing at least amino acid residues 78 and 79, 152 to 175, 235,
and 296 of the amino acid sequence as shown in SEQ ID NO: 2 and
that has D-lactate dehydrogenase activity.
3. A DNA construct that comprises a DNA segment having DNA
according to claim 1 and a DNA segment having DNA that encodes a
promoter or a homolog thereof
4. The DNA construct according to claim 3, wherein the promoter is
of the pyruvate decarboxylase gene.
5. The DNA construct according to claim 3 or 4, wherein the
promoter is of the pyruvate decarboxylase 1 gene of
Saccharomyces.
6. The DNA construct according to claim 5, wherein the promoter is
of the pyruvate decarboxylase 1 gene of Saccharomyces
cerevisae.
7. A transformant that carries the DNA according to claim 1 in an
expressible manner in a host.
8. The transformant according to claim 7, wherein the host is a
microorganism selected from the group consisting of eukaryotic
microorganisms including yeast and fungi and prokaryotic
microorganisms including lactic acid bacteria, Escherichia
bacteria, and Bacillus bacteria.
9. The transformant according to claim 7 or 8, wherein the DNA is
carried in an expressible manner under the control of a promoter or
a homolog thereof
10. The transformant according to claim 9, wherein the promoter is
of the pyruvate decarboxylase gene.
11. The transformant according to claim 9 or 10, wherein the
promoter is of the pyruvate decarboxylase 1 gene of
Saccharomyces.
12. The transformant according to claim 11, wherein the DNA is
carried in an expressible manner under the control of the promoter
of the host pyruvate decarboxylase 1 gene.
13. The transformant according to any one of claims 7 to 12,
wherein the host microorganism is Saccharomyces cerevisae.
14. A method for producing D-lactic acid comprising steps of:
culturing the transformant according to any one of claims 7 to 13;
and recovering at least one member selected from the group
consisting of D-lactic acid, a salt thereof, and a derivative
thereof from the culture product.
15. A method for producing a lactic acid polymer comprising steps
of: culturing the transformant according to any one of claims 7 to
13; recovering at least one member selected from the group
consisting of D-lactic acid, a salt thereof, and a derivative
thereof from the culture product; and producing a lactic acid
polymer using the recovered D-lactic acid or a derivative thereof
as at least one polymerization material.
Description
TECHNICAL FIELD
[0001] The present invention relates to D-lactic acid and a
technique for producing a polymer utilizing D-lactic acid. More
particularly, the present invention relates to a protein having
D-lactate dehydrogenase activity that is suitable for allowing
yeast to produce D-lactic acid, DNA having a nucleotide sequence
encoding such protein, and uses thereof.
BACKGROUND ART
[0002] With advances in recombinant DNA technology, a technique for
obtaining gene products of interest has been developed. Such
technique is realized by allowing foreign genes to be expressed in
hosts such as microorganisms, molds, animals, plants, or insects
and by allowing the resulting transformants to multiply. When a
technique of yeast culturing is employed, for example, a large
quantity of gene products of interest can be produced via
fermentative production.
[0003] Effective production of lactic acid, which is a starting
material of plant-derived plastics, has been awaited in recent
years from the viewpoint of "carbon neutrality."
[0004] Lactic acid is classified into L-lactic acid and D-lactic
acid, which are optical isomers. A technique for fermentatively
producing D-lactic acid has been known, whereby D-lactic acid is
fermentatively produced using lactic acid bacteria capable of
producing D-lactic acid in a medium containing brewers' yeast (JP
Patent Publication (Kokai) No. 58-16688 A (1983)). Also, a
technique that involves the use of Lactobacillus bulgaricus has
been known (JP Patent Publication (Kokai) No. 58-36394 A (1983)). A
technique for obtaining D-lactic acid with high optical purity has
also been disclosed (JP Patent Publication (Kokai) Nos. 61-293387 A
(1986), 4-271787 A (1992), and 2001-29063 (A)). Such microorganisms
capable of producing lactic acid have not been suitable for
industrial production of lactic acid due to the slow rates of
production and the necessity for complicated medium
compositions.
[0005] Another technique has also been disclosed, whereby a lactate
dehydrogenase gene is incorporated into a yeast strain lacking the
capacity for ethanol production or having a reduced capacity for
ethanol production, and the resulting recombinant product is used
to produce lactic acid (JP Patent Publication (Kokai) No.
2001-516584 (A)). This technique, however, merely discloses the
production of L-lactic acid. Further, a technique for producing
D-lactic acid has been disclosed, whereby a D-lactate dehydrogenase
gene is introduced into yeast that has a high capacity for
producing pyruvic acid (JP Patent Publication (Kokai) No.
2002-136293 (A)). This technique is, however, focused on highly
concentrated pyruvic acid in a yeast strain having a high capacity
for producing pyruvic acid. Accordingly, production by general
yeast is not described therein.
[0006] The present invention is directed to providing a
polynucleotide encoding a protein that can be utilized for
producing D-lactic acid having lactate dehydrogenase activity, and
it is also directed to providing such protein. In addition, the
present invention is directed to providing an excellent system for
producing D-lactic acid utilizing such polynucleotide and a
technique for effectively producing D-lactic acid.
DISCLOSURE OF THE INVENTION
[0007] In order to attain the above objects, the present inventors
have searched for D-lactate dehydrogenase and genes that allow
expression of such enzyme. As a result, they discovered a
transformed line that exhibits an excellent capacity for producing
D-lactic acid. More specifically, they discovered a protein that
can positively affect the production of D-lactic acid and that has
D-lactate dehydrogenase activity, DNA that encodes a protein having
such enzyme activity, and a transformant prepared from such DNA.
Further, they discovered a method for producing D-lactic acid using
the same and a method for producing polylactic acid.
[0008] On the basis of the above findings, the present invention
provides the following.
[0009] (1) A polynucleotide as described in any of the following
(a) to (g):
[0010] (a) a polynucleotide comprising the nucleotide sequence as
shown in SEQ ID No: 1;
[0011] (b) a polynucleotide that hybridizes under stringent
conditions with a probe comprising all or part of the nucleotide
sequence as shown in SEQ ID NO: 1 or a complementary strand thereof
and that encodes a protein having D-lactate dehydrogenase
activity;
[0012] (c) a polynucleotide that encodes a protein consisting of
the amino acid sequence shown in SEQ ID NO: 2;
[0013] (d) a polynucleotide that encodes a protein consisting of an
amino acid sequence derived from the amino acid sequence as shown
in SEQ ID NO: 2 by substitution, deletion, insertion, or addition
of one or several amino acid residues and having D-lactate
dehydrogenase activity;
[0014] (e) a polynucleotide that encodes a protein having 70% or
higher homology to the amino acid sequence as shown in SEQ ID NO: 2
and having D-lactate dehydrogenase activity;
[0015] (f) a polynucleotide that encodes a protein having an amino
acid sequence derived from the amino acid sequence as shown in SEQ
ID NO: 4 by substitution of one or more amino acid residues
selected from the list of amino acid residue substitutions shown in
Table 1 and having D-lactate dehydrogenase activity: TABLE-US-00001
TABLE 1 List of amino acid residue substitutions Substitution type
Position of substitution Amino acid substituent 1 40 Valine (Val) 2
112 Isoleucine (Ile) 3 131 Histidine (His) 4 139 Isoleucine (Ile) 5
181 Glutamic acid (Glu) 6 266 Glycine (Gly) 7 267 Leucine (Leu) 8
268 Phenylalanine (Phe) 9 269 Asparagine (Asn) 10 270 Glutamic acid
(Glu) 11 271 Aspartic acid (Asp) 12 272 Tryptophan (Trp) 13 273
Serine (Ser) 14 274 Glycine (Gly) 15 276 Glutamic acid (Glu) 16 277
Phenylalanine (Phe) 17 287 Serine (Ser) 18 292 Leucine (Leu) 19 293
Valine (Val)
wherein positions of substitution are indicated as the positions
from methionine, which corresponds to the initiation codon; or
[0016] (g) a polynucleotide that encodes a protein having an amino
acid sequence containing at least amino acid residues 78 and 79,
152 to 175, 235, and 296 of the amino acid sequence as shown in SEQ
ID NO: 2 and having D-lactate dehydrogenase activity.
[0017] (2) A protein as described in any of the following (h) to
(l):
[0018] (h) a protein that consists of the amino acid sequence as
shown in SEQ ID NO: 2 of the Sequence Listing;
[0019] (i) a protein that consists of an amino acid sequence
derived from the amino acid sequence as shown in SEQ ID NO: 2 by
substitution, deletion, insertion, or addition of one or several
amino acid residues and that has D-lactate dehydrogenase
activity;
[0020] (j) a protein that has 70% or higher homology to the amino
acid sequence as shown in SEQ ID NO: 2 and that has D-lactate
dehydrogenase activity;
[0021] (k) a protein that has an amino acid sequence derived from
the amino acid sequence as shown in SEQ ID NO: 4 by substitution of
one or more amino acid residues selected from the list of amino
acid residue substitutions shown in Table 1 and that has D-lactate
dehydrogenase activity; or
[0022] (l) a protein that has an amino acid sequence containing at
least amino acid residues 78 and 79, 152 to 175, 235, and 296 of
the amino acid sequence as shown in SEQ ID NO: 2 and that has
D-lactate dehydrogenase activity.
[0023] (3) A DNA construct that comprises a DNA segment comprising
DNA according to (1) and a DNA segment comprising DNA that encodes
a promoter or a homolog thereof.
[0024] (4) The DNA construct according to (3), wherein the promoter
is of the pyruvate decarboxylase gene.
[0025] (5) The DNA construct according to (3) or (4), wherein the
promoter is of the pyruvate decarboxylase 1 gene of
Saccharomyces.
[0026] (6) The DNA construct according to (5), wherein the promoter
is of the pyruvate decarboxylase 1 gene of Saccharomyces
cerevisae.
[0027] (7) A transformant that carries the DNA according to (1) in
an expressible manner in a host.
[0028] (8) The transformant according to (7), wherein the host is a
microorganism selected from the group consisting of eukaryotic
microorganisms including yeast and fungi and prokaryotic
microorganisms including lactic acid bacteria, Escherichiai
bacteria, and Bacillus bacteria.
[0029] (9) The transformant according to (7) or (8), wherein the
DNA is carried in an expressible manner under the control of a
promoter or a homolog thereof
[0030] (10) The transformant according to (9), wherein the promoter
is of the pyruvate decarboxylase gene.
[0031] (11) The transformant according to (9) or (10), wherein the
promoter is of the pyruvate decarboxylase 1 gene of
Saccharomyces.
[0032] (12) The transformant according to (11), wherein the DNA is
carried in an expressible manner under the control of the promoter
of the host pyruvate decarboxylase 1 gene.
[0033] (13) The transformant according to any one of (7) to (12),
wherein the host microorganism is Saccharomyces cerevisae.
[0034] (14) A method for producing D-lactic acid comprising steps
of:
[0035] culturing the transformant according to any one of (7) to
(13); and
[0036] recovering at least one member selected from the group
consisting of D-lactic acid, a salt thereof, and a derivative
thereof from the culture product.
[0037] (15) A method for producing a lactic acid polymer comprising
steps of:
[0038] culturing the transformant according to any one of (7) to
(13);
[0039] recovering at least one member selected from the group
consisting of D-lactic acid, a salt thereof, and a derivative
thereof from the culture product; and
[0040] producing a lactic acid polymer using the recovered D-lactic
acid or a derivative thereof as at least one polymerization
material.
[0041] This description includes part or all of the contents as
disclosed in the description and/or drawings of Japanese Patent
Application No. 2003-145085, which is a priority document of the
present application.
BRIEF DESCRIPTION OF THE DRAWINGS
[0042] FIG. 1A shows the homology data of the nucleotide sequence
as shown in SEQ ID NO: 1 and the nucleotide sequence as shown in
SEQ ID NO: 3.
[0043] FIG. 1B is a continuation of FIG. 1A, which shows the
homology data of the nucleotide sequence as shown in SEQ ID NO: 1
and the nucleotide sequence as shown in SEQ ID NO: 3.
[0044] FIG. 2 shows the homology data of the amino acid sequence as
shown in SEQ ID NO: 2 and the amino acid sequence as shown in SEQ
ID NO: 4.
[0045] FIG. 3 shows a part of a procedure for constructing a
pBTRP-PDC1-DLDHME vector.
[0046] FIG. 4 shows a part of a procedure for constructing a
pBTRP-PDC1-DLDHME vector.
[0047] FIG. 5 shows a part of a procedure for constructing a
pBTRP-PDC1-DLDHME vector.
[0048] FIG. 6 shows the final step of a procedure for constructing
a pBTRP-PDC1-DLDHME vector.
[0049] FIG. 7 shows a part of the chromosome structure of a diploid
transformed yeast strain obtained in Example 3.
[0050] FIG. 8 is a graph showing the results of the fermentation
test of D-lactate (calcium salt) produced by the parent strain and
the transformant having chromosomally integrated transgenes, which
shows the amount of D-lactate and ethanol produced by each
strain.
[0051] FIG. 9 is a graph showing the results of the fermentation
test of free D-lactic acid produced by the parent strain, the
transformant having chromosomally integrated transgenes, and the
self-replicating plasmid transformant, which shows the amount of
D-lactic acid and ethanol produced by each strain.
[0052] FIG. 10 is a graph showing the results of the fermentation
test of free D-lactic acid produced by the strain into which 2
copies of the D-LDHME genes had been introduced and the strain into
which 4 copies of the D-LDHME genes had been introduced, which
shows the amount of D-lactic acid and ethanol produced by each
strain.
EMBODIMENTS OF THE INVENTION
[0053] The polynucleotide according to the present invention
comprises a nucleotide sequence that encodes a protein having
D-lactate dehydrogenase (D-LDH) activity.
[0054] Use of the polynucleotide according to the present invention
can result in the production of a transformant that allows the
expression of the aforementioned protein and a transformant that
produces D-lactic acid. Also, D-lactic acid can be produced via an
enzyme reaction system that involves the use of a protein encoded
by the polynucleotide according to the present invention.
[0055] According to the present invention, a novel source for a
starting material of D-lactic acid can be provided. Also, D-lactic
acid can be produced with high selectivity or high efficiency
according to the present invention.
[0056] Accordingly, the present invention can provide a technique
for producing a lactic acid polymer with the use of an enzyme
reaction system that involves the use of the protein according to
the present invention or D-lactic acid produced by a transformant
that allows the expression of the protein according to the present
invention.
[0057] Hereafter, the polynucleotides, the protein, the
transformant, the method for producing D-lactic acid according to
the present invention, or the like are described.
[0058] The polynucleotide according to the present invention can
include any of the following.
[0059] That is:
[0060] (a) a polynucleotide that comprises the nucleotide sequence
as shown in SEQ ID NO: 1;
[0061] (b) a polynucleotide that hybridizes under stringent
conditions with a probe comprising all or part of the nucleotide
sequence as shown in SEQ ID NO: 1 or a complementary strand thereof
and that encodes a protein having D-lactate dehydrogenase (D-LDH)
activity;
[0062] (c) a polynucleotide that encodes a protein having the amino
acid sequence as shown in SEQ ID NO: 2;
[0063] (d) a polynucleotide that encodes a protein consisting of an
amino acid sequence derived from the amino acid sequence as shown
in SEQ ID NO: 2 by substitution, deletion, insertion, or addition
of one or several amino acid residues and having D-lactate
dehydrogenase activity;
[0064] (e) a polynucleotide that has encodes a protein having 70%
or higher homology to the amino acid sequence as shown in SEQ ID
NO: 2 and having D-lactate dehydrogenase activity;
[0065] (f) a polynucleotide that encodes a protein having an amino
acid sequence derived from the amino acid sequence as shown in SEQ
ID NO: 4 by substitution of one or more amino acid residues
selected from the list of amino acid residue substitutions shown in
Table 1 and having D-lactate dehydrogenase activity; or
[0066] (g) a polynucleotide that encodes a protein having an amino
acid sequence containing at least amino acid residues 78 and 79,
152 to 175, 235, and 296 of the amino acid sequence as shown in SEQ
ID NO: 2 and having D-lactate dehydrogenase activity.
[0067] The polynucleotide that consists of the nucleotide sequence
as shown in SEQ ID NO: 1 according to the present invention encodes
a protein that has the amino acid sequence as shown in SEQ ID NO:
2. Also, such polynucleotide is derived from Leuconostoc
mesenteroides, which is a lactic acid bacterium. More specifically,
such polynucleotide is derived from the Leuconostoc mesenteroides
strain IF03426 (registered with the Institute for
Fermentation).
[0068] The sequence of this polynucleotide differs from the
sequence registered with GenBank (GenBank Accession No. L29327) in
terms of 27-bp nucleotides. As a result, the amino acid sequence
(SEQ ID NO: 2) that is encoded by the polynucleotide differs from
the amino acid sequence (SEQ ID NO: 4) based on the registered
nucleotide sequence in terms of 19 amino acid residues. The
nucleotide sequence as shown in SEQ ID NO: 1 shows 97.3% homology
to the nucleotide sequence as shown in SEQ ID NO: 3. The amino acid
sequence as shown in SEQ ID NO: 2 shows 94.3% homology to the amino
acid sequence as shown in SEQ ID NO: 4. Such homology is calculated
using Genetyx-mac ver.10.1 (Software Development Co., Ltd.).
[0069] The present invention relates to a polynucleotide that
encodes a protein having D-LDH activity. In the present invention,
such polynucleotide may be a naturally occurring one such as DNA or
RNA. Alternatively, such polynucleotide may contain an artificially
synthesized nucleotide derivative. It may be single-stranded or it
may have a complementary strand.
[0070] According to an embodiment of the present invention, the
polynucleotide has the nucleotide sequence as shown in SEQ ID NO:
1. The polynucleotide as shown in SEQ ID NO: 1 encodes a protein
having the amino acid sequence as shown in SEQ ID NO: 2.
[0071] According to another embodiment of the polynucleotide
according to the present invention, a polynucleotide encodes a
protein having the amino acid sequence as shown in SEQ ID NO: 2. A
polynucleotide having a nucleotide sequence that encodes the amino
acid sequence as shown in SEQ ID NO: 2 is sufficient.
[0072] According to a further embodiment of the present invention,
the polynucleotide consists of an amino acid sequence derived from
the amino acid sequence as shown in SEQ ID NO: 2 by substitution,
deletion, insertion, or addition of one or several amino acid
residues and has a nucleotide sequence encoding a protein having
D-LDH activity. A polynucleotide that has an amino acid sequence
derived from the amino acid sequence as shown in SEQ ID NO: 2 by
substitution, deletion, insertion, or addition of one or several
amino acid residues is deduced to have D-LDH activity equivalent to
that of a polynucleotide that has the amino acid sequence as shown
in SEQ ID NO: 2. This is because the D-LDH activity resulting from
the amino acid sequence as shown in SEQ ID NO: 2 is deduced to be
equivalent to that resulting from an amino acid sequence derived
from the former by substitution, deletion, insertion, or addition
of one or several amino acid residues. A person skilled in the art
can ordinarily select a protein having D-LDH activity, which can be
sufficiently utilized in the present invention, by such amino acid
substitution or the like.
[0073] A person skilled in the art can adequately introduce
mutation such as substitution, deletion, insertion, and/or addition
into a polynucleotide having the nucleotide sequence as shown in
SEQ ID NO: 1 via site-directed mutagenesis (Nucleic Acid Res. 10,
pp. 6487, 1982; Methods in Enzymol., 100, pp. 448, 1983; Molecular
Cloning, 2nd Ed., Cold Spring Harbor Laboratory Press, 1989; PCR: A
Practical Approach, IRL Press, pp. 200, 1991) or via other means.
Preferably, "several amino acids" are approximately 2 to 10 amino
acid residues, and more preferably approximately 2 to 4 amino acid
residues.
[0074] According to a further embodiment of the present invention,
the polynucleotide hybridizes under stringent conditions with a
probe comprising all or part of the nucleotide sequence as shown in
SEQ ID NO: 1 or a complementary strand thereof and encodes a
protein having D-lactate dehydrogenase activity. A probe that can
hybridize under stringent conditions with such polynucleotide
comprises at least one DNA sequence constituted by any of 20 or
more, and preferably 30 or more (for example, 40, 60, or 100)
continuous nucleotides, of the sequence as shown in SEQ ID NO: 1.
The polynucleotide can hybridize under stringent conditions with
DNA as shown in SEQ ID NO: 1 or such probe. Under stringent
conditions, for example, hybridization is carried out in the
presence of 50% formaldehyde at approximately 37.degree. C. Under
more stringent conditions, hybridization is carried out at
approximately 42.degree. C. Under further stringent conditions,
hybridization is carried out in the presence of formaldehyde at
approximately 65.degree. C.
[0075] An example of the polynucleotide that can hybridize under
stringent conditions with DNA consisting of the nucleotide sequence
as shown in SEQ ID NO: 1 or a probe is a polynucleotide that has a
nucleotide sequence similar to the nucleotide sequence as shown in
SEQ ID NO: 1. Such polynucleotide is highly likely to encode a
protein that has functions equivalent to those of a protein
consisting of the amino acid sequence as shown in SEQ ID NO: 2.
[0076] According to a further embodiment of the present invention,
the polynucleotide encodes a protein that shows 70% or higher,
preferably 80% or higher, more preferably 90% or higher, and
further preferably 95% or higher homology to the amino acid
sequence as shown in SEQ ID NO: 2, and that has D-lactate
dehydrogenase activity. Protein homology search can be carried out
using the gene-analyzing programs BLAST (hyperlink:
http://blast.genome.ad.jp http://blast.genome.ad.jp), FASTA
(hyperlink: http://fasta.genome.ad.jp/SIT/FASTA.html hyperlink:
http://fasta.genome.ad.jp/SIT/FASTA.html), or the like. The term
"homology" used herein refers to the identity observed through
these programs.
[0077] The polynucleotide according to the present invention
encodes a protein that has an amino acid sequence derived from the
amino acid sequence as shown in SEQ ID NO: 4 by substitution of one
or more amino acid residues selected from the list of amino acid
residue substitutions shown in Table 1 and that has D-lactate
dehydrogenase activity. The amino acid sequence as shown in SEQ ID
NO: 4 is the sequence of D-LDH of Leuconostoc mesenteroides
registered with GenBank (GenBank Accession No. L29327). The amino
acid sequence as shown in SEQ ID NO: 2 is derived from the amino
acid sequence as shown in SEQ ID NO: 4 via all substitutions
indicated in the list of amino acid residue substitutions shown in
Table 1. Even if the amino acid sequence does not have all the
substitutions, however, a protein having D-LDH activity that may be
utilized in the present invention can be obtained. The number of
substitutions is preferably 2 or more, more preferably 11 or more,
and further preferably 19 or more.
[0078] In the list of amino acid residue substitutions shown in
Table 1, the amino acid substitutions indicated by substitution
types 1 to 19 are preferable. The amino acid substitutions
indicated by substitution types 6 to 16 are more preferable.
[0079] According to a further embodiment of the present invention,
the polynucleotide encodes a protein that has an amino acid
sequence containing at least amino acid residues 78 and 79, 152 to
175, 235, and 296 of the amino acid sequence as shown in SEQ ID NO:
2 and that has D-LDH activity. Amino acid residues 78 and 79, 152
to 175, 235, and 296 are considered to be characteristic of the
amino acid sequence as shown in SEQ ID NO: 2. When a polynucleotide
contains such amino acid sequence and has D-LDH activity, it can be
said to be a preferable polynucleotide according to the present
invention. More preferably, a polynucleotide has a histidine
residue at position 296 as an active center and the coenzyme NADH
binding domain constituted by amino acid residues 152 to 175 of the
amino acid sequence as shown in SEQ ID NO: 2.
[0080] As described above, a polynucleotide of the present
invention may be a polynucleotide that has functions equivalent to
those of a protein encoded by the aforementioned protein, except
the polynucleotide consisting of or comprising the nucleotide
sequence as shown in SEQ ID NO: 1
[0081] The polynucleotide of the present invention can be obtained
via various techniques as mentioned above. In addition, it can be
chemically synthesized, or it can be obtained via PCR cloning,
hybridization, or the like from other organisms based on the
nucleotide sequence as shown in SEQ ID NO: 1. For example, a
polynucleotide can be isolated from a protein derived from
prokaryotic organisms such as lactic acid bacteria, Escherichia
coli bacteria, Bacillus subtilis bacteria, or fungi or proteins
derived from eukaryotic organisms such as yeast or octopus.
Alternatively, the method of Fujimoto et al. that has been known as
a method for synthesizing long-chain DNA can be adopted (Hideya
Fujimoto, "Gousei idenshi no sakuseihott (Production of synthetic
genes)," Shokubutsu saibo kogaku (Plant Cell Technology), Series 7,
Shokubiltsit no PCR jikken purotokoru (Protocol of plant PCR
experiments), 1997, pp. 95-100, Shujunsha).
[0082] In the present invention, the polynucleotide may not be
necessarily a homolog of the polynucleotide consisting of the
nucleotide sequence as shown in SEQ ID NO: 1. This is because the
transformant according to the present invention into which such
polynucleotide has been introduced is useful for producing D-LDH
and D-lactic acid.
[0083] For example, regardless of whether or not a polynucleotide
has the nucleotide sequence as shown in SEQ ID NO: 1 or is a
homolog thereof, a polynucleotide that encodes a protein having
D-LDH activity derived from prokaryotic organisms such as lactic
acid bacteria, Escherichia coli bacteria, Bacillus subtilis
bacteria, or fungi or proteins having D-LDH activity derived from
eukaryotic organisms such as yeast or octopus can also be
employed.
(Protein)
[0084] The protein according to the present invention consists of
or comprises the amino acid sequence as shown in SEQ ID NO: 2. Such
protein is a preferable embodiment of the present invention.
[0085] According to another embodiment of the present invention,
the protein consists of an amino acid sequence derived from the
amino acid sequence as shown in SEQ ID NO: 2 by substitution,
deletion, insertion, or addition of one or several amino acid
residues and has D-lactate dehydrogenase (D-LDH) activity.
According to a further embodiment, the protein shows 70% or higher,
preferably 80% or higher, more preferably 90% or higher, and
further preferably 95% or higher homology to the amino acid
sequence as shown in SEQ ID NO: 2 and has D-LDH activity.
[0086] According to a further embodiment, the protein has an amino
acid sequence derived from the amino acid sequence as shown in SEQ
ID NO: 4 by substitution of one or more amino acid residues
selected from the list of amino acid residue substitutions shown in
Table 1 and has D-LDH activity. The amino acid sequence as shown in
SEQ ID NO: 4 is the sequence of D-LDH of Leuconostoc mesenteroides
registered with GenBank (GenBank Accession No. L29327). The amino
acid sequence as shown in SEQ ID NO: 2 is derived from the amino
acid sequence as shown in SEQ ID NO: 4 via all substitutions
indicated in the list of amino acid residue substitutions shown in
Table 1. Even if the amino acid sequence does not have all the
substitutions, however, a protein having D-LDH activity that may be
utilized in the present invention can be obtained. The number of
substitutions is preferably 2 or more, more preferably 11 or more,
and further preferably 19 or more.
[0087] The amino acid sequence preferably has amino acid
substitutions indicated by substitution types 1 to 19, and more
preferably those indicated by substitution types 6 to 16, in the
list of amino acid residue substitutions shown in Table 1.
[0088] According to a further embodiment of the present invention,
the protein comprises an amino acid sequence containing at least
amino acid residues 78 and 79, 152 to 175, 235, and 296 of the
amino acid sequence as shown in SEQ ID NO: 2 and has D-lactate
dehydrogenase (D-LDH) activity. Amino acid residues 78 and 79, 152
to 175, 235, and 296 are considered to be characteristic of the
amino acid sequence as shown in. SEQ ID NO: 2. When a protein
contains such amino acid sequence and has D-LDH activity, it can be
said to be a preferable protein according to the present invention.
More preferably, a protein has a histidine residue at position 296
as an active center and the coenzyme NADH binding domain
constituted by amino acid residues 152 to 175 of the amino acid
sequence as shown in SEQ ID NO: 2.
[0089] The protein of the present invention does not necessarily
consist of the nucleotide sequence as shown in SEQ ID NO: 2, as
long as it has D-LDH activity.
[0090] The protein of the present invention can be obtained by
culturing the Leuconostoc mesenteroides strain IFO3426.
Specifically, the protein can be obtained as a culture product
thereof Such strain can be cultured via a conventional bacterial
culture technique. The protein of the present invention can be
purified from the culture product in accordance with a conventional
technique. Alternatively, a culture product itself or bacteria may
be recovered and then used as the substance having enzyme activity
of the present invention. Bacteria, purified enzymes, or crudely
purified enzymes can be used as they are, or they may be
immobilized.
[0091] The protein of the present invention can be obtained by
adequately introducing mutation such as substitution, deletion,
insertion, and/or addition into the amino acid sequence as shown in
SEQ ID NO: 2 or another amino acid sequence via, for example,
site-directed mutagenesis (Current Protocols in Molecular Biology,
edited by Ausubel et al., Sections 8.1-8.5, 1987, John Wily &
Sons). Such modification is not limited to artificial mutagenesis
or synthesis. It also includes a product resulting from amino acid
mutation in nature on the basis of artificial mutation, but it is
not limited thereto.
[0092] Further, a polynucleotide consisting of the nucleotide
sequence as shown in SEQ ID NO: 1 or DNA as a homolog thereof may
be obtained, such DNA may be introduced into a host strain to
prepare a transformant, and the resulting transformant may then be
cultured. Thus, a homolog protein can be obtained.
[0093] In the present invention, for example, known proteins having
D-LDH activity can be employed. Examples of proteins that can be
employed include proteins derived from prokaryotic organisms such
as Lactobacillus, Escherichia coli, Bacillus subtilis, or fungi or
proteins derived from eukaryotic organisms such as yeast or
octopus.
[0094] Such protein does not necessarily have the amino acid
sequence as shown in SEQ ID NO: 2. It is not necessarily a homolog
of the protein consisting of such amino acid sequence. The
transformant of the present invention that carries a protein having
D-LDH activity in an expressible manner is useful for producing
D-LDH and D-lactic acid.
[0095] Cells that are preferable for obtaining the polynucleotide
or protein of the present invention, the transformant of the
present invention and that are gene resources of polynucleotides or
proteins are not limited to naturally occurring organisms.
Microorganisms or cells that were obtained via mutation or the like
may be employed as gene resources.
[0096] The protein that is employed in the present invention has
D-LDH activity. Such activity can be measured using, for example, a
commercialized kit (lactate dehydrogenase (LDH/LD) test-UV
(Sigma)).
(DNA Construct)
[0097] The isolated polynucleotide that encodes a protein having
D-LDH activity (DNA, it may be hereinafter referred to as
"D-LDH-DNA" when the polynucleotide is DNA) may be used to prepare
a DNA construct having such DNA segment. This DNA construct can be
used as an expression vector in such state or by being introduced
into a suitable vector. A host cell is transformed using such DNA
construct. Thus, a transformant that produces a protein having
D-LDH activity can be obtained. Further, this transformant may be
cultured to produce a protein having D-LDH activity. Also, D-lactic
acid can be produced.
[0098] Transformation of a host cell involves the use of a DNA
construct that allows expression of a DNA segment consisting of
D-LDH-DNA in a host cell. Embodiments of a DNA construct for
transformation are not particularly limited. In accordance with the
form of the foreign gene introduced (extrachromosomal or
intrachromosomal) or the type of host cell, plasmid (DNA),
bacteriophage (DNA), retrotransposon (DNA), or an artificial
chromosome (e.g., YAC, PAC, BAC, or MAC) can be selected.
Accordingly, this DNA construct can comprise a constitutive segment
of a vector according to any of the aforementioned embodiments in
addition to the DNA of interest. Preferable prokaryotic vectors,
eukaryotic vectors, animal cell vectors, and plant cell vectors are
known in the art.
[0099] Examples of plasmid DNA include: YCp Escherichia coli-yeast
shuttle vectors, such as pRS413, pRS415, pRS416, YCp50, pAUR112, or
pAUR123; YEp Escherichia coli-yeast shuttle vectors, such as pYES32
or YEp13; YIp Escherichia coli-yeast shuttle vectors, such as
pRS403, pRS404, pRS405, pRS406, pAUR101, or pAUR135; plasmids
derived from Escherichia coli (e.g., ColE plasmids, such as pBR322,
pBR325, pUC18, pUC19, pUC119, pTV118N, pTV119N, pBluescript,
pHSG298, pHSG396, or pTrc99A; p1A plasmids, such as pACYC177 or
pACYC184; or pSC101 plasmids, such as pMW118, pMW119, pMW218, or
pMW219); and plasmids derived from Bacillus subtilis, such as
pUB110 or pTP5. Examples of phage DNA include .lamda. phage (e.g.,
Charon4A, Charon21A, EMBL3, EMBL4, .lamda.gt100, gt11, or zap),
.phi.X174, M13mp18, and M13mp19. An example of retrotransposon is a
Ty factor. An example of YAC is pYACC2.
[0100] The DNA construct of interest can be prepared by, for
example, cleaving a fragment containing D-LDH-DNA with an adequate
restriction enzyme and-inserting the fragment into a restriction
site or a multicloning site of the vector DNA to be used.
[0101] According to the first embodiment of the present invention,
the DNA construct comprises a promoter segment to which a DNA
segment consisting of D-LDH-DNA is ligated in an expressible
manner. Specifically, this DNA segment is ligated to a site located
downstream of the promoter, so that the promoter can control such
DNA segment.
[0102] A protein having D-LDH activity is preferably expressed in
yeast. Accordingly, use of a promoter that is capable of expressing
such protein in yeast is preferable. Examples of a promoter that
can be preferably used include a pyruvate decarboxylase gene
promoter, a gall promoter, a gal10 promoter, a heat shock protein
promoter, an MF.alpha.1 promoter, a PH05 promoter, a PGK promoter,
a GAP promoter, an ADH promoter, and an AOX1 promoter. A promoter
of the pyruvate decarboxylase 1 gene derived from Saccharomyces is
particularly preferable, and use of the promoter of the pyruvate
decarboxylase 1 gene derived from Saccharomyces cerevisae is more
preferable. The expression of proteins induced by these promoters
is enhanced in the ethanol fermentation pathway of Saccharomyces
(cerevisae). The promoter sequence of interest can be isolated by
PCR amplification wherein the genomic DNA of the pyruvate
decarboxylase 1 gene of the yeast Saccharomyces is used as a
template. For example, the nucleotide sequence of the promoter
derived from Saccharomyces cerevisae is shown in SEQ ID NO: 5. The
promoter segment in the DNA construct can be DNA consisting of the
nucleotide sequence as shown in SEQ ID NO: 5, DNA consisting of a
nucleotide sequence derived from the nucleotide sequence as shown
in SEQ ID NO: 5 by deletion, substitution, insertion, and/or
addition of one or several nucleotides and having promoter
activity, or DNA that can hybridize under stringent conditions with
DNA comprising all or part of the nucleotide sequence as shown in
SEQ ID NO: 5 or a complementary strand thereof and having promoter
activity (i.e., a homolog of such promoter). Also, promoters of the
pyruvate decarboxylase gene or the pyruvate decarboxylase 1 gene
derived from other types of yeast or other types of the yeast
Saccharomyces cerevisae can also be used.
[0103] According to the second embodiment of the present invention,
the DNA construct comprises a DNA segment for homologous
recombination of a host chromosome in addition to DNA. The DNA
segment for homologous recombination has a DNA sequence that is
homologous to the DNA sequence in the vicinity of the target site
in a host chromosome into which the DNA of interest is to be
introduced. The DNA construct comprises at least 1, and preferably
2, DNA segments for homologous recombination. For example, DNA
sequences homologous to DNA located at sites upstream and
downstream of the target site on the chromosome are provided as 2
DNA segments for homologous recombination, and the DNA of interest
is preferably ligated to a site between these DNA segments.
[0104] When the DNA of interest is introduced into a host
chromosome via homologous recombination, such DNA can be introduced
in a manner such that the promoter on the host chromosome is able
to control the DNA. In such a case, introduction of the target gene
can also disrupt the endogenous gene that should be controlled by
the promoter and can allow expression of foreign D-LDH-DNA instead
of the endogenous gene. It is particularly useful when such
promoter is capable of enhanced expression in a host cell.
[0105] In order to create such expression system on a host
chromosome, genes capable of enhanced expression are targeted in
the host chromosome, and D-LDH-DNA is preferably introduced into a
site downstream of the promoter that controls the gene of interest
in a manner such that the D-LDH-DNA can be controlled by such
promoter. When an ethanol fermentation microorganism such as yeast
is used as a host, the pyruvate decarboxylase gene (particularly
the pyruvate decarboxylase 1 gene) is targeted, and DNA that
encodes a protein having LDH activity can be introduced under the
control of the promoter of the endogenous pyruvate decarboxylase
gene. In such a case, a DNA segment for homologous recombination
can be homologous to a sequence in the LDH structural gene domain
of the pyruvate decarboxylase 1 gene or a sequence in the vicinity
thereof (including a sequence in the vicinity of the initiation
codon, a sequence upstream of the initiation codon, a sequence in
the structural gene, and the like). A segment of the promoter of
the pyruvate decarboxylase gene can be included in the DNA
construct.
[0106] Preferably, the yeast Saccharomyces (particularly
Saccharomyces cerevisae) is used as a host, and a DNA construct
that targets the pyruvate decarboxylase 1 gene of this host is
prepared. Such DNA construct can disrupt the pyruvate decarboxylase
1 gene and substitute this structural gene portion with D-LDH by
using a single vector. Pyruvate decarboxylase 1 is an enzyme that
mediates the irreversible reaction from pyruvic acid to
acetaldehyde. Disruption of the gene thereof can inhibit the
conversion of pyruvic acid to acetaldehyde and then to ethanol.
Also, generation of D-lactic acid by D-LDH can be accelerated with
the use of pyruvic acid as a substrate.
[0107] The DNA construct according to the first embodiment can also
be a DNA construct for homologous recombination by comprising a DNA
segment for homologous recombination with a host chromosome. In the
case of the DNA construct according to the first embodiment, a
promoter segment thereof can also serve as a DNA segment for
homologous recombination with a host chromosome. For example, a DNA
construct that has a promoter of a Saccharomyces cerevisae host
chromosome, such as a promoter of the pyruvate decarboxylase 1 gene
as a promoter segment, constitutes a targeting vector having the
gene of the host as a target site. In such a case, a DNA construct
preferably comprises a sequence homologous to the sequence of the
structural gene domain located downstream of the pyruvate
decarboxylase 1 gene.
[0108] According to need, a cis element such as an enhancer, a
splicing signal, a poly A addition signal, a selection marker, or a
ribosome binding sequence (SD sequence) can be ligated to the DNA
construct, in addition to a terminator. A selection marker is not
particularly limited, and various conventional selection marker
genes, including drug-resistant genes and auxotrophic genes, can be
used. For example, the dihydrofolate reductase gene, the hygromycin
B gene, and the neomycine resistant gene can be used.
(Transformation Using DNA Construct)
[0109] Once a DNA construct is prepared, such DNA construct can be
introduced into an adequate host cell via any adequate techniques
such as transformation, transfection, conjugation, protoplast
fusion, electroporation, lipofection, the lithium acetate method,
the particle gun method, calcium phosphate precipitation, the
agrobacterium method, the PEG method, or direct microinjection.
After the DNA construct has been introduced, the recipient cell is
then cultured in a selection medium.
[0110] Examples of host cells include: bacteria such as Escherichia
coli and Bacillus subtilis; yeast such as Saccharomyces cerevisae,
Schizosaccharomydces pombe, and Pichia pastoris; insect cells such
as sf9 and sf21; animal cells such as COS cells and Chinese hamster
ovary (CHO) cells; and plant cells such as sweet potato and
tobacco. Preferably, host cells are alcohol fermentation
microorganisms or acid resistant microorganisms such as yeasts.
Examples thereof include the yeast Saccharomyces, such as
Saccharomyces cerevisae. Specific examples thereof include the
Saccharomyces cerevisae strains IF02260 and YPH.
[0111] The transformant prepared from the DNA construct of interest
comprises a constituent of such DNA construct in the chromosome or
extrachromosomal element (including an artificial chromosome). When
the DNA construct is maintained extrachromosomally or integrated
into a chromosome via random integration, genes of another type of
enzymes that act on the pyruvic acid as a substrate, which is also
a substrate of LDH, such as the pyruvate decarboxylase gene (and
the pyruvate decarboxylase 1 gene in the case of the yeast
Saccharomyces cerevisae) are preferably knockout via a targeting
vector
[0112] If the aforementioned DNA construct capable of homologous
recombination is introduced, a ligated D-LDH-DNA is located at a
site downstream of a desired promoter or the promoter substituted
by the desired promoter or a homolog thereof in a manner such that
such promoter can control D-LDH-DNA. A transformant of the yeast
Saccharomyces preferably comprises on the host chromosome D-LDH-DNA
at a site downstream of the promoter of the pyruvate decarboxylase
1 gene or the promoter substituted by the promoter or a homolog in
a manner such that such promoter can control D-LDH-DNA. In general,
a homologous recombinant comprises a selection marker gene or a
part of the disrupted structural gene (a site corresponding to a
homologous sequence on the DNA construct) in its site downstream of
D-LDH-DNA.
[0113] As a result of introduction of the DNA construct, a protein
encoded by D-LDH-DNA is generated. Upon disruption of the pyruvate
decarboxylase gene of yeast, D-LDH is introduced under the control
of the promoter of the gene or a homolog thereof. This can result
in the production of D-LDH in a type of yeast that does not
originally produce D-lactic acid, which in turn results in the
production of D-lactic acid.
[0114] More particularly, introduction of D-LDH-DNA into a site
downstream of the promoter of the pyruvate decarboxylase 1 gene of
yeast (specifically, Saccharomyces, and typically Saccharomyces
cerevisae) or a homolog thereof can result in selective production
of D-lactic acid. It is deduced that if D-LDH-DNA encodes a protein
having D-LDH activity, the expression of D-LDH is enhanced with the
aid of such promoter, which also positively affects the enhanced
production of D-lactic acid. In contrast, D-LDH-DNA encodes a
protein consisting of the amino acid sequence as shown in SEQ ID
NO: 2. This is deduced to result in the enhanced and/or selective
production of D-lactic acid.
[0115] Whether or not D-LDH-DNA has been introduced into a site
downstream of a desired promoter can be confirmed via PCR, Southern
hybridization, or other means. For example, DNA may be prepared
from a transformant, such DNA may be subjected to PCR using a
primer for site-directed mutagenesis, and the PCR product may be
then subjected to electrophoresis to detect an expected band.
Alternatively, confirmation can be made via PCR using a primer
labeled with a fluorescent dye or the like. Confirmation can also
be made based on the protein produced by the transformant. These
techniques are known in the art.
[0116] It is preferable to prepare a transformant into which
multicopy D-LDH genes have been introduced via introduction of a
DNA construct. When a yeast transformant is prepared, for example,
at least 2 copies and preferably 4 to 10 copies of D-LDH-DNAs are
introduced. A transformant into which multicopy D-LDH genes have
been introduced has significantly improved capacity for D-lactic
acid production. That is, the use of a transformant into which
multicopy D-LDH genes have been introduced can result in
significantly improved D-lactic acid productivity.
(Production of D-Lactic Acid)
[0117] Culturing of the transformant into which the DNA construct
of the present invention has been introduced leads to the
generation of D-LDH, which is the expression product of a foreign
gene, in the culture product. It further leads to the generation of
D-lactic acid. Lactic acid can be obtained by performing a step of
separating lactic acid from the culture product. In the present
invention, examples of the culture product include a cultured cell
or bacterium and a disrupted cell or bacterium in addition to the
culture supernatant.
[0118] In the present invention, D-lactic acid can be selectively
produced using, for example, a type of yeast cell that does not
originally produce D-lactic acid as a host. Use of fermentation
microorganisms is particularly effective for obtaining D-lactic
acid. A transformant resulting from a yeast host is effective for
enhanced production of D-lactic acid because of its fast growth
rate. With the use of such transformant having the capacity for
selective production of D-lactic acid, the need for separating an
optical isomer is eliminated, which in turn results in more
effective production of D-lactic acid.
[0119] The transformant of the present invention can be cultured
under adequate conditions in accordance with the type of
transformant. Such adequate conditions are known in the art.
[0120] As a medium for culturing the transformant obtained from a
microorganism host such as E. Coli or yeast, either a natural or
synthetic medium may be used as long as it contains carbon sources,
nitrogen sources, and inorganic salts assimilable by the
microorganism and is capable of efficiently culturing the
transformant. Examples of carbon sources include: carbohydrates
such as glucose, fructose, sucrose, starch, and cellulose; organic
acids such as acetic acid and propionic acid; and alcohols such as
ethanol and propanol. Examples of nitrogen sources include:
ammonia; ammonium salts of inorganic or organic acids such as
ammonium chloride, ammonium sulfate, ammonium acetate, and ammonium
phosphate; other nitrogen-containing compounds; peptone; meat
extract; and corn steep liquor. Examples of inorganic substances
include: monopotassium phosphate, magnesium phosphate, magnesium
sulfate, sodium chloride, iron(I) sulfate, manganese sulfate,
copper sulfate, and calcium carbonate.
[0121] Usually, culture is carried out under aerobic conditions,
such as shake culture or aeration agitation culture, at 30.degree.
C. for an adequate period of time. For example, culture can be
carried out for 6 to 120 hours. During the culture, the pH is
preferably maintained at 2.0 to 6.0. The pH can be adjusted with an
inorganic or organic acid, an alkali solution, or the like.
[0122] Examples of media for culturing a transformant obtained from
an animal host cell include common RPMI 1640 medium, DMEM medium,
and a medium prepared by adding fetal bovine serum or the like to
the aforementioned medium. Usually, culture is carried out in the
presence of 5% CO.sub.2 at 37.degree. C. for 1 to 30 days. During
the culture, an antibiotic such as kanamycin or penicillin may be
added to the medium, if necessary.
[0123] Culture may be carried out via a batch or continuous system.
Culture may be carried out by a method wherein the transformant is
subjected to neutralization with an alkali such as ammonium or
calcium salt to obtain lactates such as ammonium-D-lactate or
calcium D-lactate. Alternatively, a culture product may be free
D-lactic acid.
[0124] After the completion of the culture, D-lactic acid, which is
the gene product, can be separated from the culture product via
suitable combinations of common purification techniques and the
like. When D-lactic acid is produced in transformed cells, for
example, cells are disrupted via conventional techniques such as
ultrasonic disintegration, grinding, or pressure disruption to
separate the gene product from such cells. In such a case, protease
is added according to need. When D-lactic acid is produced in the
culture supernatant, this solution is subjected to filtration,
centrifugation, or other means to remove solid components.
[0125] After the completion of the step of culturing, for example,
a culture solution can be subjected to a step of solid-liquid
separation via at least one of belt press, centrifugation, or
filter press. The separated filtrate is preferably subjected to a
step of purification. In the step of purification, for example, a
lactic acid-containing filtrate can be subjected to electrodialysis
to remove organic acids other than lactic acid and saccharides.
Thus, an aqueous solution of lactic acid or ammonium lactate can be
prepared. In the case of an ammonium lactate solution, ammonia is
degraded via a bipolar membrane or the like to separate an aqueous
solution of lactic acid from aqueous ammonia. When the content of
organic acids other than lactic acid or that of saccharides in the
filtrate is relatively small, electrodialysis is not to be
performed. In such a case, the filtrate may be concentrated by
vaporizing its moisture content according to need, and ammonia can
be degraded via a bipolar membrane.
[0126] The temperature of a solution at the time of electrodialysis
is generally in the range of 20.degree. C. to 45.degree. C. and
preferably in the range of 35.degree. C. to 40.degree. C. Amino
acids, inorganic ions (e.g., K, Ca, or Mg), and organic acids
(e.g., citric acid or malic acid) that were not removed via
electrodialysis can be removed with the use of a chromatographic
separator or ion exchanger at a later stage. Further, the resulting
lactic acid solution can be concentrated, if necessary. For
example, moisture in the solution can be evaporated to obtain a 50%
to 90% lactic acid solution.
[0127] Techniques for separating and purifying D-lactic acid or a
salt thereof from the culture solution or the crude extract are not
limited to the aforementioned. A variety of purification and
separation techniques, such as separation and extraction or
distillation with the use of an organic solvent, can be employed to
separate and purify D-lactic acid or a salt thereof If necessary,
the culture solution, the crude extract, and a purified product
thereof can be subjected to esterification, lactide conversion,
oligomerization, prepolymerization, or the like. Thus, various
D-lactic acid derivatives can be obtained. According to need, one
or more of D-lactic acid, a salt thereof, and a derivative thereof
can be recovered from a solution of fermented lactic acid.
[0128] D-lactic acid can also be produced via an enzyme reaction
system instead of a culture system with the use of the protein
having D-LDH activity of the present invention. D-lactic acid can
be produced under any enzyme reaction conditions as long as they
allow D-lactic acid to be produced. A variety of induction
techniques can be applied to D-lactic acid obtained by such
techniques.
(Production of Lactic Acid Polymer)
[0129] The obtained D-lactic acid, a salt thereof, and a derivative
thereof can be used as at least one type of polymerization material
to produce a lactic acid polymer. Examples of polymerization
materials that can be used include monomers such as D-lactic acid
or derivatives thereof and prepolymers or oligomers resulting from
polymerization of such monomers to adequate lengths. Further,
L-lactic acid or derivatives thereof and prepolymers or oligomers
thereof can also be used.
[0130] Examples of lactic acid polymers include homopolymers of
D-lactic acids, heteropolymers of D-lactic acid and L-lactic acid,
hetero-block polymers, and various types of heteropolymers of
lactic acids and other polymerization materials.
[0131] These lactic acid polymerization materials, or a lactic acid
polymerization material and another polymerization material, may be
allowed to react with an adequate polymerization initiator to
produce lactic acid polymers.
[0132] According to the present invention, selective and/or
enhanced production of D-lactic acid is possible. Thus, D-lactic
acid can be effectively obtained, which results in effective
production of lactic acid polymers that comprise D-lactic acid as a
polymerization material.
EXAMPLES
[0133] Hereafter, the examples of the present invention are
described, although the present invention is not limited thereto. A
variety of modification can be made within the scope of the present
invention.
Example 1
Isolation of D-Lactate Dehydrogenase Gene
[0134] The D-lactate dehydrogenase genes derived from prokaryotic
lactic acid bacteria, Leuconostoc mesenteroides (D-LDH genes,
hereafter they may be simply referred to as "D-LDHME genes"), were
isolated.
[0135] The genomic DNA of the strain IFO3426 (registered with the
Institute for Fermentation) was used as a template to isolate the
gene resource via PCR amplification. The genomic DNA of this strain
was prepared using a genome DNA preparation kit (Fast DNA Kit, Bio
101) in accordance with the protocol included with the kit. The
prepared genomic DNA was subjected to DNA concentration measurement
using the Ultrospec 3000 spectrophotometer (Amersham Pharmacia
Biotech).
[0136] The KOD Plus DNA Polymerase (Toyobo Co., Ltd.), which is
perceived as having high accuracy, was used as the amplification
enzyme in PCR. The reaction solution (50 .mu.l in total) comprising
50 ng of the previously prepared genomic DNA, 50 pmol of .times.2
primer DNA, 5 .mu.l of 10.times. buffer for KOD enzyme reaction, 2
.mu.l of 25 mM MgSO.sub.4, 5 .mu.l of 2 mM dNTP mix, and 1.0 unit
of KOD plus DNA polymerase was subjected to DNA amplification using
a PCR amplification apparatus (Gene Amp PCR system 9700, PE Applied
Biosystems).
[0137] PCR was carried out under the following conditions. Heat
treatment was first carried out at 96.degree. C. for 2 minutes,
followed by a 3-stage temperature change cycle of 96.degree. C. for
30 seconds, 53.degree. C. for 30 seconds, and 72.degree. C. for 90
seconds. This cycle was repeated 25 times, and the temperature was
reduced to 4.degree. C. at the end. The reaction sample (5 .mu.l)
was electrophoresed on 1% TBE agarose gel (containing 0.5 .mu.g/ml
ethidium bromide), and DNA bands were detected by irradiating the
gel with ultraviolet rays of 254 nm (Funakoshi) to confirm the
amplified gene fragment.
[0138] The synthetic DNA primers (Sawady Technology) were used for
the reaction, and the DNA sequences of these primers are as shown
below. TABLE-US-00002 DLDEME-U (21 mer, Tm value of 57.2.degree.
C.) (SEQ ID NO: 6) 5'-ATG AAG ATT TTT GCT TAC GGC-3' DLDEME-U (24
mer, Tm value of 54.7.degree. C.) (SEQ ID NO: 7) 5'-ATC TTA ATA TTC
AAC AGC AAT AGC-3'
[0139] The PCR-amplified fragment was subcloned into the
pBluescriptII SK+ vector (Toyobo Co., Ltd.). The reaction was
carried out in accordance with a general technique for DNA
subcloning. Specifically, the amplified gene fragment obtained in
Example 1 was ligated to the aforementioned vector, which was
processed with the EcoRV restriction enzyme (Takara Shuzo Co.,
Ltd.) and the Alkaline Phosphatase dephosphorylation enzyme (Takara
Shuzo Co., Ltd.), using T4 DNA ligase. The reaction using T4 DNA
ligase was carried out using LigaFast Rapid DNA Ligation (Promega)
in accordance with the protocol included therewith.
[0140] Subsequently, the ligation reaction solution was transformed
into E. coli competent cells. JM109 competent E. coli cells (Toyobo
Co., Ltd.) were used, and transformation was carried out in
accordance with the protocol included therewith. Colony selection
was carried out on an LB plate that contains 100 .mu.g/ml
ampicillin, plasmid DNA was prepared from each selected colony, and
the plasmid DNA was subjected to PCR using the aforementioned
primer DNA to subclone the D-LDH gene of interest. Ethanol
precipitation, restriction enzyme treatment, and the like were
carried out in accordance with the instructions described in
Molecular Cloning: A Laboratory Manual, 2nd Ed., Maniatis et al.,
Cold Spring Harbor Laboratory Press, 1989.
[0141] The nucleotide sequence of the D-LDH gene thus obtained was
determined. The ABI PRISM 310 Genetic Analyzer (PE Applied
Biosystems) was used as an apparatus for nucleotide sequence
analysis, and the method for sample preparation, the use of
apparatus, and other conditions were determined in accordance with
the instructions included with the apparatus. The vector DNA
containing the isolated D-LDH gene was prepared by the alkaline
extraction method, then the vector DNA was subjected to column
purification using the GFX DNA Purification kit (Amersham Pharmacia
Biotech), followed by the DNA concentration was measured using the
Ultrospec 3000 spectrophotometer (Amersham Pharmacia Biotech), and
the vector DNA, the concentration of which had been adjusted, was
used.
[0142] The DNA sequence determined via sequence analysis is shown
in SEQ ID NO: 1, and the corresponding amino acid sequence is shown
in SEQ ID NO: 2.
[0143] The nucleotide sequence of the D-LDHME gene isolated in the
present example was compared with the sequence of the D-LDH gene
that has been already registered with GenBank (GenBank Accession
No. L29327, derived from the lactic acid bacteria Leuconostoc
mesenteroides, SEQ ID NO: 3). This revealed that these sequences
were different from each other in terms of 19 amino acid residues
at the amino acid sequence level and in terms of 27-bp nucleotides
at the nucleotide sequence level.
[0144] FIG. 1A and FIG. 1B show the homology data of the nucleotide
sequence (SEQ ID NO: 1) of the D-LDHME gene that was obtained in
the present example and the nucleotide sequence (SEQ ID NO: 3) of
the D-LDH gene that has been registered with GenBank. The upper
sequence shows the nucleotide sequence of the D-LDHME gene that was
obtained in the present example, and the lower sequence shows the
nucleotide sequence of the D-LDH gene that has been registered with
GenBank.
[0145] FIG. 2 shows the homology data of the amino acid sequence
(SEQ ID NO: 2) corresponding to the nucleotide sequence of the
D-LDHME gene that is obtained in the present example and the amino
acid sequence (SEQ ID NO: 4) corresponding to the nucleotide
sequence of the D-LDH gene that has been registered with GenBank.
The upper sequence shows the amino acid sequence corresponding to
the D-LDHME gene that was obtained in the present example, and the
lower sequence shows the amino acid sequence corresponding to the
D-LDH gene that has been registered with GenBank.
[0146] Based on the amino acid sequences, the amino acid sequences
of the genes obtained in the present example were found to have
amino acid residue substitutions as shown in Table 2.
TABLE-US-00003 TABLE 2 List of amino acid residue substitutions
Substitution type Position of substitution Amino acid substituent 1
40 Valine (Val) 2 112 Isoleucine (Ile) 3 131 Histidine (His) 4 139
Isoleucine (Ile) 5 181 Glutamic acid (Glu) 6 266 Glycine (Gly) 7
267 Leucine (Leu) 8 268 Phenylalanine (Phe) 9 269 Asparagine (Asn)
10 270 Glutamic acid (Glu) 11 271 Aspartic acid (Asp) 12 272
Tryptophan (Trp) 13 273 Serine (Ser) 14 274 Glycine (Gly) 15 276
Glutamic acid (Glu) 16 277 Phenylalanine (Phe) 17 287 Serine (Ser)
18 292 Leucine (Leu) 19 293 Valine (Val)
[0147] In this table, positions of substitution are indicated as
the positions from methionine, which corresponds to the initiation
codon.
Example 2
Construction of Recombinant Vector
[0148] A chromosomally integrated vector capable of expressing a
target gene, i.e., the D-LDHME gene obtained in Example 1, was
constructed. This vector is capable of expressing the target gene
under the control of the promoter sequence of the pyruvate
decarboxylase 1 gene (PDCI) derived from Saccharomyces cerevisae.
Such newly constructed and chromosomally integrated vector was
designated as the pBTRP-PDC1-DLDHME vector. Construction of the
vector was carried out in accordance with the general technique for
DNA subcloning.
[0149] Hereafter, the process of vector construction in the present
example is described in detail with reference to FIGS. 3 to 6.
[0150] All the enzymes used for vector construction were
manufactured by Takara Shuzo Co., Ltd. It should be noted that the
possible vector construction processes are not limited to this
process.
1. Isolation of a Promoter Fragment of the PDC1 Gene (PDC1P) and a
Downstream Fragment of the PDC1 gene (PDC1D)
[0151] The 971-bp promoter fragment of the PDC1 gene (PDC1P) and
the 518-bp downstream fragment of the PDC1 gene (PDC1D), which were
essential for vector construction, were isolated from the gene
resource, i.e., the Saccharomyces cerevisae strain IFO2260, via PCR
amplification that used the genomic DNA of this strain as a
template. The strain IFO2260 is registered with the Institute for
Fermentation. The genomic DNA of this strain was prepared using a
genome DNA preparation kit (Fast DNA Kit, Bio 101) in accordance
with the protocol included with the kit. The prepared genomic DNA
was subjected to DNA concentration measurement using the Ultrospec
3000 spectrophotometer (Amersham Pharmacia Biotech).
[0152] The KOD Plus DNA Polymerase (Toyobo Co., Ltd.), which is
perceived as having high accuracy, was used as the amplification
enzyme in PCR. The reaction solution (50 .mu.l in total) comprising
50 ng of the previously prepared genomic DNA of the strain IFO2260,
50 .mu.mol of primer DNA.times.2, 5 .mu.l of 10.times. buffer for
KOD enzyme reaction, 2 .mu.l of 25 mM MgSO.sub.4, 5 .mu.l of 2 mM
dNTP mix, and 1.0 unit of KOD plus DNA polymerase was subjected to
DNA amplification using a PCR amplification apparatus (Gene Amp PCR
system 9700, PE Applied Biosystems).
[0153] PCR was carried out under the following conditions. Heat
treatment was first carried out at 96.degree. C. for 2 minutes,
followed by a 3-stage temperature change cycle of 96.degree. C. for
30 seconds, 53.degree. C. for 30 seconds, and 72.degree. C. for 90
seconds. This cycle was repeated 25 times, and the temperature was
reduced to 4.degree. C. at the end. The reaction sample (5 .mu.l)
was electrophoresed on 1% TBE agarose gel (containing 0.5 .mu.g/ml
ethidium bromide), and DNA bands were detected by irradiating the
gel with ultraviolet rays of 254 nm (Funakoshi) to confirm the
amplified gene fragment.
[0154] The synthetic DNA primers (Sawady Technology) were used for
the reaction, and the DNA sequences of these primers are as shown
below. TABLE-US-00004 [Primers for PDC1P fragment amplification]
PDC1P-LDH-U (31 mer, Tm value of 58.3.degree. C.) (SEQ ID NO: 8)
5'-ATA TAT GGA TCC GCG TTT ATT TAC CTA TCT C-3' (underlined
portion: BamHI site) PDC1P-LDH-D (31 mer, Tm value of 54.4.degree.
C.) (SEQ ID NO: 9) 5'-ATA TAT GAA TTC TTT GAT TGA TTT GAC TGT G-3'
(underlined portion: EcoRI site) [Primers for PDC1D fragment
amplification] PDC1D-LDH-U (31 mer, Tm value of 55.3.degree. C.)
(SEQ ID NO: 10) 5'-ATA TAT CTC GAG GCC AGC TAA CTT CTT GGT CGA C-
3' (underlined portion: XhoI site) PDC1D-LDH-D (31 mer, Tm value of
65.2.degree. C.) (SEQ ID NO: 11) 5'-ATA TAT GGG CCC CCC CTC GAG GTC
CCC CCT C-3' (underlined portion: ApaI site)
[0155] The amplified fragments of the PDC1P and PDC1D genes
obtained in the above reaction were purified via ethanol
precipitation, and the amplified PDC1P fragment and the amplified
PDC1D fragment were cleaved with the BamHI and EcoRI restriction
enzymes and the XhoI and ApaI restriction enzymes, respectively.
Ethanol precipitation and restriction enzyme cleavage were carried
out in accordance with the instructions described in Molecular
Cloning: A Laboratory Manual, 2nd ed., Maniatis et al., Cold Spring
Harbor Laboratory Press, 1989.
2. Construction of pBPDC1P Vector
[0156] The PDC1P fragment, which was amplified by PCR and cleaved
with restriction enzymes, was ligated to the pBluescriptll SK+
vector (Toyobo Co., Ltd.), which was processed with the restriction
enzymes BamHI and EcoRI (Takara Shuzo Co., Ltd.) and the Alkaline
Phosphatase dephosphorylation (BAP) enzyme (Takara Shuzo Co.,
Ltd.), using T4 DNA ligase (FIG. 3, upper portion). The reaction
using T4 DNA ligase was carried out using LigaFast Rapid DNA
Ligation (Promega) in accordance with the protocol included
therewith.
[0157] Subsequently, the ligation reaction solution was transformed
into E. coli competent cells. JM109 competent cells (Toyobo Co.,
Ltd.) were used, and transformation was carried out in accordance
with the protocol included therewith. The resulting culture
solution was sowed on an LB plate containing 100 .mu.g/ml of
antibiotic ampicillin, and culture was conducted overnight. The
grown colony was subjected to colony PCR using primer DNA, and the
prepared plasmid DNA solution via miniprep method was subjected to
restriction enzyme processing to confirm the inserted fragment and
then the target pBPDC1P vector was isolated (FIG. 3, middle
portion).
3. Construction of pBPDC1P-LDHI Vector
[0158] As shown in FIG. 3, a fragment of the LDH gene (derived from
Bifidobacterium longum) that can be obtained by treating the pYLD1
vector constructed by Toyota Motor Corporation (JP Patent
Publication (Kokai) No. 2001-204468 (A)) with the EcoRI and AatII
restriction enzymes and the end-modification enzyme, i.e., T4 DNA
polymerase, was subcloned into the pBPDC1P vector that has been
similarly processed with the EcoRI restriction enzyme and the
end-modification enzyme, i.e., T4 DNA polymerase, in the manner as
described above. Thus, the pBPDC1P-LDHI vector was prepared (FIG.
3, middle to lower portions). The aforementioned pYLD1 vector was
introduced into E. coli (name: "E. coli pYLD1") and deposited
internationally under the Budapest Treaty at the International
Patent Organism Depositary of the National Institute of Advanced
Industrial Science and Technology (Tsukuba Central 6, 1-1-1
Higashi, Tsukuba, Ibaraki, 305-8566, Japan) as of Oct. 26, 1999,
under the accession number: FERM BP-7423 (the original
deposit).
4. Construction of pBPDC1P-LDH Vector
[0159] As shown in FIG. 4, this vector was processed with the XhoI
and ApaI restriction enzymes, and the amplified PDC1D fragment,
which had been similarly processed with restriction enzymes, was
ligated thereto to prepare the pBPDC1P-LDHII vector (FIG. 4, upper
portion).
[0160] Subsequently, the pBPDC1P-LDHII vector was processed with
EcoRV and T4 DNA poymerase, and the Trp marker fragment, which was
obtained by processing the pRS404 vector (Promega) with AatII and
SspI and T4 DNA polymerase, was ligated thereto to prepare the
pBTRP-PDC1-LDH vector (FIG. 4, lower portion).
5. Construction of pBTRP-PDC1PII Vector
[0161] As shown in FIG. 5, the existing pBPDC1P vector was
processed with the HincII restriction enzyme and the Alkaline
Phosphatase dephosphorylation enzyme. The pBTrp-PDC1-LDH vector was
processed with the ApaI and AJ7I1 restriction enzymes and then with
the end-modification enzyme, i.e., T4 DNA polymerase, to produce a
fragment containing trp marker. This fragment was ligated to the
processed pBPDC1P vector to construct the pBTRP-PDC1PII vector.
6. Construction of pBTRP-PDC1-DLDHME vector
[0162] As shown in FIG. 6, the pBTRP-PDC1PII vector was processed
with the EcoRV restriction enzyme and then with the
end-modification enzyme, i.e., T4 DNA polymerase, and the fragment
of the D-LDHME gene isolated in Example 1 was ligated thereto to
construct the final vector, i.e., the chromosomally integrated
pBTRP-PDC1-DLDHME vector.
[0163] As other D-LDH genes, oligonucleotides were synthesized in
accordance with the gene sequence (SEQ ID NO: 3) of the GenBank
Accession No. L29327 in the database of the D-LDH genes derived
from lactic acid bacteria Leuconostoc mesenteroides. These
oligonucleotides were successively ligated to fully synthesize the
target D-LDH genes. The resulting gene fragments were subjected to
the same procedures as those in the present example to construct a
chromosomally integrated vector.
Example 3
Transformation of Yeast
[0164] The yeast host strain IFO2260 (registered with the Institute
for Fermentation) lacking the capacity for tryptophan synthesis was
cultured in 10 ml of YPD medium at 30.degree. C. until the
logarithmic growth phase, and cells were collected and washed with
TE buffer. Subsequently, 0.5 ml of TE buffer and 0.5 ml of 0.2 M
lithium acetate were added, the resultant was subjected to shake
culture at 30.degree. C. for 1 hour, and pBTRP-PDC1-DLDHME that had
been processed with the ApaI and SpeI restriction enzymes (Takara
Shuzo Co., Ltd.) was then added.
[0165] The resulting suspension was subjected to shake culture at
30.degree. C. for 30 minutes, 150 .mu.l of 70% polyethylene glycol
4000 (Wako Pure Chemical Industries, Ltd.) was added thereto, and
the mixture was thoroughly stirred. The resultant was further
subjected to shake culture at 30.degree. C. for 1 hour, the culture
product was treated with a heat shock of 42.degree. C. for 5
minutes, and the cells were then cultured in 1 ml of YPD culture
medium at 30.degree. C. for 12 hours. The culture solution was
washed and then suspended in 200 .mu.l of sterilized water. The
resulting suspension was then smeared on a tryptophan selection
medium.
[0166] The resulting colony was isolated again in a new tryptophan
selection medium, and strains that maintained the capacity for
tryptophan synthesis were selected as candidate transformants.
These strains were cultured in an YPD culture solution and genomic
DNA thereof was prepared using a genome DNA preparation kit (Fast
DNA Kit, Bio 101). The genomic DNA was subjected to PCR to confirm
the presence or absence of the transgenes. As a result, strains
having the D-LDHME genes introduced downstream of the PDC1 promoter
were found.
[0167] The obtained strains into which transgenes had been
introduced were smeared on a sporulation-inducing medium, and
sporulation was induced at 30.degree. C. for 4 days. Cells were
collected from the medium, 5 units of Zymolyase (Zymo Research
Corp.) were added thereto, the resultant was subjected to enzyme
reaction at 37.degree. C. for 1 hour, and spores were then
separated on YPD medium using a microscope (Olympus Corporation)
and a micromanipulator (Narishige Scientific Instrument
Laboratory). The progeny strains of the obtained spores were
inspected for the capacity for tryptophan marker selection, and PCR
was carried out to confirm that they showed 2:2 segregation. Thus,
the target diploid strains were obtained. Diploid strains were the
TC14-6-1A, TC14-6-2A, and TC14-6-3A strains. The obtained diploid
strains had the structures shown in FIG. 7 in the yeast
chromosomes.
[0168] Concerning the chromosomally integrated vector of other
D-LDH genes constructed in Example 2, gene introduction into the
strain IFO2260 lacking the capacity for tryptophan synthesis was
similarly carried out, and the TC20-1-1A strains having
chromosomally integrated D-LDH were found via PCR.
[0169] As the control, DLDHME was introduced into the pYPD1
plasmid, which is a yeast self-replicating 2.mu. plasmid vector, to
construct a self-replicating vector. The resulting vector was
similarly introduced into the strain IFO2260, and the strain TC21-1
was found to have plasmids containing D-LDH genes via PCR.
Example 4
Confirmation of Production of D-Lactic Acid in a Transformant
[0170] The 5 types of transformants prepared in Example 3 and the
parent strain IFO2260 were subjected to the fermentation test.
These strains were inoculated into 5 ml of YPD liquid medium,
subjected to shake culture at 30.degree. C. and 130 rpm overnight,
and cells necessary for fermentative production were prepared.
[0171] The inoculated cells were collected, the collected cells
were inoculated into YPD liquid medium containing 10% glucose to a
cell concentration of 0.5%, and stationary fermentation was carried
out at 30.degree. C. for 4 days. In this fermentation test, 2 types
of production forms were tested. That is, production with the use
of D-lactate prepared by adding 2.5% calcium carbonate (Nacalai
Tesque Inc.) to a fermented mash and production with the use of
free D-lactic acid without calcium carbonate were tested.
[0172] The fermented mash was collected 4 days after the initiation
of fermentation, and the amounts of D-lactic acid and ethanol
contained in the mash were measured using a multifunctional
biosensor BF-4 (Oji Scientific Instruments). D-lactic acid was
measured using the kit for measuring D-lactic acid (Oji Scientific
Instruments) in accordance with the instructions included with the
kit. The results are shown in FIG. 8 and FIG. 9.
[0173] As shown in FIG. 8 and FIG. 9, the parent yeast strain
(IFO2260) into which no genes had been introduced produced ethanol
but did not produce D-lactic acid. In contrast, the 4 types of
transformed yeast strains having chromosomally integrated
transgenes prepared in Example 3 exhibited lower ethanol production
than the parent strain but produced D-lactic acid.
[0174] Specifically, 4 types of strains, i.e., TC14-6-1A,
TC14-6-2A, and TC14-6-3A, which are transformants into which
D-LDHME genes had been introduced downstream of the PDC1 promoter
on the yeast chromosome, and TC20-1-1A into which the D-LDH genes
had been introduced, produced D-lactic acid at concentrations of 4%
to 6% and ethanol at concentrations of 2% to 3%. These
transformants having chromosomally integrated transgenes did not
produce L-lactic acid.
[0175] In contrast, the strains (TC21-1) into which D-LDHME genes
had been introduced with the aid of self-replicating plasmids
produced a very small amount of D-lactic acid. The amount of
ethanol produced was the same as that of the strain IFO2260.
[0176] Accordingly, transformants having chromosomally integrated
transgenes were found to be effective for D-LDH expression and
D-lactic acid production. In particular, introduction of D-LDH
genes (including D-LDHME genes) under the control of the PDC1
promoters was found to be effective for the enhanced production of
D-lactic acids.
[0177] Among the 4 aforementioned types of transformants having
chromosomally integrated transgenes, 3 strains into which the
D-LDHME genes had been incorporated downstream of the PDC1
promoters exhibited high D-lactic acid productivity of 5% or 6% but
low ethanol productivity of 2%, i.e., the productivity of D-lactic
acid was 2 or 3 times as high as that of ethanol. In the case of
the transformant into which the D-LDH gene (GenBank Accession No.
L29327) had been incorporated downstream of the PDC1 promoter,
D-lactic acid productivity was as low as approximately 4%, which
was substantially the same as or approximately 1.3 times higher
than ethanol productivity. Accordingly, D-LDHME genes were found to
be effective for the enhanced production of D-lactic acid in
transformants having chromosomally integrated transgenes.
[0178] The level of enhanced D-lactic acid production was
approximately the same in the case of D-lactate (calcium) and in
the case of free L-lactic acid.
Example 5
Gene Recombinant Yeast Strain Having Increased D-LDHME Copy
Numbers
[0179] Among the D-lactic-acid-producing yeast strains prepared in
Example 4, the strain TC14-6-3A was used to prepare a gene
recombinant yeast strain having an increased copy number of the
D-LDHME genes introduced. The D-LDHME genes were introduced into
the strain TC14-6-3A in accordance with the method described in
Example 3. Specifically, the D-LDHME genes were introduced into the
strain TC14-6-3A in the same manner as in Example 3, except that
the strain TC14-6-3A was used instead of the host yeast strain
IFO2260.
[0180] The obtained strains into which genes had been introduced
were designated as the strains TD1-10-1B, TD1-10-3B, TD1-10-6A, and
TD1-10-7D. The copy numbers of D-LDHME genes introduced in these 4
strains were inspected in the same manner as in Example 3. As a
result, the copy number of the D-LDHME genes introduced was found
to be 4.
[0181] Subsequently, these 4 types of strains were subjected to the
fermentation test in accordance with the method described in
Example 4 to inspect the amount of D-lactic acid produced. The
results are shown in FIG. 10. As shown in FIG. 10, the amounts of
D-lactic acids produced by the strains TD1-10-1B, TD1-10-3B,
TD1-10-6A, and TD1-10-7D obtained in the present example were
increased from the amount of D-lactic acid produced (4.65%) by the
parent strain TC14-6-3A. All of the 4 strains obtained in the
present example exhibited a decreased amount of ethanol production,
compared to the parent strain.
[0182] This indicates that the capacity for D-lactic acid
production of the transformed yeast strains could be improved by
increasing the copy number of the D-LDHME genes introduced. In the
present example, the transformed yeast strains into which 4 copies
of D-LDHME genes had been introduced were examined. It should be
noted that introduction of a larger copy number of D-LDHME genes
can result in enhanced production of D-lactic acid. More
specifically, the present example revealed that transformed yeast
strains having superior capacity for D-lactic acid production can
be prepared by increasing the copy number of the D-LDHME genes
introduced.
[0183] All publications, patents, and patent applications cited
herein are incorporated herein by reference in their entirety.
INDUSTRIAL APPLICABILITY
[0184] The present invention provides a technique for effectively
producing D-lactic acid.
Sequence Listing Free Text
SEQ ID NOs: 6 to 11: artificial DNAs (primers)
* * * * *
References