U.S. patent application number 14/178496 was filed with the patent office on 2014-09-11 for gene expression cassette and a transformant, and a method for manufacturing 2-deoxy-scyllo-inosose and a method for purifying 2-deoxy-scyllo-inosose using said transformant.
This patent application is currently assigned to MITSUI CHEMICALS, INC.. The applicant listed for this patent is MITSUI CHEMICALS, INC., NIIGATA BIO-RESEARCH PARK, INC.. Invention is credited to Katsumi Ajisaka, Masao Hirayama, Takahisa Kogure, Tatsuo Miyazaki, Masamichi TAKAGI, Hiroaki Takaku, Naoki Wakisaka.
Application Number | 20140256960 14/178496 |
Document ID | / |
Family ID | 37086767 |
Filed Date | 2014-09-11 |
United States Patent
Application |
20140256960 |
Kind Code |
A1 |
TAKAGI; Masamichi ; et
al. |
September 11, 2014 |
GENE EXPRESSION CASSETTE AND A TRANSFORMANT, AND A METHOD FOR
MANUFACTURING 2-DEOXY-SCYLLO-INOSOSE AND A METHOD FOR PURIFYING
2-DEOXY-SCYLLO-INOSOSE USING SAID TRANSFORMANT
Abstract
A transformant is prepared to insert at least a gene expression
cassette comprising a gene involved in the synthesis of
2-deoxy-scyllo-inosose into E. coli as host cells. A
2-deoxy-scyllo-inosose is synthesized from D-glucose,
oligosaccharide, polysaccharide, starch and rice bran, using the
transformant. A culture solution containing the
2-deoxy-scyllo-inosose is treated with a mixed bed or double bed
type column comprising a hydrogen form of strong acidic cation
exchange resin and an organic ion form of basic anion exchange
resin. The 2-deoxy-scyllo-inosose as purified is reacted with
trimethoxymethane to convert into 2-deoxy-scyllo-inosose
dimethylketal, and the dimethylketal is crystallized and purified.
Then, DOI is highly purified through hydrolyzing the dimethylketal
in the presence of acid.
Inventors: |
TAKAGI; Masamichi;
(Fuchu-shi, JP) ; Kogure; Takahisa; (Kashiwa-shi,
JP) ; Wakisaka; Naoki; (Niigata-shi, JP) ;
Takaku; Hiroaki; (Niigata-shi, JP) ; Ajisaka;
Katsumi; (Niigata-shi, JP) ; Miyazaki; Tatsuo;
(Niigata-shi, JP) ; Hirayama; Masao; (Niigata-shi,
JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
MITSUI CHEMICALS, INC.
NIIGATA BIO-RESEARCH PARK, INC. |
Tokyo
Niigata-shi |
|
JP
JP |
|
|
Assignee: |
MITSUI CHEMICALS, INC.
Tokyo
JP
NIIGATA BIO-RESEARCH PARK, INC.
Niigata-shi
JP
|
Family ID: |
37086767 |
Appl. No.: |
14/178496 |
Filed: |
February 12, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
11887445 |
Sep 28, 2007 |
8758741 |
|
|
PCT/JP2006/305782 |
Mar 23, 2006 |
|
|
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14178496 |
|
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Current U.S.
Class: |
549/292 ;
435/124 |
Current CPC
Class: |
C12Y 503/01009 20130101;
C12N 9/90 20130101; C07D 309/30 20130101; C12P 7/26 20130101; C12P
17/06 20130101; C12N 9/88 20130101; C12N 9/92 20130101 |
Class at
Publication: |
549/292 ;
435/124 |
International
Class: |
C12P 17/06 20060101
C12P017/06; C07D 309/30 20060101 C07D309/30 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 30, 2005 |
JP |
PCT/JP2005/006002 |
Claims
1.-14. (canceled)
15. A method for manufacturing 2-deoxy-scyllo-inosose, the method
comprising: contacting an isolated host cell with carbon source,
wherein the host cell comprises a gene expression cassette having a
gene encoding 2-deoxy-scyllo-inosose synthase with at least one
disrupted gene selected from the group consisting of pgi gene
encoding phosphoglucose isomerase, zwf gene encoding
glucose-6-phosphate 1-dehydrogenase, pgm gene encoding
phosphoglucomutase, and rmf gene encoding ribosome modulation
factor involved in modification of protein synthesis during
stationary phase, and wherein the host cell is Escherichia coli,
and 2-deoxy-scyllo-inosose is synthesized within the host cell.
16. The method for manufacturing 2-deoxy-scyllo-inosose according
to claim 15, wherein said carbon source is at least one type of
carbon sources selected from the group consisting of D-glucose,
oligosaccharide, polysaccharide, starch, cellulose, rice bran and
molasses and biomasses capable of obtaining D-glucose.
17. A composition comprising 2-deoxy-scyllo-inosose obtained from
the method for manufacturing 2-deoxy-scyllo-inosose according to
claim 15.
18. A method for purifying 2-deoxy-scyllo-inosose, the method
comprising: contacting an isolated host cell with a carbon source
to obtain a composition containing 2-deoxy-scyllo-inosose, wherein
the host cell comprises a gene expression cassette having a gene
encoding 2-deoxy-scyllo-inosose synthase with at least one
disrupted gene selected from the group consisting of pgi gene
encoding phosphoglucose isomerase, zwf gene encoding
glucose-6-phosphate 1-dehydrogenase, pgm gene encoding
phosphoglucomutase, and rmf gene encoding ribosome modulation
factor involved in modification of protein synthesis during
stationary phase, and wherein the host cell is Escherichia coli,
and 2-deoxy-scyllo-inosose is synthesized within the host cell; and
treating said composition with mixed bed column or double bed
column comprising a hydrogen ion form of a strong-acid cation
exchange resin and an organic acid ion form of a basic anion
exchange resin.
19. The method for purifying 2-deoxy-scyllo-inosose according to
claim 18, wherein said organic acid ion form of the basic anion
exchange resin is acetate ion form of anion exchange resin.
20. A composition of 2-deoxy-scyllo-inosose obtained from the
method for purifying 2-deoxy-scyllo-inosose according to claim
18.
21. A method for purifying 2-deoxy-scyllo-inosose according to
claim 18, further comprising: reacting the composition comprising
2-deoxy-scyllo-inosose with trialkoxymethanes to obtain
2-deoxy-scyllo-inosose dialkylketals; and hydrolyzing said
2-deoxy-scyllo-inosose dialkylketals in the presence of acid.
22. The method for purifying 2-deoxy-scyllo-inosose according to
claim 21, wherein said trialkoxymethanes are trimethoxymethane.
23. A composition comprising 2-deoxy-scyllo-inosose obtained from
the method for purifying 2-deoxy-scyllo-inosose according to claim
22.
Description
RELATED ART
[0001] The present invention relates to a gene expression cassette
and a transformant, and a method for manufacturing
2-deoxy-scyllo-inosose and a method for purifying
2-deoxy-scyllo-inosose using the transformant.
PRIOR ART
[0002] The six-membered carbocyclic compound has been manufactured
from petroleum as starting materials in petrochemistry. On the
other hand, 2-deoxy-scyllo-inosose synthase (DOI synthase) was
found in the butirosin-producer Bacillus circulans, wherein the
2-deoxy-scyllo-inosose synthase catalyzes a reaction of
synthesizing 2-deoxy-scyllo-inosose (hereinafter, referred also to
as DOI), which is one of six-membered carbocyclic compound, from
glucose-6-phosphate (G-6-P) as a substrate (see FIG. 1, and
Patent-related Document 1).
[0003] The DOI synthase is an enzyme involved in the biosynthesis
of the aminoglycoside antibiotics containing 2-deoxystreptamine as
an aglycon. DOI which is a product of the DOI synthase is a useful
substance as starting materials for medicine and
chemical-industrial resources. The method for chemically
synthesizing DOI is necessary to employ the multi-step reaction,
and use toxic or costly metals. On the other hand, by using the DOI
synthase, it is possible to efficiently and shortly manufacture
DOI. The method for shortly producing DOI from glucose-6-phosphate
has been established, using a recombinant DOI synthase obtained
from E. coli expressing the DOI synthase (see Patent-related
document 1). Further, it is known that DOI can be synthesized by
the two-steps enzyme reaction of acting glucose with hexokinase and
the DOI synthase, or by the one-step enzyme reaction of acting
glucose-6-phosphate with the DOI synthase (see Patent-related
document 1, and Non-Patent-related document 1). In addition, it has
been reported that DOI can be transformed into catechol by
concentrating the enzymatic reacting solution and acting it with
hydriodic acid in an acetic acid, without purifying DOI (see
Patent-related document 1).
[0004] However, a method for synthesizing DOI with the fermentation
from the biomass-derived D-glucose as starting materials using E.
coli introduced the DOI synthase has not been proposed as an
issue.
[0005] In addition, a method for purifying and isolating DOI itself
has not still been reported, although it has been reported that DOI
is synthesized by the enzymatic reaction and that DOI contained in
the reaction mixture is transformed. Further, there is not any
information involved in that DOI is purified from the nutrient
medium of microorganism containing amino acids derived from peptone
and several types of metallic ions in addition to several types and
large amounts of constituent elements of any medium, and glucose as
carbon source. That is, it has not been reported that DOI is
purified under the circumstance of contaminating substances
including the enzymatic reacting solution and the nutrient medium
of microorganisms. Still more, it has never been established an
industrially-applicable method for purifying DOI.
[0006] In order to purify DOI under the circumstance of the
contaminating substance including the nutrient medium of
microorganisms, a batch off by HPLC or a method using a charcoal
column chromatography is known as laboratorial methods. However, it
goes without saying that the batch off by HPLC is not useful for an
industrial production. In addition, in the method using the
charcoal column chromatography, after organic compounds in the
medium are adsorbed in the charcoal, it will be sequentially eluted
by utilizing the difference of adsorbability of substances, with
changing the concentration of organic solvents such as alcohols.
Therefore, in the production of large amounts of DOI, it is
necessary to use a large amount of charcoal enough to adsorb an
enormous proportion of organic compounds in the medium.
Accordingly, such a method is also unsuitable for a large-scale
purification. For such reasons, it has not been established a
suitable method for purifying DOI with an industrially-applicable
method. [0007] [Patent-related document 1] Japanese Patent
Application Publication No. 2000-236881 (JPB3122762) [0008]
[Non-patent-related document 1] K. Kakinuma, E. Nango, F. Kudo, Y.
Matsushima and T. Eguchi, Tetrahedron Letters, 2000, vol. 41, p.
1935-1938 [0009] [Non-patent-related document 2] Ota, Y. et al., J.
Antibiot., 2000, vol. 53, p. 1158-1167 [0010] [Non-patent-related
document 3] Kudo, F. et al., J. Antibiot., 1999, vol. 52, p.
559-571
DISCLOSURE OF THE INVENTION
A Problem to be Solved by the Invention
[0011] The present invention is made in view of the above-mentioned
problems. That is, the present invention is to provide a gene
expression cassette which enables a system for manufacturing DOI
with industrial-scale, a transformant comprising the gene
expression cassette, and a method for manufacturing
2-deoxy-scyllo-inosose and a method for purifying
2-deoxy-scyllo-inosose.
Means for Solving the Problem
[0012] The gene expression cassette according to the present
invention is characterized in:
[0013] A gene expression cassette comprising a gene involved in a
synthesis of 2-deoxy-scyllo-inosose.
[0014] In the gene expression cassette according to the present
invention, said gene involved in a synthesis of
2-deoxy-scyllo-inosose is 2-deoxy-scyllo-inosose synthase.
Herewith, it is possible to obtain a transformant which can
industrially manufacture DOI
[0015] In addition, the transformant according to the present
invention is characterized in:
[0016] A transformant comprising the above-mentioned gene
expression cassette transformed in a host cell.
[0017] In the transformant according to the present invention, said
host cell is a host cell selected from the groups consisting of
Escherichia coli and any of host cells stated in the GILSP
genetically-modified microorganisms list on March, 2006. Herewith,
it is possible to perform an industrial method for producing
DOI.
[0018] In the transformant according to the present invention, said
host cell is a host cell disrupted at least one of genes selected
from the group consisting of pgi gene encoding phosphoglucose
isomerase, zwf gene encoding glucose-6-phosphate 1-dehydrogenase,
pgm gene encoding phosphoglucomutase and rmf gene encoding ribosome
modulation factor involved in modification of protein synthesis
during stationary phase. Herewith, it is possible to perform an
industrial method for manufacturing DOI with high efficiency, in
addition to the above-mentioned matter.
[0019] On the other hand, the method for manufacturing
2-deoxy-scyllo-inosose according to the present invention is
characterized in:
[0020] A method for manufacturing 2-deoxy-scyllo-inosose comprising
a step of contacting the above-mentioned transformant with carbon
source. Herewith, it is possible to industrially manufacture
DOI.
[0021] In the method for manufacturing 2-deoxy-scyllo-inosose
according to the present invention, said carbon source is at least
one type of carbon sources selected from the group consisting of
D-glucose, oligosaccharide, polysaccharide, starch, cellulose, rice
bran and molasses and biomasses capable of obtaining D-glucose.
Herewith, it is possible to generally manufacture DOI, in addition
to the above-mentioned matter.
[0022] The 2-deoxy-scyllo-inosose according to the present
invention is characterized in:
[0023] A 2-deoxy-scyllo-inosose being obtained from the
above-mentioned method for manufacturing 2-deoxy-scyllo-inosose.
Herewith, it is possible to obtain 2-deoxy-scyllo-inosose with the
attribute of the manufacturing method using the transformant.
[0024] Further, the method for purifying 2-deoxy-scyllo-inosose
according to the present invention is characterized in:
[0025] A method for purifying 2-deoxy-scyllo-inosose comprising the
steps of:
[0026] contacting the above-mentioned transformant with a carbon
source to obtain a composition containing 2-deoxy-scyllo-inosose;
and
[0027] treating said composition with mixed bed column or double
bed column comprising a hydrogen ion form of a strong-acid cation
exchange resin and an organic acid ion form of a basic anion
exchange resin. Herewith, it is possible to industrially obtain a
highly purified DOI.
[0028] In the method for purifying 2-deoxy-scyllo-inosose according
to the present invention, said organic acid ion form of the basic
anion exchange resin is acetate ion form of anio exchange resin.
Herewith, the method can be practically and preferably used, since
acetic acid will be removed by concentration procedure.
[0029] The 2-deoxy-scyllo-inosose according to the present
invention is characterized in:
[0030] A 2-deoxy-scyllo-inosose being obtained from the
above-mentioned method for purifying 2-deoxy-scyllo-inosose.
Herewith, it is possible to obtain a high purity of
2-deoxy-scyllo-inosose with the attribute of the manufacturing
method using the transformant.
[0031] The method for purifying 2-deoxy-scyllo-inosose according to
the present invention is characterized in:
[0032] A method for purifying 2-deoxy-scyllo-inosose comprising the
steps of:
[0033] reacting the above-mentioned 2-deoxy-scyllo-inosose with
trialkoxymethanes to obtain 2-deoxy-scyllo-inosose dialkylketals;
and
[0034] hydrolyzing said 2-deoxy-scyllo-inosose dialkylketals in the
presence of acid. Herewith, it is possible to efficiently separate
the contaminating substances, in addition to the above-mentioned
matter.
[0035] In the method for purifying 2-deoxy-scyllo-inosose according
to the present invention, said trialkoxymethanes are
trimethoxymethane. Herewith, the method can be practically and
preferably used, since methanol which will be formed in the
following step of hydrolysis reaction can be easily removed by
concentration procedure.
[0036] The 2-deoxy-scyllo-inosose according to the present
invention is characterized:
[0037] A 2-deoxy-scyllo-inosose 2-deoxy-scyllo-inosose being
obtained from the above-mentioned method for purifying
2-deoxy-scyllo-inosose. Herewith, it is possible to obtain
2-deoxy-scyllo-inosose which is practically superior in view of
purity.
Effect of the Invention
[0038] The six-membered carbocyclic compound which is important for
the starting material of medicine and industrial chemistry have
been manufactured by the petroleum chemistry using the petroleum as
starting materials. However, it is possible to synthesize
2-deoxy-scyllo-inosose being the six-membered carbocyclic compound
from the regenerative biomass-derived D-glucose as starting
materials by microorganisms, by means of using an art of the
present invention. In addition, it is possible to restore purified
DOI as treated with the culture medium.
BRIEF EXPLANATION OF THE DRAWING
[0039] FIG. 1 is an illustration showing reaction pathways of
forming DOI from D-glucose, and a reaction catalyzed by the DOI
synthase.
[0040] FIG. 2A is an illustration showing the structure of
pLEX-btrC.
[0041] FIG. 2B is an illustration showing the structure of
pGAP-btrC.
[0042] FIG. 2C is an illustration showing the structure of
pGAD-btrC.
[0043] FIG. 2D is an illustration showing the structure of
pGAP-btrC/pGAD-btrC.
[0044] FIG. 3A is a view showing a SDS-PAGE pattern of cell
extracts from the E. coli GI724 .DELTA.pgi strain comprising
pLEX-btrC in the case of the strain cultured in 2.times.YT medium
or the rice bran medium at the indicated time points.
[0045] FIG. 3B is a view showing a SDS-PAGE pattern of cell
extracts from the E. coli GI724.DELTA.pgi.DELTA.zwf.DELTA.pgm
strain comprising pGAP-btrC/pGAD-btrC in the case of the strain
cultured in 2.times.YT medium at the indicated time points.
[0046] FIG. 4A is a HPLC chart showing the oxime obtained from the
supernatant of the culture medium of the E. coli GI724.DELTA.pgi
strain comprising pLEX-btrC (3 L of 2.times.YT, 30.degree. C., pH
7.5, 5.5% D-glucose, 24 hours incubation).
[0047] FIG. 4B is a HPLC chart showing the oxime obtained from the
supernatant of the culture medium of the E. coli GI724.DELTA.rmf
strain comprising pLEX-btrC (10 mL of 2.times.YT, 30.degree. C., pH
7, 3% D-glucose, 48 hours incubation) (left chart at 0 hr
culture).
[0048] FIG. 5 is a graph showing time courses of (A) turbidity of
the medium, (B) D-glucose concentration and (C) DOI production
during the culture of the E. coli GI724.DELTA.pgi strain comprising
pLEX-btrC (2.times.YT medium+2% glucose (.diamond-solid.) or
2.times.YT+5% glucose (.box-solid.), 3 L, 30.degree. C., pH 7.5)
(the horizontal axes correspond to the elapsed time after addition
of glucose).
[0049] FIG. 6 is a graph showing time courses of (A) turbidity of
the medium, (B) D-glucose concentration and (C) DOI production
during the culture of the E. coli GI724.DELTA.pgi.DELTA.zwf strain
comprising pLEX-btrC (2.times.YT medium+3% mannitol+5% D-glucose, 3
L, 30.degree. C., pH 7.5) (the horizontal axes correspond to the
elapsed time after addition of glucose).
[0050] FIG. 7 is a graph showing time courses of (left) turbidity
derived from microorganisms, (center) D-glucose concentration and
(right) DOI production during the culture of the E. coli
GI724.DELTA.pgi.DELTA.zwf.DELTA.pgm strain comprising pLEX-btrC
(2.times.YT medium+5% D-glucose+0.5% mannitol, 3 L, 30.degree. C.,
pH 7) (the horizontal axes correspond to the elapsed time after
addition of glucose).
[0051] FIG. 8 is a graph showing time courses of (left) turbidity
derived from microorganisms, (center) D-glucose concentration and
(right) DOI production during the culture of the E. coli
GI724.DELTA.pgi.DELTA.zwf.DELTA.pgm strain (.diamond-solid.)
comprising pLEX-btrC, and the E. coli
GI724.DELTA.pgi.DELTA.zwf.DELTA.pgm strain (.box-solid.) comprising
pGAP-btrC/pGAD-btrC (2.times.YT medium+5% D-glucose+0.5% mannitol,
3 L, 25.degree. C., pH 6-7) (the horizontal axes correspond to the
elapsed time after addition of glucose).
[0052] FIG. 9 is a graph showing time courses of (left) turbidity
derived from microorganisms, (center) D-glucose concentration in
the medium and (right) DOI production during the culture of the
wild type E. coli (.diamond-solid.) comprising pLEX-btrC, and the
E. coli GI724.DELTA.rmf strain (.box-solid.) comprising pLEX-btrC
(2.times.YT medium+3% D-glucose, 10 mL, 30.degree. C., pH 7) (the
horizontal axes correspond to the elapsed time after addition of
glucose).
[0053] FIG. 10 is a graph of pH, conductivity and DOI concentration
at each fraction obtained from the eluent wherein the cultured
medium is passed through the ion exchange resin to obtain the
eluent, in accordance with Example 8.
[0054] FIG. 11 is .sup.13C-NMR spectrum of DOI as purified and
obtained in accordance with the method of Example 8.
[0055] FIG. 12 is a graph of pH, conductivity and DOI concentration
at each fraction obtained from the eluent wherein the cultured
medium is passed through the ion exchange resin to obtain the
eluent, in accordance with Example 9.
[0056] FIG. 13 is .sup.13C-NMR spectrum of DOI as purified and
obtained, in accordance with the method of Example 9.
[0057] FIG. 14 is an illustration showing a principle of the second
aspect of the method for purifying 2-deoxy-scyllo-inosose according
to the present invention.
[0058] FIG. 15 is .sup.1H-NMR spectrum of DOI as purified and
obtained, in accordance with the method of Example 10.
[0059] FIG. 16 is .sup.1H-NMR spectrum of DOI as purified and
obtained, in accordance with the method of Example 11.
[0060] FIG. 17 is .sup.1H-NMR spectrum of DOI as purified and
obtained, in accordance with the method of Example 12.
BEST MODE FOR CARRYING OUT THE PRESENT INVENTION
[0061] In view of the property of the DOI synthase, there is a
possibility that the useful resource DOI can be efficiently and
shortly produced from the biomass-derived and abundant D-glucose as
starting materials by means of microorganism as introduced the
enzyme.
[0062] Here, the inventors were made the present invention, in
order to develop a method for producing DOI by fermentation of E.
coli as host cells with easy procedure and low cost, as a novel
method for synthesizing DOI.
[0063] In addition, in the case that a system which industrially
purify DOI will be considered, preferred examples of methods
include several methods, one of which is to apply the culture
medium containing DOI from top of a column and to elute off the
purified DOI from the bottom of the column, the other of which is
to crystallize DOI or DOI derivative and to separate thereof, and
yet the other of which is combined said one of methods with said
other of methods. In view of these, the present invention is made
by means of searching column materials which adsorb substrates
other than DOI, do not adsorb DOI and primarily elute off DOI, and
by means of searching DOI derivatives which are capable of
efficiently obtaining DOI after the crystallization and
separation.
[0064] Reference will be made with regard to embodiments of the
present invention.
[0065] <The Gene Expression Cassette According to the Present
Invention>
[0066] The gene expression cassette according to the present
invention is characterized in:
[0067] A gene expression cassette comprising a gene involved in a
synthesis of 2-deoxy-scyllo-inosose. That is, the gene expression
cassette according to the present invention is not limited,
provided that the gene expression cassette according to the present
invention comprises gene(s) involved in a synthesis of
2-deoxy-scyllo-inosose and gene(s) capable of expressing the gene
involved in a synthesis of 2-deoxy-scyllo-inosose.
[0068] (The Gene Involved in a Synthesis of
2-Deoxy-Scyllo-Inosose)
[0069] In the gene expression cassette according to the present
invention, the gene involved in a synthesis of
2-deoxy-scyllo-inosose may be a gene encoding the well-know
proteins which synthesize the 2-deoxy-scyllo-inosose. The examples
thereof includes btrC gene encoding 42 kDa subunit of the DOI
synthase derived from Bacillus circulans wherein the DOI synthase
synthesizes DOI from D-glucose (see Patent-related document 1,
Non-patent-related document 2, and Genbank No. AB066276 etc.). Of
course, genes derived from any organisms other than Bacillus
circulans can be also utilized, provided that the genes encode an
enzyme having an activity of synthesizing DOI. In addition, the
nucleotide sequences of the above-mentioned genes may comprise any
mutations including deletion, substitution and insertion of the
nucleotide(s), provided that said genes are a gene expressing an
enzyme having an activity of synthesizing DOI.
[0070] (Construction of the Gene Expression Cassette According to
the Present Invention)
[0071] In construction of the gene expression cassette according to
the present invention, a gene which can express the gene involved
in the 2-deoxy-scyllo-inosose in the below-mentioned host cells may
be used. The gene constitution of the gene expression cassette
includes a promoter, sequence related to the transcriptional
activation, RBS (ribosome binding site), and a terminator. For
example, in a system for abundant expression of proteins in E. coli
as host cells, DNA sequences including the promoter, the sequence
related to the transcriptional activation and RBS (ribosome binding
site) may be linked at 5'-upstream region of the gene, and DNA
sequences including the terminator may be linked at 3'-downstream
region of the gene. These DNA sequences may be used, as long as the
sequences can properly act in E. coli. There is a promoter which
constitutively or inductively expresses the target genes. Any
promoters may be used in the present invention. The promoter that
the expression of the target gene can be controlled is preferable.
It should be noted that, in the gene expression in E. coli as host
cells, high cost inducer(s) for the gene expression including IPTG
(isopropyl-thio-galactopyranoside) have typically been used.
[0072] In the present invention, it is desirable that high
expression system for the target gene is used, without using the
above-mentioned high cost inducer such as IPTG. For this purpose,
the expression system using the GAP promoter, the GAD promoter, the
P.sub.L Expression System (Invitrogen) can be utilized.
[0073] In the P.sub.L Expression System, the gene expression
introduced in the vector (pLEX, ampicilin-resistant marker) is
controlled by the P.sub.L promoter which is derived from lambda
phage and which constitutively and strongly induces the expression
of the target gene. In addition, the P.sub.L Expression System is
constituted such that the expression is controlled depending on the
tryptophan concentration in the culture medium and that the
expression is induced in the medium containing high concentration
of tryptophan.
[0074] On the other hand, in the systems of either a system using
gapA (a gene encoding glyceraldehyde-3-phosphate dehydrogenase A)
promoter, or a system using gadA (a gene encoding glutamate
decarboxylase A) promoter or a system using both promoters, the
target gene introduced in the vector (pUC origin,
ampicilin-resistant marker) is constitutively and strongly
expressed in the logarithmic phase or the stationary phase of the
cells. The expression system such as gapA promoter and gadA
promoter is constituted such that the expression is induced in
ordinary culture condition, without using the particular kinds of
reagents and handlings.
[0075] On the other hand, in the P.sub.L Expression System, there
is enough amounts of tryptophan in the complete medium as normally
used in the culture of E. coli to induce the promoter activity. So,
it is not necessary to further add tryptophan in order to induce
the expression. In addition, it is not necessary to add the inducer
for inducing the expression in the systems using gapA promoter or
gadA promoter. That is, it is possible to highly express the target
gene without using high cost inducer.
[0076] <The Transformant According to the Present
Invention>
[0077] The transformant according to the present invention is
characterized in:
[0078] A transformant comprising the above-mentioned gene
expression cassette transformed in a host cell. Hereinafter, the
host cells of the present invention will be explained.
[0079] (The Host Cells)
[0080] Host cells which can be used in the transformant according
to the present invention are not limited, and any types of host
cells can be used, including microorganisms which are deposited in
depository institutions of strains such as IFO and ATCC. Examples
include E. coli. In addition, the host cells as stated in GILSP
(Good Industrial Large-Scale Practice) can be used, including the
host cells as stated in GILSP on March, 2006 such as Bacillus
amyloliquefaciens, Bacillus brevis HPD31, Bacillus brevis HPD31-M3,
Bacillus licheniformis DN2461, Bacillus licheniformis DN2717,
Bacillus subtilis K2A1, Bacillus subtilis Marburg 168,
Corynebacterium glutamicum, Escherichia coli K12, Geobacillus
stearothermophilus.
[0081] (The Gene-Disrupted Strain of the Present Invention)
[0082] In the transformant according to the present invention, the
host cells as disrupted the chromosomal genes and/or the plasmid
genes can be used, in view of the synthesis of the
2-deoxy-scyllo-inosose. In the preferred aspects of the present
invention, either, both or all genes of pgi gene, zwf gene and pgm
gene in the host cells, wherein these three gene are genes encoding
phosphglucose isomerase, glucose-6-phosphate 1-dehydrogenase and
phosphoglucomutase, respectively and wherein these enzymes are
relevant to the metabolism of glucose-6-phosphate, are disrupted
(i.e. in the case that one of genes is disrupted, pgi gene, zwf
gene, or pgm gene; in the case that two of genes are disrupted, pgi
and zwf genes, or pgi and pgm genes; in the case that three of
genes are disrupted, pgi, zwf and pgm genes). Further, in the
preferred aspects of the present invention, the gene encoding RMF
protein involved in modification of protein synthesis during
stationary phase (rmf gene) in the host cells is singly disrupted,
or the rmf gene in the above-mentioned strains which genes encoding
enzymes relevant to the metabolism of glucose-6-phosphate is
disrupted. This leads to the suppression of the degradation of
glucose-6-phosphate, which is direct substrate for production of
DOI, in the microorganisms. In addition, the productivity of DOI
was dramatically increased due to the protein synthesis in the
stationary phase.
[0083] [Gene-Disrupted Strain Wherein the Gene is Related to the
Degradation of Glucose-6-Phosphate]
[0084] In order to synthesize DOI with possibly high yield, it is
necessary to be considered to possibly suppress the catabolism of
D-glucose in the microorganisms. In E. coli, genes encoding enzymes
related to the catabolism of D-glucose include three genes wherein
the genes are: pgi gene encoding phosphoglucose isomerase which
relates to transform glucose-6-phosphate into fructose-6-phosphate
in the glycolytic system; zwf gene encoding glucose-6-phosphate
dehydrogenase which relates to transform glucose-6-phosphate into
phosphogluconolactone in the pentose phosphate pathway; and pgm
gene encoding phosphoglucomutase which relates to transform
glucose-6-phosphate into glucose-1-phosphate. It can be considered
that, by disrupting these genes, it is possible to suppress the
catabolism of glucose-6-phosphate as substrate for the DOI
synthase, along with the growth of the microorganisms.
[0085] [Gene-Disrupted Strain Wherein the Gene Relates to the
Protein Synthesis During the Stationary Phase]
[0086] In order to cancel the prevention of the BtrC protein
synthesis during the stationary phase, it can be considered that it
is necessary to suppress the production of RMF protein related
thereto. Here, it can be considered that rmf gene encoding RMF
protein is disrupted to continue the synthesis of the BtrC protein
after the stationary phase, thereby continuing the synthesis of
DOI.
[0087] (Manufacture of the Gene-Disrupted Strain in the Present
Invention)
[0088] In the present invention, the well-known method can be used
for manufacturing the gene-disrupted strain in which the particular
gene of the host cells is disrupted. For example, examples of such
a method include a method for inducing the mutation (a method of
natural breeding, addition of mutagen, UV irradiation, radiation),
a method utilizing random mutation (methods using insertion
sequence (IS) or transposon (Tn)) and the site-specific
gene-disrupting method (single or double cross over method). Among
these, it is preferable to use the site-specific gene-disrupting
method which can insert fragments comprising the drug-resistant
gene into the target gene, in view of screening to obtain the
desired gene-disrupted strain. It should be noted that the host
cells of the present invention is not limited as mentioned below,
although the gene-disrupted host cells which are used for the
transformant according to the present invention are manufactured by
using Quick and Easy BAC Modification Kit (Gene Bridges).
[0089] (Method for Manufacturing the Transformant According to the
Present Invention)
[0090] The transformant according to the present invention can be
manufactured to introduce the above-mentioned gene expression
cassette into the host cells. The method for introducing includes
the competence method and endocytosis through any receptors.
[0091] <The Method for Manufacturing 2-Deoxy-Scyllo-Inosose
According to the Present Invention>
[0092] The method for manufacturing 2-deoxy-scyllo-inosose
according to the present invention is characterized to contact the
above-mentioned transformant with a carbon source in any medium
suitable to grow the transformant.
[0093] As the carbon source, D-glucose, nitrogen-containing
monosaccharides such as D-glucosamine and D-galactosamine, and
monosaccharides derived from sugars or oligosaccharides comprising
two or more of monosaccharides, or carbohydrate (e.g. starch, rice
bran and molasses) can be used.
[0094] As the medium for carrying out the method for manufacturing
2-deoxy-scyllo-inosose according to the present invention, the form
of the medium such as solid medium, liquid medium is not limited,
provided that the host cells can be proliferated and/or grown in
the well-known medium. Examples of such medium include the agar
medium, RMG medium, 2.times.YT medium, LB medium, M9 minimum medium
and SOB medium. These medium may contain carbon sources, nitrogen
sources, inorganic salts and other organic nutrient sources. The
carbon sources may be as substances as set forth in Table 1 such as
mannitol, in addition to the above-mentioned substances. Examples
of the nitrogen sources include ammonium chloride, casamino acid,
peptone and yeast extract. Examples of the inorganic salts include
sodium hydrogen phosphate, potassium dihydrogen phosphate,
magnesium chloride and sodium chloride. As mentioned above, it is
not necessary to use the high cost inducer, in the case of using
the GAP-GAD expression system and the P.sub.L Expression System
(Invitrogen) as the host cells-vector system.
[0095] On the other hand, the medium may contain any suitable
additives, depending on the growth of the host cells. In order to
express the gene involved in the synthesis of the
2-deoxy-scyllo-inosose as introduced in the gene expression
cassette, the medium may contain compounds increasing the promoter
activity such as IPTG, tryptophan. Especially, the inducer may be
added to the medium, in the case that the expression of the gene by
the promoter is inducibly performed.
[0096] In the method for manufacturing 2-deoxy-scyllo-inosose
according to the present invention, conditions of contacting the
transformant with the carbon source, including temperature,
duration and circumstance, are not limited, provided that the
conditions are suitable for the growth of the transformant. For
example, the temperature is more preferable in the range of 20 to
37.degree. C. In addition, the duration may be in the range of 1 to
7 days, although the duration is not limited thereto.
[0097] For example, in the method for manufacturing
2-deoxy-scyllo-inosose according to the present invention, the
transformant is firstly contacted with any materials being capable
of being assimilated in E. coli as the carbon source. Thereafter,
DOI is recovered from the obtained culture supernatant. In this
way, it is possible to industrially obtain DOI by the method for
manufacturing 2-deoxy-scyllo-inosose according to the present
invention using the transformant.
[0098] DOI can be obtained with high yield from D-glucose by the
actions of hexokinase and the DOI synthase, since the method for
manufacturing 2-deoxy-scyllo-inosose according to the present
invention has the above-mentioned constitution. In addition, by
means of culturing the transformed strains including the
GI724.DELTA.rmf transformed strain, the GI724.DELTA.pgi.DELTA.rmf
transformed strain, the GI724.DELTA.pgi transformed strain, the
GI724.DELTA.pgi.DELTA.zwf transformed strain and the
GI724.DELTA.pgi.DELTA.zwf.DELTA.pgm transformed strain comprising
the above-mentioned plasmids of pGAP-btrC, pGAD-btrC,
pGAP-btrC/pGAD-btrC and pLEX-btrC, DOI is produced from D-glucose
via glucose-6-phosphate through five steps of reaction catalyzed by
the DOI synthase, as shown in FIG. 1.
[0099] In the method for manufacturing 2-deoxy-scyllo-inosose
according to the present invention, the transformant is contacted
with the carbon source, thereby obtaining a composition comprising
2-deoxy-scyllo-inosose (e.g. the medium containing
2-deoxy-scyllo-inosose and the host cells). For example, DOI may be
collected from the culture supernatant, in the case that the
culture medium containing 2-deoxy-scyllo-inosose is used as the
starting material of 2-deoxy-scyllo-inosose. In the present
invention, as a method for recovering DOI from the culture
solution, 2-deoxy-scyllo-inosose may be recovered by the well-known
extraction method, in accordance with, for example,
physical/chemical characteristics of 2-deoxy-scyllo-inosose and
compositions of the culture medium. For example, the following
method can be used. That is, after finishing the culture, the
culture medium is firstly centrifuged by the centrifugal separator
and the filtrator to remove the microorganism from the medium,
thereby obtaining a culture supernatant. Thereafter, the culture
supernatant is further filtrated to remove any solid materials
including the microorganisms, the filtrate is applied to ion
exchange resins and an elution is performed with the distilled
water. Fractions not containing any impurities are collected
through monitoring the refractive index, pH, the conductivity, and
solvents of the so obtained solution are removed to recover DOI.
Analysis of the so obtained DOI is performed by any methods
including the high-performance liquid chromatography and the
nuclear magnetic resonance method.
[0100] <First Aspect of the Method for Purifying
2-Deoxy-Scyllo-Inosose According to the Present Invention>
[0101] The method for purifying 2-deoxy-scyllo-inosose according to
the present invention is characterized in:
[0102] A method for purifying 2-deoxy-scyllo-inosose comprising the
steps of:
[0103] contacting the above-mentioned transformant with a carbon
source to obtain a composition containing 2-deoxy-scyllo-inosose;
and
[0104] treating said composition with mixed bed column or double
bed column comprising a hydrogen ion form of a strong-acid cation
exchange resin and an organic acid ion form of a basic anion
exchange resin. In the method for purifying 2-deoxy-scyllo-inosose
according to the present invention, the step of contacting the
transformant with the carbon source is performed in accordance with
the method for manufacturing 2-deoxy-scyllo-inosose according to
the present invention. This step will afford a composition
containing 2-deoxy-scyllo-inosose. This composition comprises the
above-mentioned medium. Hereinafter, reference will be made, in the
case that culture medium is used as the above-mentioned medium.
[0105] There are several components of the culture medium in the
culture solution after finishing the culture of the transformant in
the medium containing the carbon source, in addition to the
remained glucose as the carbon source. Examples of these several
components include amino acids, peptides and metal ions. It is
necessary to remove these several components in order to obtain
DOI. The method for purifying 2-deoxy-scyllo-inosose according to
the present invention will provide to solve the above-mentioned
matter.
[0106] Impurities to be removed in the culture medium includes the
remained D-glucose as the carbon source, amino acids, peptides and
the metal ions, as mentioned above. It has found that it is
possible to continue the culture until D-glucose is completely
consumed, in accordance with the examination of the culture
condition. Amino acids, peptides and metal ions were remained in
the medium as the remained impurities. The amino acids include
basic amino acids comprising a plurality of amino groups such as
lysine, histidine and tryptophan, acidic amino acids comprising a
plurality of carboxyl groups such as glutamic acid and aspartic
acid. In addition, the metal ions include cations, and the counter
ions including chloride ion and sulfate ion is present in the
culture medium. Therefore, column materials will be searched such
that all of such impurities are adsorbed on the material and DOI is
not adsorbed on the material.
[0107] The present inventors have examined the search of the column
material and condition for the elution, in accordance with such a
concept. As the result, DOI was not purified in good yield with a
method using general ion exchange resins including sodium ion or
hydrogen ion form of strong-acid cation exchange resin and chloride
ion or hydroxylate ion form of basic anion exchange resin. That is,
the above-mentioned purpose cannot be achieved by using a double
bed column connecting the above-mentioned resins or a mixed bed
column by mixing the resins. Amino acids bind to either of the
above-mentioned two ion exchange resins. In addition, metal ions
bind to the cation exchange resin, while DOI has a property of not
binding both of the exchange resins since DOI does not have ionic
functional groups. Therefore, it can be presumed that the amino
acids and the metallic salts bind to the ion exchange resins, and
DOI is eluted off without being adsorbed. However, the obtained
result is not so. The yield of DOI was lower than 50%, and the
yield was greatly changed depending on the use conditions of the
ion exchange resin. More yields and stable performances are
required in order to fit an industrial method in mass scale.
[0108] The present inventors have further examined the column
material and the conditions for the purification. As the result,
the present inventors have found that DOI can be efficiently
purified by using the mixed bed column or double bed column
comprising organic acid ion as counter ion of the anion exchange
resin, that is, organic acid ion form of basic anion exchange resin
and hydrogen ion form of strong-acid cation exchange resin.
Examples of the organic acid include acetic acid, propionic acid
and oxalic acid, especially, acetic acid is preferably used. Acetic
acid as the organic acid is preferably used, since DOI is easily
and extremely degraded over pH 8, and DOI is only stable in the
weak acidic range of pH 3 to 5. That is, if DOI was completely
stable at both ranges of acidic range and basic range, it would be
possible to remove the impurities, even in a method to elute the
culture medium through the double bed column of separate columns
filled with the anion exchange resin and the cation exchange resin
as generally-used. In addition, it would be possible to purify DOI
with the mixed bed column in the both ranges if DOI had similar
stability to that of fructose. The present inventors are the first
person who found that DOI is unstable in the alkaline range such
that DOI is degraded even by using the mixed bed column along with
the double bed column.
[0109] In order to solve this problem, the present inventors have
examined a selection of the counter ion binding to the ion
exchanged resin to be used. As the result, the present inventors
have found a method using the organic acid ion as the counter ion
for the anion exchange resin. This will lead that the organic acid
is remained in the eluent and is contaminated in fractions
containing DOI. This is effective to maintain pH of the eluent as
around 4. Among the organic acid, acetic acid has an advantage of
capable of being removed by the concentration procedure.
[0110] It has found that DOI can be purified by means of passing
the culture solution through an ion exchange column comprising a
mixture of hydrogen form of cation exchange resin and organic acid
ion form of anio exchange resin. Therefore, the present invention
has been completed. This is a desired method for industrially
manufacturing in mass scale.
[0111] Charged impurities, amino acids, peptides and metal ions
which are derived from starting materials as remained in the
culture medium which has completely consumed glucose can be
adsorbed and removed in the method for purifying using mixed bed or
double bed form of ion exchange resin column. Therefore, DOI which
is neutral and is not adsorbed can be eluted off to purify it. It
should be noted that there is a possibility to contaminate any
neutral materials into the fraction containing DOI, depending on
the condition of the culture. Accordingly, a further purification
method of DOI was examined to obtain the highly purified DOI eluted
off from the ion exchange column. It is presumed that DOI is an
equilibrium mixture of keto and hydrate forms, on the basis of NMR
analysis. So, it can be understood that it is difficult to
crystallize DOI. It has not been reported the crystallization of
DOI and DOI derivatives. Hereinafter, a method for highly purifying
DOI will be described.
[0112] <Second Aspect of the Method for Purifying
2-Deoxy-Scyllo-Inosose According to the Present Invention>
[0113] The method for purifying 2-deoxy-scyllo-inosose according to
the present invention is characterized in:
[0114] A method for purifying 2-deoxy-scyllo-inosose comprising the
steps of:
[0115] reacting the above-mentioned 2-deoxy-scyllo-inosose with
trialkoxymethanes to obtain 2-deoxy-scyllo-inosose dialkylketals;
and
[0116] hydrolyzing said 2-deoxy-scyllo-inosose dialkylketals in the
presence of acid. Examples of the 2-deoxy-scyllo-inosose which can
be used in this aspect of the present invention include the
2-deoxy-scyllo-inosose as obtained in the first aspect of the
present invention, in addition to the above-mentioned composition
containing 2-deoxy-scyllo-inosose. Hereinafter, the second aspect
will be described.
[0117] The present inventors have examined the search of materials
and the examination of a method for purifying complying with a
principle wherein the principle is based on that DOI as eluted is
converted into crystallizable material(s), the materials is
crystallized, the crystallized material is separated and purified,
and the crystallizable material is efficiently restored into DOI.
FIG. 14 shows an illustration of the principle. FIG. 14 is an
illustration showing a principle of the second aspect of the method
for purifying 2-deoxy-scyllo-inosose according to the present
invention.
[0118] In order to comply with these requirements, it is necessary
that reactions for converting DOI and for restoring it into DOI can
be performed in an acidic condition which does not degrade DOI,
that the material(s) as converted derivative is easily crystallized
and restored into DOI, and that these procedure(s) can be
industrially performed. It has been examined several methods
complying with these requirements. As the result, it has been found
that a method for reacting DOI with alkoxymethanes in an acidic
condition in which DOI is stable to convert it into
2-deoxy-scyllo-inosose dialkylketals (DOI-dak), and for
crystallizing and purifying DOI-dak.
[0119] In alkoxymethane ((RO).sub.3CH), examples of R are not
limited, provided that examples include alkanes having 1 to 4 of
carbon atoms, such as methane, ethane, propane and butane. In the
present invention, a most preferable example of trialkoxymethanes
is trimethoxymethane, since an alcohol which is formed in the
following step of hydrolyzing can be easily removed. It should be
noted that deoxy-scyllo-inosose dimethylketal (DOI-dmk) will be
obtained in the case of using trimethoxymethane.
[0120] The crystallization of 2-deoxy-scyllo-inosose alkylketals
may be performed such that 2-deoxy-scyllo-inosose alkylketals are
dissolved in an appropriate solvent (e.g. methanol, ethanol and
water), and a liquid medium which decrease the hydrophilicity of
the liquid phase thereof (e.g. chloroform, hexane and ethers) is
used. So, crystals of 2-deoxy-scyllo-inosose alkylketals will be
obtained.
[0121] The so obtained 2-deoxy-scyllo-inosose alkylketals will be
hydrolyzed in the presence of acid to be restored into
2-deoxy-scyllo-inosose. The hydrolysis reaction may be performed in
an appropriate catalyst such as p-toluenesulfonic acid,
hydrochloric acid and sulfuric acid. After hydrolysis,
2-deoxy-scyllo-inosose will be obtained.
[0122] Therefore, the method for purifying 2-deoxy-scyllo-inosose
according to the present invention is a desirable method for
industrial manufacturing in mass scale.
Embodiment
[0123] An explanation will be made with regard to the present
invention in accordance with embodiments. It should be noted that
the scope of the present invention is not limited to these
embodiments.
Example 1
DOI Synthase Gene
[0124] btrC gene was utilized for hydrocarbon-cyclizing enzyme gene
wherein the btrC gene encodes 42 kDa subunit protein of
2-deoxy-scyllo-inosose (DOI) synthase derived from Bacillus
circulans (see Patent-related document 1, Genbank No. AB066276
and
[0125] Non-patent-related document 2).
Example 2
Construction of Recombinant Plasmid and Recombinant Strain
[0126] <btrC Gene and pLEX-btrC>
[0127] Plasmid pDS4 comprising full length of btrC gene
(Non-patent-related document 3) as the template, primer 1 stated as
Sequence No. 1, and primer 2 stated as Sequence No. 2 were used.
KOD polymerase (Toyobo) was used for PCR amplification of btrC
gene. 30 cycles of PCR reaction were performed wherein the cycle
consists of a cycle of reacting at 94.degree. C. for 30 seconds, at
52.degree. C. for 30 seconds and 68.degree. C. for 1 minute. DNA
fragment so obtained by the PCR amplification was digested with Nde
I and XbaI, and was inserted into the NdeI-XbaI site of the
multicloning site located on a vector pLEX to constitute pLEX-btrC
(FIG. 2A).
[0128] <pGAP-btrC/p-GAD-btrC>
[0129] A gapA promoter gene was PCR-amplified with chromosomal DNA
of E. coli as the template and a primer stated as Sequence No. 17
and a primer stated as Sequence No. 18. The PCR amplification of
the gapA promoter gene was performed with KOD polymerase in the
following reacting condition to obtain the gapA promoter
fragments.
[0130] 30 cycles, the cycle of reacting:
[0131] at 94.degree. C. for 30 seconds;
[0132] at 50.degree. C. for 30 seconds; and
[0133] at 68.degree. C. for 1 minute
[0134] A gadA promoter gene was PCR-amplified with chromosomal DNA
of E. coli as the template, a primer stated as Sequence No. 19 and
a primer stated as Sequence No. 20. The PCR amplification of the
gadA promoter gene was performed with KOD polymerase in the
following reacting condition to obtain the gadA promoter
fragments.
[0135] 30 cycles, the cycle of reacting:
[0136] at 94.degree. C. for 30 seconds;
[0137] at 52.degree. C. for 30 seconds; and
[0138] at 68.degree. C. for 1 minute
[0139] A aspA terminator gene was PCR-amplified with a plasmid pLEX
(invitrogen) comprising the aspA terminator gene as the template
and a primer stated as Sequence No. 21 and a primer stated as
Sequence No. 22. The PCR amplification of the aspA promoter gene
was performed with KOD polymerase (TOYOBO) in the following
reacting condition to obtain the aspA terminator fragments.
[0140] 30 cycles, the cycle of reacting:
[0141] at 94.degree. C. for 30 seconds;
[0142] at 55.degree. C. for 30 seconds; and
[0143] at 68.degree. C. for 1 minute
[0144] The PCR-amplified gadA promoter fragments were reacted with
the above-mentioned btrC fragment at 16.degree. C. for 30 minutes,
using 2.times. Ligation mix (TAKARA). The so obtained ligation
product as the template, a primer stated as Sequence No. 2 and a
primer stated as Sequence No. 5 were used to be PCR-amplified,
using KOD polymerase, in the following conditions, thereby
obtaining a gadA-btrC fragment ligated with the above-mentioned two
fragments.
[0145] 30 cycles, the cycle of reacting:
[0146] at 94.degree. C. for 30 seconds;
[0147] at 52.degree. C. for 30 seconds; and
[0148] at 68.degree. C. for 1 minute
[0149] Digested fragment with XbaI was inserted into XbaI site of
the multicloning site located on the vector pLEX (Invitrogen).
[0150] Next, the PCR-amplified gapA promoter fragments were reacted
with the above-mentioned btrC fragment and the aspA terminator
fragment at 16.degree. C. for 30 minutes, using the 2.times.
Ligation mix. The so obtained ligation product as the template, a
primer stated as Sequence No. 8 were used to be PCR-amplified,
using KOD polymerase, in the following conditions, thereby
obtaining a gapA promoter-btrC-aspA terminator fragment ligated
with the above-mentioned three fragments.
[0151] 30 cycles, the cycle of reacting:
[0152] at 94.degree. C. for 30 seconds;
[0153] at 50.degree. C. for 30 seconds; and
[0154] at 68.degree. C. for 1 minute
[0155] Digested fragment with BamHI was inserted into BamHI site of
the multicloning site located on the vector pLEX (Invitrogen) which
was inserted the gadA-btrC to constitute pGAP-btrC/pGAD-btrC (FIG.
5).
Example 3
Preparation of Host Cells
[0156] The disruption of genes was performed with Quick and Easy
BAC Modification Kit (Gene Bridges), in accordance with the method
as stated in the manufacturer's protocol of the kit. Sequences of
primer sets for PCR which are used to manufacture the cassette
using the single disruption of the pgi gene are shown in Sequence
No. 3 and Sequence No. 4. Sequences of primer sets for the
amplification of the cassette using the single disruption of the
zwf gene are shown in Sequence No. 5 and Sequence No. 6. Sequences
of primer sets for manufacturing the cassette using the single
disruption of the pgm gene are shown in Sequence No. 7, Sequence
No. 8, Sequence No. 9 and Sequence No. 10. Sequences of primer sets
which are used to amplify the cassette for disrupting the pgi gene
against the single-gene-disrupted strain of the zwf gene for
obtaining the double gene-disrupted strain of the pgi and zwf genes
are shown in Sequence No. 11 and Sequence No. 12. Sequences of
primer sets which are used to amplify the cassette for disrupting
the pgm gene against the single-gene-disrupted strain of the pgi
gene for obtaining the double gene-disrupted strain of the pgi and
pgm genes are shown in Sequence No. 7, Sequence No. 8, Sequence No.
9 and Sequence No. 10. Further, sequences of primer sets which are
used to amplify the cassette for disrupting the zwf gene against
the double gene-disrupted strain of the pgi and pgm genes for
obtaining the triple gene-disrupted strain of the pgi, zwf and pgm
genes are shown in Sequence No. 5 and Sequence No. 6. In addition,
sequences of primers for a disruption cassette of the rmf gene are
shown in Sequence No. 13, Sequence No. 14, Sequence No. 15 and
Sequence No. 16.
[0157] A vector pSC101-BAD-gbaA-tetra was introduced into E. coli
as the host cells, wherein the vector encodes group of enzymes
which enhance the homologous recombination in E. coli (GI724), as
enclosed in the Kit. The strain d in the LB medium supplemented
with 3 .mu.g/ml of tetracycline for overnight. Precultured
microorganisms were inoculated in the LB medium added with 3
.mu.g/ml of tetracycline at 1% concentration, and then grown at
30.degree. C. until O.D.=0.2. At the time, arabinose was added at
final concentration of 0.2%, and further cultured at 37.degree. C.
for 1 hour to inductively express the group of enzymes involved in
the enhancement of the homologous recombination. Further, the
transformation was performed with the cassette for the gene
disruption to induce the gene disruption for the target genes. The
so obtained transformant was selected at 37.degree. C. with the LB
medium supplemented with one, two or three of chloramphenicol,
neomycin (kanamycin) or streptomycin to obtain the desired
gene-disrupted strains including pgi gene-disrupted strain
(.DELTA.pgi strain), zwf gene-disrupted strain (.DELTA.zwf strain),
pgm gene-disrupted strain (.DELTA.pgm strain), pgi/zwf double
gene-disrupted strain (.DELTA.pgi.DELTA.zwf strain), pgi/pgm
double-gene disrupted strain (.DELTA.pgi.DELTA.pgm strain) and
pgi/zwf/pgm triple gene-disrupted strain
(.DELTA.pgi.DELTA.zwf.DELTA.pgm strain). rmf gene-disrupted strains
for each of the above-mentioned gene-disrupted strains were
obtained in accordance with the above-mentioned procedure. Growth
and development of wild type strain and each of gene-disrupted
strains in any one of the carbon sources are shown in Table 1.
[0158] The growth rates of the .DELTA.pgi, .DELTA.zwf and
.DELTA.pgm strains were remarkably delayed in comparison with the
wile type, even though these gene-disrupted strains can utilize
glucose as the carbon source. It can be understood that the
consumption of D-glucose is suppressed in the microorganisms. In
addition, the growth of the .DELTA.pgi.DELTA.zwf,
.DELTA.pgi.DELTA.pgm and .DELTA.pgi.DELTA.zwf.DELTA.pgm strains
were extremely difficult in the medium containing D-glucose as the
single carbon source. It can be understood that the degradation of
glucose-6-phosphate is almost completely suppressed. This will
promise that DOI production and the conversion rate into DOI are
more increased in comparison with the wild type, by means of using
these gene-disrupted strains. In addition, it is possible to
improve the growth of the double- and triple-gene disrupted strains
by supplementarily adding non-fermented carbon source such as
mannitol and gluconate (see Table 1). In addition, with regard to
the carbon source in the rmf-gene disrupted strain, the same growth
was observed similar to the strains in which the
glucose-6-phosphate metabolic enzyme genes were disrupted.
TABLE-US-00001 TABLE 1 carbon source wild type .DELTA.pgi
.DELTA.zwf .DELTA.pgm .DELTA.pgi.DELTA.zwf .DELTA.gi.DELTA.pgm
.DELTA.pgi.DELTA.zwf.DELTA.pgm D-glucose ++ + ++ ++ - - - fructose
++ ++ ++ ++ + + + glycerol +/- + ++ ++ + + + mannitol ++ + ++ ++ ++
++ ++ gluconate - ++ ++ ++ ++ ++ ++ (in table 1, a mark "++"
indicates that the cells are extremely grown, a mark "+" indicates
that the cells are well grown, a mark "+/-" indicates that the
cells are slightly grown, and a mark "-" indicates that the cells
are less grown.)
Example 4
The Transformant
[0159] The host cells of E. coli (GI724) were transformed with
pLEC-btrC as obtained, and selected in the medium containing
ampicillin to obtain GI724/pLEX-btrC strain, in accordance with the
protocol as stated in the manufacturer's protocol of the Kit made
by Gene Bridges. In addition, the host cells of E. coli (GI724)
were transformed with pGAP-btrC/pGAD-btrC as obtained, and selected
in the medium containing ampicillin to obtain
GI724/pGAP-btrC/pGAD-btrC strain, in a similar way. Further,
several strains which were gene-disrupted including
GI724.DELTA.rmf, GI724.DELTA.pgi, GI724.DELTA.zwf, GI724.DELTA.pgm,
GI724.DELTA.pgi.DELTA.rmf, GI724.DELTA.pgi.DELTA.zwf,
GI724.DELTA.pgi.DELTA.pgm and GI724.DELTA.pgi.DELTA.zwf.DELTA.pgm
were also transformed with pGAP-btrC/pGAD-btrC to obtain
GI724.DELTA.rmf/pGAP-btrC/pGAD-btrC strain,
GI724.DELTA.pgi/pGAP-btrC/pGAD-btrC strain,
GI724.DELTA.zwf/pGAP-btrC/pGAD-btrC strain,
GI724.DELTA.pgm/pGAP-btrC/pGAD-btrC strain,
GI724.DELTA.pgi.DELTA.rmf/pGAP-btrC/pGAD-btrC strain,
GI724.DELTA.pgi.DELTA.zwf/pGAP-btrC/pGAD-btrC strain,
GI724.DELTA.pgi.DELTA.pgm/pGAP-btrC/pGAD-btrC strain and
GI724.DELTA.pgi.DELTA.zwf.DELTA.pgm/pGAP-btrC/pGAD-btrC strain.
Example 5
Confirmation for the Expression of the DOI Synthase
[0160] <GI724.DELTA.pgi/pLEX-btrC Strain>
[0161] The GI724.DELTA.pgi/pLEX-btrC strain was grown at 30.degree.
C. in an induction medium (6% sodium hydrogen phosphate, 3%
potassium dihydrogen phosphate, 0.5% sodium chloride, 1% ammonium
chloride, 0.2% casamino acid, 0.5% D-glucose, 1 mM magnesium
chloride) until O.D. 600 nm was reached to 0.7. Then, the stain was
transferred into 2.times.YT medium (complete medium for E. coli,
1.6% tryptophan, 1% yeast extract, 0.5% sodium chloride) and
further cultured 37.degree. C. for 6 hours. Then, the strain was
collected. The expression of BtrC protein was confirmed with 12%
SDS polyacrylamide gel electrophoresis. The result is shown in FIG.
3A. It was confirmed that the BtrC protein were abundantly
expressed in the culture of 2.times.YT medium for 6 hours. It
should be noted that the following items were electrophored in each
lane of FIG. 3A.
[0162] Lane M:
[0163] Molecular weight marker (Precision Plus Protein Standards
(BIO-RAD), Catalog No. 161-0374, hereinafter, by the same
token)
[0164] Lane 1:
[0165] Cell extract with 1.times.SDS Loading buffer (hereinafter,
by the same token) from the strain after the culture of GI724
strain comprising pLEX-btrC for 6 hours in the minimum nutritive
medium (0.6% sodium hydrogen phosphate, 0.3% potassium dihydrogen
phosphate, 0.05% sodium chloride, 0.1% ammonium chloride)
(1.times.SDS Loading Buffer: 50 mM Tris-HCl (pH6.8); 2% SDS; 0.1%
Bromophenolblue; 10% Glycerol; 100 mM Dithiothreitol solution)
[0166] Lane 2:
[0167] Cell extract from the strains after the culture of GI724
strain comprising pLEX-btrC for 6 hours in the induction medium
supplemented with tryptophan
[0168] Lane 3:
[0169] Cell extract from the strains after the culture of GI724
strain comprising pLEX-btrC for 6 hours in the induction medium not
supplemented with tryptophan
[0170] Lane 4:
[0171] Cell extract from the strains after the culture for 2 hours
in a rice bran medium (composition: containing 20% of rice bran
treated with an enzyme)
[0172] Lane 5:
[0173] Cell extract from the strains after the culture for 6 hours
in a rice bran medium (composition: containing 20% of rice bran
treated with an enzyme) supplemented with tryptophan
[0174] The BtrC protein was abundantly expressed in the strain
cultured in the 2.times.YT medium even though the medium was not
supplemented with tryptophan. In addition, the BtrC protein was
abundantly expressed in the strain cultured in the rice bran
medium. Therefore, it was shown that the DOI synthase gene can be
highly expressed in these expression systems without using the high
cost inducer.
[0175] <The
GI724.DELTA.pgi.DELTA.zwf.DELTA.pgm/pGAP-btrC/pGAD-btrC
Strain>
[0176] The GI724.DELTA.pgi.DELTA.zwf.DELTA.pgm/pGAP-btrC/pGAD-btrC
strain was cultured in a pre-culture medium (0.6% disodium hydrogen
phosphate, 0.3% potassium dihydrogen phosphate, 0.05% sodium
chloride, 0.1% ammonium chloride, 2% casamino acid, 1% glycerin, 1
mM magnesium chloride) at 30.degree. C. for overnight. 1% of the
strain in the pre-culture medium were added in 2.times.YT medium
(complete medium for E. coli, 1.6% tryptone, 1% yeast extract, 0.5%
sodium chloride) and cultured at 30.degree. C. until O.D. 600 nm
was reached to 0.7. Then, the strain was collected. The expression
of BtrC protein was confirmed with 12% SDS polyacrylamide gel
electrophoresis. The result is shown in FIG. 3B. It should be noted
that the following items were electrophored in each lane of FIG.
3B.
[0177] Lane 1: Molecular weight marker
[0178] Lane 2: Cell extract of the strain obtained from the culture
at 0 hour
[0179] Lane 3: Cell extract of the strain obtained from the culture
after 12 hours
[0180] Lane 4: Cell extract of the strain obtained from the culture
after 36 hours
[0181] Lane 5: Cell extract of the strain obtained from the culture
after 72 hours
[0182] It was confirmed that the BtrC protein was abundantly
expressed in the strain cultured in the 2.times.YT medium for 6
hours, as shown in FIG. 3B. Therefore, it was shown that the DOI
synthase gene can be highly expressed in these expression systems
without using the high cost inducer.
Example 6
Synthesis of DOI
[0183] <Synthesis of DOI Using the GI724.DELTA.pgi Strain
Comprising pLEX-btrC>
[0184] The GI724.DELTA.pgi/pLEX-btrC strain was inoculated in 35 mL
of a pre-culture solution (RMG medium, 0.6% sodium hydrogen
phosphate, 0.3% potassium dihydrogen phosphate, 0.05% sodium
chloride, 0.1% ammonium chloride, 2% casamino acid, 1% glycerin, 1
mM magnesium chloride) contained in 300 mL conical flask, and
cultured for 15 hours. Next, 1% of the strain was inoculated in 3 L
of 2.times.YT medium contained in 10 L of jarfermenter. The strain
was cultured in a condition of 30.degree. C. of culture
temperature, 300 rpm of mixing speed, 10 L air per minute and pH
7.7 until O.D. 600 nm was reached to 0.7, and then 2% or 5% of
D-glucose was added in the culture, and the strain was further
cultured for 48 hours. The culture solution at the indicated time
points was centrifuged to remove the strain, thereby collecting a
culture supernatant 1.
[0185] <Synthesis of DOI Using the GI724.DELTA.pgi.DELTA.zwf
Strain Comprising pLEX-btrC>
[0186] The GI724.DELTA.pgi.DELTA.zwf/pLEX-btrC strain was
inoculated in 35 mL of a RMG medium containing 1% mannitol (0.6%
sodium hydrogen phosphate, 0.3% potassium dihydrogen phosphate,
0.05% sodium chloride, 0.1% ammonium chloride, 2% casamino acid, 1%
glycerin, 1 mM magnesium chloride), and cultured for overnight.
Next, 1% of the strain was inoculated in 3 L of 2.times.YT medium
containing 3% mannitol contained in 10 L jarfermenter, and were
cultured in a condition of 30.degree. C. of culture temperature,
300 rpm of mixing speed, 10 L air per minute and pH 7.7 until O.D.
600 nm was reached to 0.7, and then 3% of D-glucose was added in
the culture, and the culture was continued. The culture solution at
the indicated time points was centrifuged to remove the strain,
thereby collecting a culture supernatant 2.
[0187] <Synthesis of DOI Using the
GI724.DELTA.pgi.DELTA.zwf.DELTA.pgm Strain Comprising
pLEX-btrC>
[0188] The GI724.DELTA.pgi.DELTA.zwf.DELTA.pgm strain comprising
pLEX-btrC was inoculated in 50 mL of a preculture medium contained
in 300 mL conical flask (0.6% disodium hydrogen phosphate, 0.3%
potassium dihydrogen phosphate, 0.05% sodium chloride, 0.1%
ammonium chloride, 2% casamino acid, 1% glycerol, 1 mM magnesium
chloride), and cultured for 15 hours. Next, 1% of the strain of the
preculture solution was inoculated in 3 L of 2.times.YT medium
contained in 10 L jarfermenter (MDL-6C, made by Marubishi
Bioengineering). The strain was cultured in a condition of
25.degree. C. of culture temperature, 300 rpm of mixing speed, 10 L
air per minute and pH 7.0 until O.D. 600 nm was reached to 0.7, and
then 5% of D-glucose was added in the culture, and the culture was
further cultured for 72 hours. The culture solution at the
indicated time points time was centrifuged to remove the strain,
thereby collecting a culture supernatant 3.
[0189] <Synthesis of DOI Using the
GI724.DELTA.pgi.DELTA.zwf.DELTA.pgm Strain Comprising
pGAP-btrC/pGAD-btrC>
[0190] The GI724.DELTA.pgi.DELTA.zwf.DELTA.pgm strain comprising
pGAP-btrC/pGAD-btrC was inoculated in 50 mL of a preculture
solution contained in 300 mL conical flask (0.6% disodium hydrogen
phosphate, 0.3% potassium dihydrogen phosphate, 0.05% sodium
chloride, 0.1% ammonium chloride, 2% casamino acid, 1% glycerol, 1
mM magnesium chloride), and cultured for 15 hours. Next, 1% of the
strain of the preculture solution was inoculated in 3 L of
2.times.YT medium contained in 10 L jarfermenter, and was cultured
in a condition of 25.degree. C. of culture temperature, 300 rpm of
mixing speed, 10 L air per minute and pH 6.0 until O.D. 600 nm was
reached to 0.7, and then 5% of D-glucose was added in the culture,
and the culture was further cultured for 72 hours. The culture
solution at the indicated time points was centrifuged to remove the
strain, thereby collecting a culture supernatant 4.
[0191] <Synthesis of DOI Using the GI724.DELTA.rmf Strain
Comprising pLEX-btrC>
[0192] The GI724.DELTA.rmf strain comprising pLEX-btrC was
inoculated in 3 mL of a preculture solution contained in test tube
(0.6% disodium hydrogen phosphate, 0.3% potassium dihydrogen
phosphate, 0.05% sodium chloride, 0.1% ammonium chloride, 2%
casamino acid, 1% glycerol, 1 mM magnesium chloride), and cultured
for 15 hours. 1% of the strain of the preculture solution was
inoculated in 10 mL of 2.times.YT medium contained in L-shaped test
tube. The strain was cultured in a condition of 30.degree. C. of
culture temperature, 160 rpm of mixing speed and pH 7.0 until O.D.
600 nm was reached to 0.7, and then 3% of D-glucose was added in
the culture, and the culture was further cultured for 72 hours. The
culture solution at the indicated time points was centrifuged to
remove the strain, thereby collecting a culture supernatant 5.
Example 7
Measurement of DOI Production
[0193] <Oximation of DOI>
[0194] Measurement of DOI accumulated in the culture supernatant
was performed as follows. That is, with regard to the culture
supernatants 1 and 2, the culture solution(s) at the indicated time
points were collected. One volume of water, two volumes of methanol
and 1.5 mg/ml of O-(4-nitrobenzyl)hydroxylamine (NBHA), with regard
to the volume of the supernatant(s) were added to the
supernatant(s) and mixed, and incubated at 60.degree. C. for 1 hour
to obtain oxime derivative of DOI.
[0195] With regard to the culture supernatants 3, 4 and 5, 9
volumes of distilled water was added to the supernatant(s), and
added one volume of methanol with regard to this solution, and then
30 mg/ml of O-(4-nitrobenzyl)hydroxylamine hydrochloride salt
(NBHA) was added and mixed, and incubated at 60.degree. C. for 1
hour to obtain oxime derivative of DOI.
[0196] <Detection of DOI>
[0197] The solvent was evaporated from the oxime of DOI so obtained
with Speed Vac System (ISS110, made by Thermo), the obtained oxime
derivative of DOI was dissolved in an appropriate volume of
methanol, the aliquot was analyzed with HPLC (high-performance
liquid chromatography) to perform the detection and quantification
of DOI. In the HPLC, LC-10AT (Shimadzu), and Luna 5u C18 column
(Phrnomenex, column length 150 mm, column diameter 4.6 mm) were
used, 20% methanol was used as an eluent, and the ultraviolet
absorption at 262 nm was measured. Amount of the oxime derivative
of DOI with O-(4-nitrobenzyl) was quantified with standard curve
thereof. It should be noted that the quantification of glucose was
performed with glucose assay procedure kit (Megazyme).
[0198] As shown in FIGS. 4A and 4B, the peak corresponding to the
oxime derivative of DOI was confirmed in the HPLC analysis. In
addition, FIGS. 5 to 7 show a time course of the DOI production,
turbidity of the medium and the D-glucose concentration.
Reference Example 1
Synthesis of DOI Using the GI724.DELTA.pgi Strain Comprising
pLEX-btrC
[0199] The GI724.DELTA.pgi strain comprising pLEX-btrC was
inoculated in 35 mL of a preculture solution contained in 300 mL
conical flask (RMG medium: 0.6% sodium hydrogen phosphate, 0.3%
potassium dihydrogen phosphate, 0.05% sodium chloride, 0.1%
ammonium chloride, 2% casamino acid, 1% glycerin, 1 mM magnesium
chloride), and cultured for 15 hours. Next, 1% of the strain of the
preculture solution was inoculated in 3 L of 2.times.YT medium
contained in 10 L of jarfermenter. The strain was cultured in a
condition of 30.degree. C. of culture temperature, 300 rpm of
mixing speed, 10 L air per minute and pH 7.7 until O.D. 600 nm was
reached to 0.7, and then 2% or 5% of D-glucose was added in the
culture, and the culture was further cultured for 24 hours. The
culture solution at the indicated time points was centrifuged to
remove the strain, thereby collecting a culture supernatant 11.
Reference Example 2
Synthesis of DOI Using the GI724.DELTA.pgi.DELTA.zwf.DELTA.pgm
Strain Comprising pLEX-btrC
[0200] The GI724.DELTA.pgi.DELTA.zwf.DELTA.pgm strain comprising
pLEX-btrC was inoculated in 50 mL of a preculture solution
contained in 300 mL conical flask (0.6% disodium hydrogen
phosphate, 0.3% potassium dihydrogen phosphate, 0.05% sodium
chloride, 0.1% ammonium chloride, 2% casamino acid, 1% glycerol, 1
mM magnesium chloride), and cultured for 15 hours. Next, 1% of the
strain of the preculture solution was inoculated in 3 L of
2.times.YT medium contained in 10 L of jarfermenter (MDL-6C, made
by Marubishi Bioengineering). The strain was cultured in a
condition of 25.degree. C. of culture temperature, 300 rpm of
mixing speed, 10 L air per minute and pH 7.0 until O.D. 600 nm was
reached to 0.7, and then 5% of D-glucose was added in the culture,
and the culture was further cultured for 72 hours. The culture
solutions before and after the addition of D-glucose were
collected, and were centrifuged to remove the strain, thereby
collecting culture supernatants 12 and 13.
Reference Example 3
Synthesis of DOI Using the Wild Type GI724 Strain Comprising
pLEX-btrC
[0201] The wild type GI724 strain comprising pLEX-btrC was
inoculated in 3 mL of a preculture solution contained in test tube
(0.6% disodium hydrogen phosphate, 0.3% potassium dihydrogen
phosphate, 0.05% sodium chloride, 0.1% ammonium chloride, 2%
casamino acid, 1% glycerol, 1 mM magnesium chloride), and cultured
for 15 hours. 1% of the strain of the preculture solution was
inoculated in 10 mL of 2.times.YT medium contained in L-shaped test
tube. The strain was cultured in a condition of 30.degree. C. of
culture temperature, 160 rpm of mixing speed and pH 7.0 until O.D.
600 nm was reached to 0.7, and then 3% of D-glucose was added in
the culture, and the culture was further cultured for 72 hours. The
culture solutions at the indicated time points were collected, and
were centrifuged to remove the strain, thereby collecting a culture
supernatant 14.
Reference Example 4
Measurement of DOI Production
[0202] Quantification of the DOI production accumulated in the
culture supernatants 11 to 14 as obtained was performed as
follows.
[0203] One volume of water, two volumes of methanol and 15 mg/ml of
O-(4-nitrobenzyl)hydroxylamine (NBHA), with regard to the volume of
the culture supernatant 11 were added and mixed in the culture
supernatant 11, and incubated at 60.degree. C. for 1 hour to obtain
the oxime derivative of DOI.
[0204] With regard to the culture supernatants 12, 13 and 14, 9
volumes of distilled water was added to the supernatant(s), and
added one volume of methanol with regard to this solution, and then
30 mg/ml of O-(4-nitrobenzyl)hydroxylamine hydrochloride salt
(NBHA) was added and mixed, and incubated at 60.degree. C. for 1
hour to obtain the oxime derivative of DOI.
[0205] The solvent was evaporated from the solution of the oxime
derivative of DOI so obtained with Speed Vac System (ISS110, made
by Thermo), the obtained oxime derivative of DOI was dissolved in
an appropriate volume of methanol, the aliquot was analyzed with
HPLC (high-performance liquid chromatography) to perform the
detection and determination of the amount of DOI. In the HPLC,
LC-9A (Shimadzu), and Luna 5u C18 column (Phrnomenex, column length
150 mm, column diameter 4.6 mm) were used, 20% methanol was used as
an eluent, and the ultraviolet absorption at 262 nm was measured.
Amount of the oxime derivative of DOI with 0-(4-nitrobenzyl) was
quantified with Standard curve thereof.
Example 8
A Method Using a Mixed Bed Column of Amberlite IR120 and Amberlite
IRA410
[0206] 200 mL of a hydrogen form of Amberlite IR 120 and 200 mL of
an acetate form of Amberlite IRA410 were mixed, and filled in a
column (.phi.5 cm.times.25 cm). 100 mL of the culture medium as
obtained from the reference example 1 and adjusted to pH 2.96
(containing 1.4 g of DOI) was applied to the column, an elution was
performed with water at 2 mL per minute. 6 mL of eluate per a
fraction was collected and pH and the conductivity of every other
fraction were measured. In addition, the quantity of DOI was
measured at every three fractions. The quantity measurement of DOI
was performed such that DOI was derivatized into the
O-(4-nitrobenzyl)oxime and then the peak area on the HPLC was
measured, and the quantity of DOI was estimated from the
calibration curve based on the peak area. The result so obtained is
shown in FIG. 10. In addition, after the fractions were grouped
into 4 blocks and each block was freeze-dried, the purity and
amount of DOI on each group was measured. The result is shown in
Table 2. .sup.13C-NMR spectrum of the purified DOI so obtained is
shown in FIG. 11. The .sup.13C-NMR spectrum was measured with
DPX-250 NMR apparatus (.sup.13C nucleus was resonated at 67.5 MHz)
made by Bruker with regard to the sample dissolved in deuterated
water.
Example 9
A Method Using a Mixed Bed Column of Amberlite IR200 and Amberlite
IRA410
[0207] 200 mL of a hydrogen form of Amberlite IR 200 and 200 mL of
an acetate form of Amberlite IRA410 were mixed, and filled in a
column (.phi.5 cm.times.25 cm). 100 mL of the culture medium as
obtained from the reference example 1 adjusted to pH 2.97
(containing 562 mg of DOI) was applied to the column, an elution
was performed with water at a flow rate of 2 mL per minute. 6 mL of
eluate per a fraction was collected and pH and the conductivity of
every other fraction were measured. In addition, the quantitative
of DOI was performed at every three fractions. The result so
obtained is shown in FIG. 12. In addition, after the fractions were
grouped into 4 blocks and each block was freeze-dried, the purity
and amount of DOI on each group were measured. The result is shown
in Table 3. .sup.13C-NMR spectrum of the purified DOI so obtained
is shown in FIG. 13. The measurement of quantity of DOI and
measurement of the .sup.13C-NMR spectrum were performed in
accordance with Example 8
TABLE-US-00002 TABLE 2 weight amounts of DOI purity (mg) (mg) (%)
Fr.27-47 207 42 20.3 Fr.48-60 252.4 226.3 89.7 Fr.61-95 253.9 117.8
46.4 Fr.96- 24.6 13.2 53.5 Total 737.9 399.3 54.1
TABLE-US-00003 TABLE 3 weight amounts of DOI purity (mg) (mg) (%)
Fr.26-45 85.7 71.4 83.8 Fr.46-71 120 114.1 95.1 Fr.72-95 31.6 25.1
79.5 Fr.96- 11.9 1.3 10.8 Total 249.2 211.9 85.0
Example 10
A Method Using a Double Bed Column of Amberlite 200CT and Amberlite
IRA96SB
[0208] A DOI containing culture solution (DOI amount: 22.1 g/850
mL) was applied on a cation exchange resin column (Amberlite 200CT,
a hydrogen form, 400 mL) and eluted with water. Fractions which
contain DOI in the obtained eluents analyzed by TLC were applied on
an anion exchange resin column (Amberlite IRA96SB, an acetate form,
600 mL), and eluted with water. Fractions which contain DOI in the
obtained eluents confirmed by the analysis with TLC were
concentrated in vacuo. As the result, 20.8 g of DOI which has a
purity as observed in a .sup.1H-NMR spectrum shown in FIG. 15 was
obtained. The purity is approximately same as those obtained from
the method of the purification using the above-mentioned mixed bed
column. The .sup.1H-NMR spectrum was measured with DPX-250 NMR
apparatus made by Bruker with regard to the sample dissolved in
deuterated water.
Example 11
A Method for Restoring 2-Deoxy-Scyllo-Inosose, Following to Convert
DOI into the Dimethylketal Derivative, to Cryltallize and to
Purify
[0209] The purified DOI as obtained in Example 10 was dissolved in
methanol (835 mL) and trimethoxymethane (310 mL) and
p-toluenesulfonic acid monohydrate (2.12 g) were added, and stirred
for 3 hours. Then, the reaction mixture was neutralized with sodium
bicarbonate (30.6 g), and the vacuum concentration was performed
after filtration. The residue was dissolved in methanol, three
volumes of silica gel (C-200, 60 g) were added, and the vacuum
concentration was performed to adsorb DOI onto the surface of the
silica gel. The DOI adsorbed onto the silica gel was filled in a
silica gel column with ethyl acetate and methanol (5:1). Then, DOI
was eluted from the column with mixed solvent of ethyl acetate and
methanol (5:1). The eluents which contain DOI were collected and
the vacuum concentration was performed with the collected eluents,
thereby obtaining the dimethylketal derivative of DOI (20.7 g).
Subsequently, a solution of the dimethylketal derivative dissolved
in 25 mL of methanol was added to 100 mL of chloroform and 7.5 mL
of hexane under heating, and then cooled to precipitate and
separate white crystals, thereby obtaining the crystal of
2-deoxy-scyllo-inosose dimethylketal (9.1 g). Any signals
originated from any impurities were not observed in .sup.1H-NMR as
shown in FIG. 16.
[0210] The crystals of the dimethylketal derivative (995 mg) were
dissolved in acetone (22 mL), and p-toluenesulfonic acid
monohydrate (280 mg) and distilled water (6 mL) were added, and the
solution was stirred for 5 hours. After the starting materials were
disappeared by confirming with TLC analysis, the vacuum
concentration was performed. The residue dissolved in water was
applied on an anion exchange resin column (IRA96SB, an acetate
form, 10 mL), and the eluents containing the target product were
concentrated to quantitatively obtain highly purified DOI (770 mg).
Any signals originated from any impurities were not observed in
.sup.1H-NMR as shown in FIG. 17.
[0211] [Statement of Microorganism as Deposited]
[0212] Statements of microorganism as deposited which is used in
the present invention are as follows.
[0213] Escherichia coli
[0214] GI724.DELTA.pgi.DELTA.zwf.DELTA.pgm/pGAP-btrC/pGAD-btrC
[0215] FERM AP-20809
[0216] 1. Name of organization of receipt: National Institute of
Advanced Industrial Science and Technology/International Patent
Organism Depositary
[0217] 2. Receiving date: Feb. 24, 2006
[0218] 3. Number of receipt: FERM AP-20809
[0219] Escherichia coli
[0220] GI724.DELTA.rmf.DELTA.pgi/pLEX-btrC
[0221] FERM AP-20808
[0222] 1. Name of organization of receipt: National Institute of
Advanced Industrial Science and Technology/International Patent
Organism Depositary
[0223] 2. Receiving date: Feb. 24, 2006
[0224] 3. Number of receipt: FERM AP-20808
INDUSTRIALLY APPLICABILITY
[0225] According to the present invention, it is possible to
manufacture DOI with high purity which is a starting material for
manufacturing various types of six-membered carbocyclic compounds,
by fermentation using biomass-derived starting materials such as
starch which are regenerative resources, instead of chemical
compounds derived from petroleum in the prior art.
[0226] As mentioned above, the present invention has been described
in accordance with the preferred embodiments thereof. It is obvious
that modifications and alterations can be made to the embodiments,
without departing from the spirit and scope of the present
invention as defined in the Claims, although the present invention
has been described in accordance with the specific embodiments.
That is, the present invention shall not be construed in limiting
into the detail of the embodiments and the drawing as attached.
Sequence CWU 1
1
22131DNAartificial sequenceprimer1 1gggaattcca tatgacgact
aaacaaattt g 31229DNAartificial sequenceprimer 2 2gctctagatt
acagcccttc ccggatcac 29375DNAartificial sequenceprimer 1 for pgi
expression 3atgaaaaaca tcaatccaac gcagaccgct gcctggcagg cactacagaa
tggacagcaa 60gcgaaccgga attgc 75474DNAartificial sequenceprimer 2
for pgi expression 4ttaaccgcgc cacgctttat agcggttaat cagaccattg
gtcgagctat tcagaagaac 60tcgtcaagaa ggcg 74575DNAartificial
sequenceprimer 1 for zwf expression 5atggcggtaa cgcaaacagc
ccaggcctgt gacctggtca ttttcggcgc tggacagcaa 60gcgaaccgga attgc
75674DNAartificial sequenceprimer 2 for zwf expression 6ttactcaaac
tcattccagg aacgaccatc acgggtaatc atcgccaccg tcagaagaac 60tcgtcaagaa
ggcg 74721DNAartificial sequenceprimer 1 for pgm expression
7cgcatccgac attttacggg c 21821DNAartificial sequenceprimer 2 for
pgm expression 8ttgcttgtgt cctttgtctg c 21920DNAartificial
sequenceprimer 3 for pgm expression 9cacatttaat aaaaaaaggg
201021DNAartificial sequenceprimer 4 for pgm expression
10ccaaccggga tttaaaccga c 211176DNAartificial sequenceprimer 1 for
pgi+zwf expression 11atgaaaaaca tcaatccaac gcagaccgct gcctggcagg
cactacagaa cagaataaat 60aaatcctggt gtccct 761276DNAartificial
sequenceprimer 2 for pgi+zwf expression 12ttaaccgcgc cacgctttat
agcggttaat cagaccattg gtcgagctat atccgcttat 60tatcacttat tcaggc
761320DNAartificial sequenceprimer 1 for pgi+zwf+pgm expression
13ttaccgtaac ggagtttaac 201420DNAartificial sequneceprimer 2 for
pgi+zwf+pgm expression 14gcctcgtttc cctcatactg 201521DNAartificial
sequneceprimer 3 for pgi+zwf+pgm expression 15ctgtctcttt aaaaagaaac
c 211618DNAartificial sequenceprimer 4 for pgi+zwf+pgm expression
16aatgaccgaa acgggtgg 181730DNAartificial sequenceprimer 1 for gapA
promoter 17cgcggatccg cgggaagagt gaggcgagtc 301819DNAartificial
sequenceprimer 2 for gapA promoter 18atattccacc agctatttg
191928DNAartificial sequenceprimer 1 for gadA promoter 19ctagtctaga
gtcgtttttc tgcttagg 282028DNAartificial sequenceprimer 2 for gadA
promoter 20ttcgaactcc ttaaatttat ttgaaggc 282122DNAartificial
sequenceprime 1 for aspA terminator 21taacataacg ttgtaaaaac cg
222228DNAartificial sequenceprimer 2 for aspA terminator
22cgcggatcct taagttgggt aacgccag 28
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