JP2001112488A - Method for producing gdp-fucose - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for producing GDP-fucose useful as a synthetic substrate, etc., of complex carbohydrates which are useful for application to and immunotherapy for the protection against infections by bacteria, viruses and the like and cardiovascular diseases and GDP-4-keto-6-deoxymannose (hereinafter referred to as GKDM) which is an intermediate for the GDP-fucose. SOLUTION: This method for producing GDP-fucose comprises allowing GKDM and an enzyme source to be present in an aqueous medium, wherein the enzyme source is a culture broth of a microorganism capable of converting GKDM into GDP-fucose or a treated product of the culture broth, forming and accumulating GDP-fucose in the aqueous medium and recovering the GDP- fucose therefrom.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to guanosine-5'-
Fucose diphosphate (hereinafter abbreviated as GDP-fucose) and guanosine-5'-diphosphate-4-keto-6
A method for producing deoxymannose (hereinafter abbreviated as GKDM). GDP-fucose is useful as a glycoconjugate synthetic substrate useful for protection against infection with bacteria and viruses, application to cardiovascular disorders, immunotherapy and the like. Also,
GKDM is useful as an intermediate or the like for the production of GDP-fucose.

[0002]

2. Description of the Related Art Regarding the production method of GDP-fucose,
Although a chemical synthesis method [Carbohyd. Res., 242 , 69 (1993)] is known, there are difficulties in stereoselectivity and supply of substrates. In the method using an enzyme [Agric. Biol. Chem, 48 , 823 (1984), Japanese Translation of PCT International Publication No. 7-500248, WO99 / 9180], expensive raw materials are used, and purification of the enzyme is complicated. Unsuitable for production. Also, a method utilizing the activity of a microorganism (WO98 / 12
343) has also been developed and is a useful method, but further improvement is required for use as an industrial production method. It is also known that the activity of GDP-mannose 4,6-dehydratase, which is the first enzyme of biosynthesis from GDP-mannose to GDP-fucose, is inhibited by the final product, GDP-fucose [Biochim. Biophys.
Acta, 117 , 79 (1966), FEBS Lett., 412 , 126 (199
7)].

[0003]

SUMMARY OF THE INVENTION An object of the present invention is to provide a GD
It is to provide an efficient method for producing P-fucose and GKDM.

[0004]

Means for Solving the Problems The present inventors have conducted intensive studies in order to solve the above-mentioned problems, and after producing and accumulating GKDM which is a precursor of GDP-fucose, the accumulated GKDM was obtained.
By converting KDM to GDP-fucose, GD
Inhibition of GDP-mannose 4,6-dehydratase activity by P-fucose can be avoided and G-mannose
It has been found that DP-fucose can be produced, and the present invention has been completed.

That is, the present invention relates to the following (1) to (18). (1) A culture solution of a microorganism having the ability to convert GKDM to GDP-fucose or a processed product of the culture solution is used as an enzyme source, and the enzyme source and GKDM are allowed to exist in an aqueous medium. -A process for producing GDP-fucose, comprising producing and accumulating fucose and collecting GDP-fucose from the aqueous medium.

(2) A culture solution of a microorganism capable of producing GTP from a precursor of GTP or a processed product of the culture solution, and a culture solution of a microorganism capable of producing GKDM from sugar and GTP or the culture solution Using the treated product as an enzyme source, allowing these enzyme source, GTP precursor and sugar to be present in an aqueous medium, producing and accumulating GKDM in the aqueous medium, and then converting GKDM into GDP-fucose. Is used as an enzyme source, and the accumulated GKDM is converted into GDP-fucose to produce and accumulate GDP-fucose in the aqueous medium. -A process for producing GDP-fucose, characterized by collecting fucose.

(3) A culture solution of a microorganism capable of producing GTP from a precursor of GTP or a processed product of the culture solution, and a culture solution of a microorganism capable of producing GKDM from sugar and GTP or the culture solution Using the treated product as an enzyme source, allowing these enzyme source, GTP precursor and sugar to be present in an aqueous medium, producing and accumulating GKDM in the aqueous medium, and collecting GKDM from the aqueous medium. Characteristic method of producing GKDM.

(4) The processed product of the culture solution is a concentrate of the culture solution, a dried product of the culture solution, cells obtained by centrifuging the culture solution, a dried product of the cells, and a freeze-drying of the cells. Product, a surfactant-treated product of the cells, an ultrasonic treatment of the cells, a mechanically ground product of the cells, a solvent-treated product of the cells, an enzyme-treated product of the cells, (1), (2) or (3), which is a protein fraction of a cell, an immobilized substance of the cell, or an enzyme preparation obtained by extraction from the cell.

(5) GTP precursors are guanine, xanthine, hypoxanthine, guanosine, xanthosine,
The method according to the above (2) or (3), which is inosine, guanosine-5'-monophosphate, xanthosine-5'-monophosphate, or inosine-5'-monophosphate. (6) The method of the above (2) or (3), wherein the sugar is a sugar selected from glucose, fructose and mannose.

(7) The method according to (2) or (3), wherein the microorganism capable of producing GTP from a precursor of GTP is a microorganism selected from microorganisms belonging to the genus Corynebacterium. (8) The method according to (7) above, wherein the microorganism belonging to the genus Corynebacterium is Corynebacterium ammoniagenes.

(9) The microorganism according to the above (2) or (3), wherein the microorganism having an ability to produce GKDM from sugar and GTP is composed of one or more microorganisms.
Manufacturing method. (10) The process according to (9), wherein the microorganism is one or more microorganisms selected from microorganisms belonging to the genus Escherichia and Corynebacterium.

(11) The method according to (10), wherein the microorganism belonging to the genus Escherichia is Escherichia coli. (12) The method according to (10), wherein the microorganism belonging to the genus Corynebacterium is Corynebacterium ammoniagenes. (13) Microorganisms capable of producing GKDM from sugar and GTP include glucokinase (hereinafter abbreviated as glk), phosphomannommutase (hereinafter abbreviated as manB), guanylyl mannose-1-phosphate Transferase (hereinafter abbreviated as manC), phosphoglucomutase (hereinafter abbreviated as pgm), phosphofructokinase (hereinafter abbreviated as pfk) and GDP-mannose 4,6-dehydratase (hereinafter abbreviated as gmd) The method according to (2) or (3), wherein the microorganism is a microorganism having at least one enzyme activity selected from the group consisting of:

(14) The microorganism is at least one selected from a gene encoding glk, a gene encoding manB, a gene encoding manC, a gene encoding pgm, a gene encoding pfk, and a gene encoding gmd. (13) The method according to the above (13), wherein the method comprises at least one kind of microorganism having a recombinant DNA of a DNA fragment containing the gene and a vector.

(15) The gene encoding glk, ma
The gene encoding nB, the gene encoding manC, the gene encoding pgm, the gene encoding pfk and the gene encoding gmd belong to Escherichia.
(14) characterized in that it is a gene derived from E. coli.
Manufacturing method. (16) The production of the above (1) or (2), wherein the microorganism having an ability to convert GKDM to GDP-fucose is a microorganism having a strong activity of GKDM epimerase / reductase (hereinafter abbreviated as wcaG). Law.

(17) The method according to (16) above, wherein the microorganism is a microorganism having a recombinant DNA of a vector and a DNA fragment containing a gene encoding wcaG. (18) The gene encoding wcaG is Escherichia
(17) The above-mentioned (17), which is a gene derived from E. coli.
Manufacturing method.

Hereinafter, the present invention will be described in detail.

[0017]

BEST MODE FOR CARRYING OUT THE INVENTION As a culture solution of a microorganism capable of producing GTP from a GTP precursor used in the present invention or a processed product of the culture solution, any microorganisms having the capability can be used. A culture solution or a processed product of the culture solution can also be used. Examples of the microorganism include microorganisms belonging to the genus Escherichia or Corynebacterium.

Suitable examples of the GTP precursor include well-known substances described below. Examples of the microorganism belonging to the genus Escherichia include Escherichia coli. Examples of microorganisms belonging to the genus Corynebacterium include Corynebacterium ammoniagenes and the like. Specifically, Corynebacter
ium ammoniagenes ATCC21170 and the like.

GKD is derived from sugar and GTP used in the present invention.
As a culture solution of a microorganism having the ability to produce M or a treated product of the culture solution, a culture solution of a microorganism having the ability to produce GKDM from sugar and GTP or a treated product of the culture solution may be used. A culture solution or a processed product of the culture solution can also be used. For example, glk, manB,
1 selected from manC, pgm, pfk and gmd
A culture solution of a microorganism or the like having a strong activity of more than one kind of enzyme is used.

Suitable examples of the above-mentioned sugar include well-known sugars described below. The microorganisms having a strong enzyme activity include, for example, glk, manB,
1 selected from manC, pgm, pfk and gmd
A microorganism in which the activity of more than one type of enzyme is higher than that of the parent strain. Here, the parent strain is a microorganism used as a source when constructing a mutant strain, a cell fusion strain, a transduced strain or a recombinant strain. Therefore, the microorganism in which the activity of one or more enzymes selected from the above enzymes is higher than that of the parent strain may be any of a mutant strain, a cell fusion strain, a transduced strain, or a recombinant strain.

[0021] Specific examples of the microorganism include microorganisms belonging to the genus Escherichia or Corynebacterium, and preferable specific examples include Escherichia coli or Corynebacterium ammoniagenes. Also, glk, manB, manC,
A transformant in which the activity of one or more enzymes selected from pgm, pfk and gmd has been enhanced by genetic recombination technology can also be used. As a specific example of the transformant, a glk gene derived from Escherichia coli [J. Bacter
iol., 179 , 1298 (1997)].
K11) Escherichia coli NM522 strain,
The manB gene from Escherichia coli [J. Bacterio
l., 178 , 4885 (1996)].
Escherichia coli NM522 harboring 11), a manC gene derived from Escherichia coli [J. Bacterio
l., 178 , 4885 (1996)].
Escherichia coli NM522 strain harboring pgm gene derived from Escherichia coli [J. Bacteriol.,
176 , 5847 (1994)].
Escherichia coli NM522 strain (WO98 /
12343), a pfkB gene derived from Escherichia coli [Gen
e, 28 , 337 (1984)].
Escherichia coli NM522 strain (WO98 /
12343) and gmd gene from Escherichia coli
Recombinant D containing [J. Bacteriol., 178 , 4885 (1996)]
Escherichia coli NM possessing NA (pGE19)
And 522 strains.

If the microorganism has the ability to produce GTP from a precursor of GTP and the ability to produce GKDM from sugar and GTP at the same time,
GKDM can be produced from precursors of P and sugars. In the case of a microorganism having only a part of the activity required for producing GKDM from sugar and GTP in one strain, GKDM can be produced by appropriately combining two or more microorganisms.

The culture solution of a microorganism having the ability to convert GKDM into GDP-fucose or a processed product of the culture solution used in the present invention may be a culture solution of a microorganism having the conversion activity or a processed product of the culture solution. Any of them can be used. For example, a culture solution of a microorganism having strong wcaG activity can be used. Specific examples include a microorganism belonging to the genus Escherichia or Corynebacterium, such as a culture solution of Escherichia coli or Corynebacterium ammoniagenes.

In addition, a transformant in which the activity of GKDM epimerase / reductase is enhanced by a gene recombination technique can also be used. As a specific example of the transformant,
The wcaG gene from Escherichia coli [J. Bacterio
l., 178 , 4885 (1996)].
8) and Escherichia coli NM522 strain.

GDP-using the above-described gene recombination technique
In the production of fucose and GKDM, isolation and purification of plasmid DNA from microorganisms, cleavage of plasmid DNA with restriction enzymes, isolation and purification of the cleaved DNA fragment, DN
Various procedures relating to gene recombination, such as enzymatic ligation of the A fragment and transformation using recombinant DNA, are performed by known methods [eg, Molecular Cloning, A Laboratory Manual, Seco
nd Edition, Cold Spring Harbor Laboratory Press (1
989) (hereinafter abbreviated as Molecular Cloning 2nd Edition), Current Protocols in Molecular Biology, Joh
n Wiley & Sons (1987-1997) (hereinafter abbreviated as Current Protocols in Molecular Biology)]. Polymerase chain reaction (hereinafter abbreviated as PCR) can be performed according to a known method [PCR Protocols, Academic Press (1990)].

In order to express a gene involved in the production of GDP-fucose or GKDM in a host, a DNA fragment containing the gene is combined with a DNA fragment of an appropriate length containing the gene by restriction enzymes or PCR. After that, the DNA can be inserted into a suitable expression vector downstream of the promoter, and then the expression vector into which the DNA has been inserted is introduced into a host cell suitable for the expression vector.

As the host cell, any cells such as bacteria and yeast can be used as long as they can express the target gene. As the expression vector, those which can replicate autonomously in the above-mentioned host cells or can be integrated into a chromosome, and which contain a promoter at a position where the target DNA can be transcribed are used.

When a prokaryote such as a bacterium is used as a host cell, the gene expression vector is capable of autonomous replication in the prokaryote and, at the same time, comprises a promoter, a ribosome binding sequence, a target DNA, and a transcription termination sequence. Preferably, the DNA is a recombinant DNA. A gene that controls the promoter may be included. Expression vectors include pKK223-3, pGEX-2T (all manufactured by Amersham Pharmacia Biotech), pSE280 (Invitrogen), pGEMEX-1 (Promega), pQE-30 (Qiagen), pET -3 (manufactured by Novagen), pKYP10 (JP-A-58-110600), pKYP200 [Agric. Biol. Chem., 48 , 669]
(1984)], pLSA1 [Agric. Biol. Chem., 53 , 277 (198
9)], pGEL1 [Proc. Natl. Acad. Sci., USA, 82 , 4306].
(1985)], pBluescriptII SK + (Stratagene),
pBluescript II SK- (Stratagene), pTrS30
[Prepared from E. coli JM109 / pTrS30 (FERM BP-5407)], pTrS32
[Prepared from E. coli JM109 / pTrS32 (FERM BP-5408)], pUC19
[Gene, 33 , 103 (1985)], pSTV28 (Takara Shuzo), pUC118
(Manufactured by Takara Shuzo), pPAC31 (WO98 / 12343) and the like.

Any promoter may be used as long as it functions in a host cell such as Escherichia coli. For example, trp promoter (P trp ), lac promoter (P lac ), P _L promoter, P _R promoter, P _SE
Examples of the promoter include promoters derived from Escherichia coli and phage, such as promoters, SPO1 promoter, SPO2 promoter, penP promoter and the like. In addition, artificially designed and modified promoters such as a promoter in which two Ptrps are connected in series, a tac promoter, a lacT7 promoter, and a let I promoter can also be used.

It is preferable to use a plasmid in which the distance between the Shine-Dalgarno sequence, which is a ribosome binding sequence, and the initiation codon is adjusted to an appropriate distance (for example, 6 to 18 bases). In the recombinant DNA of the present invention, a transcription termination sequence is not always necessary for expression of a target DNA, but it is preferable to arrange a transcription termination sequence immediately below a structural gene.

Examples of prokaryotes include microorganisms belonging to the genus Escherichia, Serratia, Bacillus, Brevibacterium, Corynebacterium, Microbacterium, Pseudomonas, etc., for example, Escherichia coli XL1-B
lue, Escherichia coli XL2-Blue, Escherichia coli D
H1, Escherichia coli MC1000, Escherichia coli W148
5, Escherichia coli NM522, Escherichia coli JM10
9, Escherichia coli HB101, Escherichia coli No.4
9, Escherichia coli W3110, Escherichia coli NY49,
Serratia ficaria , Serratia fonticola , Serratia liq
uefaciens , Serratia marcescens , Bacillus subtili
s , Bacillus amyloliquefaciens , Brevibacterium imma
riophilum ATCC14068, Brevibacterium saccharolyticu
m ATCC14066, Corynebacterium ammoniagenes , Coryneb
acterium glutamicum ATCC13032, Corynebacterium glu
tamicum ATCC14067, Corynebacterium glutamicum ATCC
13869, Corynebacterium acetoacidophilum ATCC1387
0, Microbacterium ammoniaphilum ATCC15354, Pseudomo
nas sp. D-0110 and the like.

As a method for introducing the recombinant DNA, any method can be used as long as it is a method for introducing the DNA into the above-mentioned host cells, for example, a method using calcium ions.
[Proc. Natl. Acad. Sci., USA, 69 , 2110 (1972)], protoplast method (JP-A-63-248394), electroporation method [Nucleic Acids Research, 16 , 6127 (198)
8)] and so on.

When a yeast strain is used as a host cell, for example, YEp13 (ATCC3711) may be used as an expression vector.
5), YEp24 (ATCC37051), YCp50 (ATCC37419), pHS1
9, pHS15 and the like can be used. Any promoter may be used as long as it functions in a yeast strain.Examples include a PHO5 promoter, a PGK promoter, a GAP promoter, an ADH promoter, a gal 1 promoter, a gal 10 promoter, and a heat shock polypeptide promoter. , MFα1 promoter, CUP1 promoter and the like.

As the host cell, Saccharomyces genus,
Yeast strains belonging to the genus Schizosaccharomyces, Kluyveromyces, Trichosporon, Siwaniomyces, Pichia, Candida, and the like, and specifically, Saccharomyces cerevisiae , Schizosaccharomyces
pombe , Kluyveromyces lactis , Trichosporon pullulan
s , Schwanniomyces alluvius , Pichia pastoris , Candi
da utilis and the like.

As a method for introducing a recombinant DNA, any method for introducing DNA into yeast can be used. For example, electroporation [Methods in
Enzymol., 194 , 182 (1990)], spheroplast method [Pro
Natl. Acad. Sci., USA, 81 , 4889 (1984)] and the lithium acetate method [J. Bacteriol., 153 , 163 (1983)].

The method for culturing the transformant of the present invention in a medium can be carried out according to a usual method used for culturing a host. A culture medium for culturing a transformant obtained by using a prokaryote such as Escherichia coli or a eukaryote such as yeast as a host contains a carbon source, a nitrogen source, inorganic salts, and the like which can be used by the organism. Either a natural medium or a synthetic medium can be used as long as the medium can efficiently culture C.

The carbon source may be any one which can be assimilated by the organism, such as glucose, fructose, sucrose, molasses containing these, carbohydrates such as starch or starch hydrolysate, and organic acids such as acetic acid and propionic acid. Acids, alcohols such as ethanol and propanol, and the like can be used. Examples of the nitrogen source include ammonium salts of inorganic or organic acids such as ammonia, ammonium chloride, ammonium sulfate, ammonium acetate, and ammonium phosphate, other nitrogen-containing compounds, peptone, meat extract, yeast extract, corn steep liquor, and casein. A hydrolyzate, soybean meal, a soybean meal hydrolyzate, various fermentation cells, and digests thereof can be used.

As the inorganic substance, monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, calcium carbonate and the like can be used. Culture is
Usually, it is performed under aerobic conditions such as shaking culture or deep aeration stirring culture. The culture temperature is preferably 15 to 40 ° C, and the culture time is
Usually 5 hours to 7 days. During the cultivation, the pH was 3.0 to 9.
Hold at 0. The pH is adjusted using an inorganic or organic acid, an alkaline solution, urea, calcium carbonate, ammonia, or the like.

[0039] If necessary, an antibiotic such as ampicillin or chloramphenicol may be added to the medium during the culture. When culturing a microorganism transformed with an expression vector using an inducible promoter as a promoter, an inducer may be added to the medium as necessary. For example, when culturing a microorganism transformed with an expression vector using the lac promoter, isopropyl-β-D-thiogalactopyranoside or the like is used. When culturing a microorganism transformed with an expression vector using the trp promoter, indole acryl is used. An acid or the like may be added to the medium.

When two or more types of microorganisms are used in the production of the present invention, the microorganisms may be individually cultured, and the culture solution may be used. After that, the culture solution may be used. Further, during or after the cultivation of any microorganism, the remaining microorganism may be inoculated and cultured, and then the culture solution may be used.

The culture solution of the microorganism obtained by the culturing and the processed product of the culture solution obtained by variously treating the culture solution can be used as an enzyme source in an aqueous medium for the production of the present invention. Examples of the processed product of the culture solution include a concentrate of the culture solution, a dried product of the culture solution, cells obtained by centrifuging the culture solution, a dried product of the cells, a freeze-dried product of the cells, and the cells. Surfactant treated product, ultrasonically treated product of the cells, mechanically milled product of the cells, solvent-processed product of the cells, enzyme-treated product of the cells, protein fractionation of the cells Products, immobilized products of the cells or enzyme preparations obtained by extraction from the cells.

The amount of microorganisms used in the production method of the present invention is 1 to 500 g / l, preferably 5 to 300 g / l, as wet cells. When the production reaction is carried out simultaneously using two or more kinds of microorganisms, the total wet bacterial mass of the microorganisms in the aqueous medium is 2 to 500 g / l, preferably 10 to 400 g / l. The GTP precursor used in the production method of the present invention includes guanine, xanthine, hypoxanthine, guanosine, xanthosine, inosine, guanosine-5′-monophosphate, xanthosine-5′-monophosphate, and inosine-5 ′. -Monophosphoric acid and the like. The precursor is a pure product, as long as the salt of the precursor and impurities do not inhibit the reaction.
The precursor-containing culture solution fermented and produced by a microorganism and the precursor crude product from the culture solution can be used. The precursor of GTP is 0.1 mmol / L to 1.0
mol / L, preferably used at a concentration of 0.01 to 0.5 mol / L.

Examples of the sugar used in the production method of the present invention include glucose, fructose, mannose and derivatives thereof. The sugar may be a pure product or may contain any of these, as long as impurities do not inhibit the reaction. The sugar may be added all at once at the start of the reaction, or may be added portionwise or continuously during the reaction, and is used at a concentration of 0.1 mmol / L to 2.0 mol / L.

In the production method of the present invention, an energy donor, a coenzyme, a chelating agent such as phosphate ion, magnesium ion, phytic acid, a surfactant, and an organic solvent may be added as necessary. As the energy donor, any can be used as long as it promotes production, but glucose, fructose, sucrose, lactose, maltose, mannitol, carbohydrates such as sorbitol, pyruvic acid, lactic acid, organic acids such as acetic acid, Glycine, alanine, aspartic acid, amino acids such as glutamic acid, molasses, starch hydrolyzate, and the like,
It is used at a concentration of 1.0 mmol / L to 2.0 mol / L.

Examples of the phosphate ions include inorganic phosphates such as orthophosphate, pyrophosphate, tripolyphosphate, polyphosphate, metaphosphate, monopotassium phosphate, dipotassium phosphate, monosodium phosphate and disodium phosphate. Examples thereof include salts and the like, and can be used at a concentration of 1.0 mmol / L to 1.0 mol / L. Examples of the magnesium ion include inorganic magnesium salts such as magnesium sulfate, magnesium nitrate, and magnesium chloride, and organic magnesium salts such as magnesium citrate, and are usually used at a concentration of 1 to 100 mmol / L.

Examples of the surfactant include nonionic surfactants such as polyoxyethylene octadecylamine (for example, Nimeen S-215, manufactured by NOF Corporation), cetyltrimethylammonium bromide, and alkyldimethylbenzylbenzyl chloride (for example, cationic F2-40E,
Accelerates formation of cationic surfactants such as Nippon Yushi Co., Ltd., anionic surfactants such as lauroyl sarcosinate, and tertiary amines such as alkyldimethylamine (eg, tertiary amine FB, Nippon Yushi Co., Ltd.) Any one of them may be used, and one kind or a mixture of several kinds may be used. The surfactant is usually 0.1 to 50 g /
Used at a concentration of 1.

As the organic solvent, xylene, toluene,
Examples thereof include aliphatic alcohol, acetone, and ethyl acetate, which are usually used at a concentration of 0.1 to 50 ml / l. As the aqueous medium used in the production method of the present invention,
Buffers such as water, phosphate, carbonate, acetate, borate, citrate and Tris; alcohols such as methanol and ethanol; esters such as ethyl acetate; ketones such as acetone; amides such as acetamide And the like. Further, a culture solution of a microorganism can be used as an aqueous medium.

The production method of the present invention is carried out in an aqueous medium at pH 5-1.
0, preferably 1 to 6 at a pH of 6 to 8 and 20 to 50 ° C.
Perform for 96 hours. Quantification of GDP-fucose and GKDM produced in the aqueous medium can be performed using HPLC or the like according to the method described in WO98 / 12343. The GDP-fucose and GKDM produced in the reaction solution are collected by a conventional method using activated carbon or ion exchange resin [C
arbohyd. Res., 242 , 69 (1993)].

An embodiment of the present invention will be described below.

[0050]

[Embodiment 1] glk, manB, manC, p
Construction of gm and pfkB Expression Strains A DNA primer having the nucleotide sequence of SEQ ID NO: 1 and a DNA primer having the nucleotide sequence of SEQ ID NO: 2 were synthesized using an 8905 DNA synthesizer manufactured by Perceptive Biosystems.

Using the above synthetic DNA as a primer,
Plasmid pNT46 containing glk gene (WO98 / 12343)
PCR was performed using DNA as a template. PCR is pNT4
6 ng DNA 1ng, each primer 0.5μmol / L,
1. Pfu DNA polymerase (Stratagene)
5 units, × 10 buffer for Pfu DNA polymerase (Stratagene) 4 μl, deoxyNTP 200 each
Using 40 μl of a reaction solution containing μmol / L, 94 ° C.-1
, 42 ° C. for 2 minutes, and 72 ° C. for 3 minutes were repeated 30 times.

After 1/10 of the reaction mixture was subjected to agarose gel electrophoresis to confirm that the target fragment was amplified, an equivalent amount of TE [10 mmol / L Tris-
HCl (pH 8.0), 1 mmol / L EDTA] saturated phenol / chloroform (1 vol / 1 vol) was added and mixed. After the mixture was centrifuged, 2 volumes of cold ethanol was added to the obtained upper layer, mixed, and left at -80 ° C for 30 minutes. The standing solution was centrifuged to obtain a precipitate of DNA.

The DNA precipitate was dissolved in 20 μl of TE. Using 5 μl of the lysis solution, the DNA was digested with the restriction enzyme Bgl II.
After digestion with SalI and separation of DNA fragments by agarose gel electrophoresis, a 1.3 kb fragment was recovered using a Geneclean II kit. manB and ma
pNK7 which is an nC expression plasmid (WO98 / 12343) DN
After 0.2 μg of A was cut with restriction enzymes Bam HI and Sal I, DNA fragments were separated by agarose gel electrophoresis, and an 8.2 kb fragment was recovered in the same manner.

The fragments of 1.3 kb and 8.2 kb were subjected to ligation reaction at 16 ° C. for 16 hours using a ligation kit. Escherichia coli using the ligation reaction solution
The NM522 strain was transformed according to the above-mentioned known method, and the transformant was spread on an LB agar medium containing 50 μg / ml of ampicillin, and then cultured at 30 ° C. overnight. A plasmid was extracted from the thus grown colonies of the transformant in accordance with the above-mentioned known method to obtain pNK11, which is a glk, manB, and manC expression plasmid. The structure of the plasmid was confirmed by restriction enzyme digestion (FIG. 1).

Using the plasmid pNK11 obtained above, Escherichia coli NM522 / pNT55 strain (WO98 / 12343) was transformed according to a known method, and the transformant was transformed with ampicillin 50 μg / ml and chloramphenicol 10
After coating on LB agar medium containing μg / ml, the cells were cultured at 30 ° C. overnight. By selecting transformants that have grown, glk, manB, manC, pgm and pfk can be selected.
Escherichia coli NM522 /, a strain that simultaneously expresses B
pNK11 / pNT55 was obtained.

Embodiment 2 FIG. Gm from Escherichia coli
Construction of d-expressing strain The Escherichia coli W3110 (ATCC27325) strain was cultured by the method described in Current Protocols in Molecular Biology, and the chromosomal DNA of the microorganism was isolated and purified. 8905 manufactured by Perceptive Biosystems
Using the DNAs having the nucleotide sequences of SEQ ID NOS: 3 and 4 synthesized using a DNA synthesizer as primers, the method described in Example 1 using 0.1 μg of chromosomal DNA of Escherichia coli W3110 (ATCC13027) as a template PCR reaction was performed according to the following.

An agarose gel electrophoresis was performed on 1/10 of the reaction solution, and after confirming that the target fragment was amplified, an equal amount of TE-saturated phenol / chloroform as the remaining reaction solution was added and mixed. . After the mixture was centrifuged, 2 volumes of cold ethanol was added to the resulting upper layer and mixed,
C. for 30 minutes. The standing solution was centrifuged to obtain a precipitate of DNA.

The DNA precipitate was dissolved in 20 μl of TE. Using 5 μl of the lysate, the DNA was digested with the restriction enzyme Hind.
After digestion with III and Xba I, the DNA fragment was separated by agarose gel electrophoresis, and then a 1.1 kb DNA fragment was recovered using a GeneClean II kit. PNT54 (WO98 / 12), a plasmid containing the trp promoter, was used as a primer with DNAs having the nucleotide sequences of SEQ ID NOS: 5 and 6, respectively, synthesized using a Model 8905 DNA synthesizer manufactured by Perceptive Biosystems.
PCR was performed according to the method described in Example 1 using the DNA of 343) as a template.

After 1/10 of the reaction solution was subjected to agarose gel electrophoresis to confirm that the target fragment was amplified, an equal amount of TE-saturated phenol / chloroform to the remaining reaction solution was added and mixed. . After the mixture was centrifuged, 2 volumes of cold ethanol was added to the resulting upper layer and mixed,
C. for 30 minutes. The standing solution was centrifuged to obtain a precipitate of DNA, and the precipitate of DNA was dissolved in 20 μl of TE.

Using 5 μl of the lysate, the DNA was
After digestion with Eco RI and Xba I and separation of DNA fragments by agarose gel electrophoresis,
The DNA fragment of b was recovered. pBluescriptII SK + DNA
Was cleaved with restriction enzymes Eco RI and Hin dIII to 0.2 [mu] g, the DNA fragments were separated by agarose gel electrophoresis to recover a DNA fragment of similarly 3.0 kb.

The 1.1 kb, 0.4 kb and 3.0 k
b, using a ligation kit at 16 ° C., 1
The ligation reaction was performed for 6 hours. Using the ligation reaction solution, Esch
E. coli NM522 was transformed according to the above-mentioned known method, and the transformant was spread on an LB agar medium containing 50 μg / ml of ampicillin, and then cultured at 30 ° C. overnight.

A plasmid was extracted from the thus grown colonies of the transformant in accordance with the above-mentioned known method to obtain an expression plasmid pGE19. The structure of the plasmid was confirmed by restriction enzyme digestion (FIG. 2). Embodiment 3 FIG. Construction of Escherichia coli-derived wcaG-expressing strain Escherichia coli W3110 (using DNAs having the nucleotide sequences of SEQ ID NOs: 7 and 8 respectively synthesized using a Perceptive Biosystems Model 8905 DNA synthesizer) as primers ATCC27325) Chromosome D of strain
A PCR reaction was performed according to the method described in Example 1 using NA as a template.

An agarose gel electrophoresis was performed on 1/10 of the reaction solution, and after confirming that the target fragment was amplified, an equal amount of TE-saturated phenol / chloroform was added to the remaining reaction solution and mixed. . After the mixture was centrifuged, 2 volumes of cold ethanol was added to the resulting upper layer and mixed,
C. for 30 minutes. The standing solution was centrifuged to obtain a precipitate of DNA, and the precipitate of DNA was dissolved in 20 μl of TE. Using 5 μl of the lysate , the DNA was purified with the restriction enzyme Cla I.
After digestion with XhoI and separation of DNA fragments by agarose gel electrophoresis, a 1.0 kb DNA fragment was recovered using a Geneclean II kit.

After digesting 0.2 μg of pPAC31 DNA with restriction enzymes ClaI and SalI , the DNA fragments were separated by agarose gel electrophoresis, and a 5.2 kb DNA fragment was recovered in the same manner. The 1.0 kb and 5.2 kb
Using a ligation kit at 16 ° C. and 16 ° C.
The ligation reaction was performed for a time.

Using the ligation reaction solution, Escherichia coli N
The M522 strain was transformed according to the above-mentioned known method, and the transformant was spread on an LB agar medium containing 50 μg / ml of ampicillin, and then cultured at 30 ° C. overnight. A plasmid was extracted from the thus grown colonies of the transformant in accordance with the above-mentioned known method to obtain an expression plasmid pGE8.
The structure of the plasmid was confirmed by restriction enzyme digestion (FIG. 3).

Embodiment 4 FIG. Production of GKDM Escherichia coli NM522 / pNK11 / pNT obtained in Example 1
55 strains were inoculated into a 1 L baffled Erlenmeyer flask containing 125 ml of LB medium containing 50 μg / ml ampicillin and 10 μg / ml chloramphenicol,
For 17 hours at 220 rpm. The culture solution 1
25 ml of glucose 10 g / l, bactotryptone (Difco) 12 g / l, yeast extract (Difco) 24 g / l, KH ₂ PO ₄ 2.3 g / l, K ₂ HPO ₄
Inoculate a 5 L culture tank containing 2.5 L of a liquid medium (without pH adjustment) having a composition of 12.5 g / L and 50 μg / mL of ampicillin, and at 30 ° C. under the conditions of 600 rpm and a flow rate of 2.5 L / min. After culturing for 4 hours, culturing was further performed at 40 ° C. for 3 hours. During the culture, the pH of the culture was maintained at 7.0 using 28% aqueous ammonia. During the culturing, glucose was added as needed. The culture was centrifuged to obtain wet cells. The wet cells may be -2 if necessary.
It could be stored at 0 ° C. and thawed before use.

Escherichia coli NM5 obtained in Example 2
The 22 / pGE19 strain was inoculated into a 1 L baffled Erlenmeyer flask containing 125 ml of LB medium containing 50 μg / ml of ampicillin, and cultured at 28 ° C. for 17 hours at 220 rpm. 125 ml of the culture solution was glucose 10 g / l, bactotryptone (Difco) 12 g / l, yeast extract (Difco) 24 g / l, KH ₂ PO ₄ 2.3 g / l.
1, K ₂ HPO ₄ 12.5 g / l, ampicillin 50 μg
Liquid medium (without pH adjustment) 2.5L
And inoculated into a 5 L culture tank containing
The culture was performed under the conditions of 0 rpm and aeration of 2.5 L / min.
During the culture, the pH of the culture was maintained at 7.0 using 28% aqueous ammonia. During the culturing, glucose was added as needed. The culture was centrifuged to obtain wet cells. The wet cells could be stored at −20 ° C. if necessary, and thawed before use.

[0068] Corynebacterium ammoniagenes ATCC21170
The strain was glucose 50 g / l, polypeptone (Nippon Pharmaceutical) 10 g / l, yeast extract (Oriental Yeast).
10 g / l, urea 5 g / l, (NH ₄ ) ₂ SO ₄ 5 g /
1, KH ₂ PO ₄ 1 g / l, K ₂ HPO ₄ 3 g / l, Mg
_{SO 4 · 7H 2 O 1g /} l, CaCl 2 · 2H 2 O 0.1
_{g / l, FeSO 4 · 7H} 2 O 10mg / l, ZnSO 4
_{· 7H 2 O 10mg / l,} MnSO 4 · 4~6H 2 O 2
0 mg / l, L-cysteine 20 mg / l, D-calcium pantothenate 10 mg / l, vitamin B1 5 m
g / l, nicotinic acid 5 mg / l, and biotin 30
A 300 ml baffled Erlenmeyer flask containing 25 ml of a liquid medium having a composition of μg / l (adjusted to pH 7.2 with 10 N NaOH) was inoculated at 28 ° C. and 220 rpm.
And cultured for 24 hours.

20 ml of the culture was inoculated into a 2 L baffled Erlenmeyer flask containing 250 ml of a liquid medium having the same composition as above, and cultured at 28 ° C. and 220 rpm for 24 hours. The obtained culture was used as a seed culture. Using 250 ml of the seed culture, 150 g / l of glucose, 5 g / l of meat extract (manufactured by Kyokuto Pharmaceutical Co., Ltd.), 10 g of KH ₂ PO _{4
/ L, K 2 HPO 4 10g} / l, MgSO 4 · 7H 2 O 1
0 g / l, CaCl ₂ .2H ₂ O 0.1 g / l, FeS
_{O 4 · 7H 2 O20mg / l} , ZnSO 4 · 7H 2 O 10
_{mg / l, MnSO 4 · 4~6H} 2 O 20mg / l ( separately sterilized), β- alanine 15mg / l (separately sterilized), L-
Cysteine 20 mg / l, biotin 100 μg / l,
A 5 L culture tank containing 2.25 L of a liquid medium consisting of urea 2 g / l and vitamin B1 5 mg / l (separately sterilized) (adjusted to pH 7.2 with 10 N NaOH) was inoculated at 32 ° C. and 600 rpm. Culturing was performed for 24 hours under the conditions of an aeration rate of 2.5 L / min. During the culture, the pH of the culture solution was maintained at 6.8 using 28% aqueous ammonia.

The culture was centrifuged to obtain wet cells. The wet cells could be stored at −20 ° C. if necessary, and thawed before use. Esch above
erichia coli NM522 / pNK11 / pNT55 strain wet cells 25 g / l,
Escherichiacoli NM522 / pGE19 strain wet cells 15 g / l, Cor
ynebacterium ammoniagenes ATCC21170 strain wet cells 150
g / l, fructose 60 g / l, mannose 30 g /
1, GMP 20 g / l, KH ₂ PO ₄ 25 g / l, Mg
_{SO 4 · 7H 2 O 5g /} l, phytic acid 5 g / l, Nymeen S-215 4g / l and xylene 10 ml /
1 ml of a reaction solution having a composition of 1 was placed in a 200 ml beaker, and the reaction solution was stirred (900 rpm) using a magnetic stirrer and reacted at 32 ° C. for 12 hours. During the reaction, the pH of the reaction solution was maintained at 7.2 using 4N NaOH, and fructose, K
H ₂ PO ₄ was added.

After completion of the reaction, the reaction product was analyzed by HPLC, and 18.6 g / l (29.4 mmol) was contained in the reaction solution.
1 / L) of GKDM (2Na salt) was confirmed to be generated and accumulated. Embodiment 5 FIG. Production of GDP-fucose Escherichia coli NM522 / pNK11 / pNT obtained in Example 1
55 strains were cultured by the method described in Example 4, and wet cells were obtained by centrifugation.

Escherichia coli NM5 obtained in Example 2
The 22 / pGE19 strain was cultured by the method described in Example 4, and wet cells were obtained by centrifugation. Escher obtained in Example 3
ichia coli NM522 / a pGE8 strain, Escherichia coli in Example 4 described except that the culture only added ampicillin instead of adding ampicillin and chloramphenicol NM5
The cells were cultured in the same manner as the 22 / pNK11 / pNT55 strain, and wet cells were obtained by centrifugation.

Corynebacterium ammoniagenes ATCC21170
The strain was cultured by the method described in Example 4, and wet cells were obtained by centrifugation. The wet cells could be stored at −20 ° C. if necessary, and thawed before use. Escherichia coli NM522 / pNK11 / pNT55 strain wet cells 25 g / l, Escherichiacoli NM522 / pGE19 strain wet cells 15 g / l, Corynebacterium ammoniagenes ATCC21170
Strain wet cells 150 g / l, fructose 60 g / l, mannose 30 g / l, GMP 30 g / l, KH ₂ PO ₄ 2
_{5g / l, MgSO 4 · 7H} 2 O5g / l, phytic acid 5
g / l, 4 ml / l of Nimeen S-215 and 10 ml / l of xylene in 30 ml of a reaction solution.
The reaction solution was stirred in a magnetic stirrer (900 rpm) and reacted at 32 ° C. for 12 hours. 12 hours after the reaction, Escherichia coli
NM522 / pGE8 strain wet cells were added at 15 g / l, and the reaction was continued for another 10 hours. During the reaction, 4N Na
The pH of the reaction was maintained at 7.2 using OH and fructose and KH ₂ PO ₄ were added as needed.

After completion of the reaction, the reaction product was analyzed by HPLC, and it was confirmed that 14.0 g / l of GDP-fucose was produced and accumulated in the reaction solution. Escherichia coli
When NM522 / pGE8 strain wet cells (15 g / l) were added at the start of the reaction and the reaction was carried out for 22 hours, the accumulated amount of GDP-fucose (2Na salt) was 3.7 g / l (5.9 mmol).
/ L).

[0075]

According to the present invention, GDP-fucose and GKDM can be efficiently produced.

[0076]

[Sequence free text] SEQ ID NO: 1-Description of artificial sequence:
Synthetic DNA SEQ ID NO: 2-Description of Artificial Sequence: Synthetic DNA SEQ ID NO: 3-Description of Artificial Sequence: Synthetic DNA SEQ ID NO: 4-Description of Artificial Sequence: Synthetic DNA SEQ ID NO: 5-Description of Artificial Sequence: Synthetic DNA SEQ ID NO: 6 Description of Artificial Sequence: Synthetic DNA SEQ ID NO: 7-Description of Artificial Sequence: Synthetic DNA SEQ ID NO: 8-Description of Artificial Sequence: Synthetic DNA

[0077]

[Sequence list] Sequence Listing <110> KYOWA HAKKO KOGYO CO., LTD. <120> PROCESS FOR PRODUCING GDP-FUCOSE <130> H12-1471A4 <140> <141> <150> JP99 / 225889 <151> 1999-08 -10 <160> 8 <170> PatentIn Ver.2.0 <210> 1 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA <400> 1 ccgcaagatc tcgtaaaaag ggtatcgata agc 33 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA <400> 2 gagctgactg ggttgaaggc 20 <210> 3 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA <400> 3 gaatctagaa tgtcaaaagt cgctctc 27 <210> 4 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA <400> 4 ctcaagctta tgactccagc gcgat 25 <210> 5 <211> 24 <212> DNA <213> Artificial Se quence <220> <223> Description of Artificial Sequence: Synthetic DNA <400> 5 caagaattct catgtttgac agct 24 <210> 6 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA <400> 6 cattctagac ctccttaatt cgcgaaaatg gatcgatacc ctttttac 48 <210> 7 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA <400> 7 gtcatcgata tgagtaaacagt <210> 8 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA <400> 8 ataaactcga gagagacaag cggag 25

[Brief description of the drawings]

FIG. 1 shows a construction process of pNK11 which is a glk, manB, and manC expression plasmid.

FIG. 2 shows a step of constructing a gmd expression plasmid, pGE19.

FIG. 3 shows the steps for constructing pGE8, a wcaG expression plasmid.

[Explanation of symbols]

Amp ^r : Ampicillin resistance gene P _L : P _L promoter P _lac : lac promoter P _trp : trp promoter cI857: cI857 repressor glk: Glucokinase gene manB: Phosphomannommutase gene manC: Mannose-1-guanylyl phosphate Transferase gene gmd: GDP-mannose 4,6-dehydratase gene wcaG: GKDM epimerase / reductase gene

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) (C12N 1/21 (C12N 1/21 C12R 1:15) C12R 1:19) (C12N 1/21 (C12P 19/32 Z C12R 1:19) C12R 1:15) (C12P 19/32 (C12P 19/32 Z C12R 1:15) C12R 1:19) (C12P 19/32 C12N 15/00 ZNAA C12R 1:19) (C12R 1:15) (72) Inventor Kazuhiko Tabata 3-6-6 Asahicho, Machida-shi, Tokyo Inside Kyowa Hakko Kogyo Co., Ltd. (72) Inventor Akio Ozaki 1-1-1 Kyowacho, Hofu City, Yamaguchi Prefecture Kyowa Hakko Kogyo Co., Ltd.

Claims (18)

[Claims] 1. A method for converting guanosine-5'-diphosphate-4-keto-6-deoxymannose (hereinafter abbreviated as GKDM) into guanosine-5'-diphosphate fucose (hereinafter referred to as G).
DP-fucose), a culture solution of a microorganism having the ability to convert the microorganism into a protein, or a processed product of the culture solution, used as an enzyme source, and the enzyme source and GKDM are allowed to exist in an aqueous medium. Generate and accumulate fucose,
A method for producing GDP-fucose, comprising collecting GDP-fucose from the aqueous medium. 2. A guanosine-5′-triphosphate (hereinafter referred to as G
Abbreviated as TP), or a culture of a microorganism having the ability to produce GTP from a precursor thereof, or a processed product of the culture, and guanosine-5'-diphosphate-4-keto-6-deoxymannose from sugar and GTP. (Hereinafter, abbreviated as GKDM) using a culture solution of a microorganism having an ability to produce or a processed product of the culture solution as an enzyme source.
After allowing the TP precursor and the sugar to exist in the aqueous medium and producing and accumulating GKDM in the aqueous medium, GKDM is converted to guanosine-5'-diphosphate fucose (hereinafter referred to as GDP-
Using a culture solution of a microorganism having the ability to convert it into fucose, or a processed product of the culture solution as an enzyme source, converting accumulated GKDM into GDP-fucose to produce GDP-fucose in the aqueous medium. A method for producing GDP-fucose, comprising accumulating and collecting GDP-fucose from the aqueous medium. 3. A guanosine-5′-triphosphate (hereinafter referred to as G
Abbreviated as TP), or a culture of a microorganism having the ability to produce GTP from a precursor thereof, or a processed product of the culture, and guanosine-5'-diphosphate-4-keto-6-deoxymannose from sugar and GTP. (Hereinafter, abbreviated as GKDM) using a culture solution of a microorganism having an ability to produce or a processed product of the culture solution as an enzyme source.
A method for producing GKDM, comprising allowing a TP precursor and a sugar to be present in an aqueous medium, producing and accumulating GKDM in the aqueous medium, and collecting GKDM from the aqueous medium. 4. A processed product of the culture solution is a concentrate of the culture solution, a dried product of the culture solution, cells obtained by centrifuging the culture solution, a dried product of the cells, and a freeze-dried product of the cells. , A treated cell with a surfactant, a sonicated cell, a mechanically ground cell, a solvent-treated cell, an enzyme-treated cell, and the cell 4. The production method according to claim 1, wherein the protein fraction is a protein fraction, an immobilized product of the cells, or an enzyme preparation obtained by extraction from the cells. 5. The precursor of GTP is guanine, xanthine, hypoxanthine, guanosine, xanthosine, inosine, guanosine-5′-monophosphate, xanthosine.
The production method according to claim 2 or 3, wherein the production method is 5'-monophosphate or inosine-5'-monophosphate. 6. The method according to claim 2, wherein the saccharide is a saccharide selected from glucose, fructose and mannose. 7. The method according to claim 2, wherein the microorganism capable of producing GTP from a precursor of GTP is a microorganism selected from microorganisms belonging to the genus Corynebacterium. 8. The method according to claim 7, wherein the microorganism belonging to the genus Corynebacterium is Corynebacterium ammoniagenes. 9. The method according to claim 2, wherein the microorganism having the ability to produce GKDM from sugar and GTP is composed of one or more microorganisms. 10. The microorganism according to claim 1, wherein the microorganism is selected from microorganisms belonging to the genus Escherichia and the genus Corynebacterium.
The method according to claim 9, wherein the microorganism is at least one kind of microorganism. 11. The method according to claim 10, wherein the microorganism belonging to the genus Escherichia is Escherichia coli. 12. The method according to claim 10, wherein the microorganism belonging to the genus Corynebacterium is Corynebacterium ammoniagenes. 13. Microorganisms capable of producing GKDM from sugar and GTP include glucokinase (hereinafter abbreviated as glk), phosphomannommutase (hereinafter abbreviated as manB), and mannose-1-phosphate. Anilyl transferase (hereinafter abbreviated as manC), phosphoglucomutase (hereinafter abbreviated as pgm), phosphofructokinase (hereinafter abbreviated as pfk) and GDP
The method according to claim 2 or 3, wherein the microorganism is a microorganism having one or more enzymes selected from mannose 4,6-dehydratase (hereinafter abbreviated as gmd). 14. The microorganism may be one or more selected from a gene encoding glk, a gene encoding manB, a gene encoding manC, a gene encoding pgm, a gene encoding pfk, and a gene encoding gmd. 14. The production method according to claim 13, comprising one or more microorganisms having a recombinant DNA of a DNA fragment containing a gene and a vector. 15. A gene encoding glk, manB
A gene encoding manC, a gene encoding manC, p
The method according to claim 14, wherein the gene encoding gm, the gene encoding pfk, and the gene encoding gmd are genes derived from Escherichia coli. 16. The method according to claim 1, wherein the microorganism capable of converting GKDM into GDP-fucose is a microorganism having a strong GKDM epimerase / reductase (hereinafter abbreviated as wcaG) activity. Law. 17. The method according to claim 16, wherein the microorganism is a microorganism having a recombinant DNA of a DNA fragment containing a gene encoding wcaG and a vector. 18. The method according to claim 17, wherein the gene encoding wcaG is a gene derived from Escherichia coli.