U.S. patent application number 13/049381 was filed with the patent office on 2011-08-25 for process for producing cmp-n-acetylneuraminic acid.
Invention is credited to TOMOKI HAMAMOTO, TOSHITADA NOGUCHI.
Application Number | 20110207179 13/049381 |
Document ID | / |
Family ID | 30767673 |
Filed Date | 2011-08-25 |
United States Patent
Application |
20110207179 |
Kind Code |
A1 |
NOGUCHI; TOSHITADA ; et
al. |
August 25, 2011 |
PROCESS FOR PRODUCING CMP-N-ACETYLNEURAMINIC ACID
Abstract
The present invention is directed to a process for producing
CMP-N-acetylneuraminic acid (CMP-NeuAc), characterized in that the
process includes adding yeast cells,
N-acetylglucosamine-6-phosphate 2-epimerase (GlcNAc-6P
2-epimerase), N-acetylneuraminic acid lyase (NeuAc lyase), and
CMP-N-acetylneuraminic acid synthase (CMP-NeuAc synthase) to a
reaction system containing N-acetylglucosamine (GlcNAc), pyruvate,
and cytidine 5'-monophosphate (CMP), and inducing reaction of the
mixture. The present invention is also directed to a process for
producing CMP-N-acetylneuraminic acid (CMP-NeuAc), characterized in
that the process includes adding yeast cells,
N-acetylglucosamine-6-phosphate 2-epimerase (GlcNAc-6P
2-epimerase), N-acetylneuraminic acid synthase (NeuAc synthase),
and CMP-N-acetylneuraminic acid synthase (CMP-NeuAc synthase) to a
reaction system containing N-acetylglucosamine (GlcNAc) and
cytidine 5'-monophosphate (CMP), and inducing reaction of the
mixture.
Inventors: |
NOGUCHI; TOSHITADA;
(CHOSHI-SHI, JP) ; HAMAMOTO; TOMOKI; (CHOSHI-SHI,
JP) |
Family ID: |
30767673 |
Appl. No.: |
13/049381 |
Filed: |
March 16, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10521576 |
Jun 29, 2005 |
7955825 |
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PCT/JP2003/000258 |
Jan 15, 2003 |
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13049381 |
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Current U.S.
Class: |
435/89 |
Current CPC
Class: |
A61P 31/12 20180101;
C12N 9/1241 20130101; C12N 9/88 20130101; C12P 19/305 20130101;
C12N 9/90 20130101 |
Class at
Publication: |
435/89 |
International
Class: |
C12P 19/30 20060101
C12P019/30 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 18, 2002 |
JP |
2002-208987 |
Claims
1. A process for producing CMP-N-acetylneuraminic acid (CMP-NeuAc),
which comprises adding yeast cells, N-acetylglucosamine-6-phosphate
2-epimerase (GlcNAc-6P 2-epimerase), N-acetylneuraminic acid lyase
(NeuAc lyase), and CMP-N-acetylneuraminic acid synthase (CMP-NeuAc
synthase) to a reaction system containing N-acetylglucosamine
(GlcNAc), pyruvate, and cytidine 5'-monophosphate (CMP), and
inducing reaction of the mixture.
2. The process according to claim 1, wherein the process comprises
adding N-acetylglucosamine-6-phosphate 2-epimerase (GlcNAc-6P
2-epimerase) and N-acetylneuraminic acid lyase (NeuAc lyase) to a
reaction system containing N-acetylglucosamine (GlcNAc) and
pyruvate, to thereby synthesize N-acetylneuraminic acid (NeuAc),
and subsequently adding, to the resultant reaction system, cytidine
5'-monophosphate (CMP), yeast cells, and cytidine 5'-monophosphate
N-acetylneuraminic acid synthase (CMP-NeuAc synthase), to thereby
synthesize CMP-N-acetylneuraminic acid (CMP-NeuAc).
3. The process according to claim 1, wherein cells (including
transformants) or processed products thereof are employed as the
GlcNAc-6P 2-epimerase, NeuAc lyase, or CMP-NeuAc synthase.
4. The process according to claim 1, which employs a transformant
of GlcNAc-6P 2-epimerase and a transformant of NeuAc lyase, said
respective transformants having enhanced activity, and a processed
product of cells as the CMP-NeuAc synthase.
5. A process for producing CMP-N-acetylneuraminic acid (CMP-NeuAc),
which comprises adding yeast cells, N-acetylglucosamine-6-phosphate
2-epimerase (GlcNAc-6P 2-epimerase), N-acetylneuraminic acid
synthase (NeuAc synthase), and CMP-N-acetylneuraminic acid synthase
(CMP-NeuAc synthase) to a reaction system containing
N-acetylglucosamine (GlcNAc) and cytidine 5'-monophosphate (CMP),
and inducing reaction of the mixture.
6. The process for producing CMP-N-acetylneuraminic acid
(CMP-NeuAc) according to claim 1, wherein cells (including
transformants) or processed products thereof are employed as the
GlcNAc-6P 2-epimerase, NeuAc synthase, or CMP-NeuAc synthase.
7. The process according to claim 1, which employs a transformant
of GlcNAc-6P 2-epimerase and a transformant of NeuAc synthase, said
respective transformants having enhanced activity, and a processed
product of cells having CMP-NeuAc synthase activity as the
CMP-NeuAc synthase.
Description
TECHNICAL FIELD
[0001] The present invention relates to an improved process for
producing CMP-N-acetylneuraminic acid (CMP-NeuAc), which is an
important material for synthesizing sugar chains.
BACKGROUND ART
[0002] In recent years, with the rapid progress of research
concerning the structures and functions of sugar chains, research
efforts have been undertaken to develop applications of
oligosaccharides, glycolipids, glycoproteins, and similar materials
having physiological activities in the fields of drugs and
functional materials. Among sugar chains, a sialic-acid-containing
sugar chain having N-acetylneuraminic acid (NeuAc) at an end
thereof plays an important role as a receptor in, for example, cell
adhesion or viral infection.
[0003] Generally, the sialic-acid-containing sugar chain is
synthesized by use of sialyltransferase as a catalyst.
Sialyltransferase is an enzyme which catalyzes the transfer of
sialic acid from CMP-N-acetylneuraminic acid (CMP-NeuAc), which
serves as a sugar donor, to an acceptor such as a sugar chain.
[0004] However, CMP-NeuAc employed as a sugar donor is very
expensive and therefore has been provided only in small amounts on
reagent levels.
[0005] In a known method for producing CMP-NeuAc, CMP-NeuAc is
synthesized from cytidine 5'-triphosphate (CTP) and NeuAc serving
as substrates by use of CMP-NeuAc synthase as a catalyst (Appl.
Microbiol. Biotechnol., 44, 59-67 (1995)). Since CTP and NeuAc are
expensive substances, direct use of these substances as starting
materials inevitably increases the cost for producing
CMP-NeuAc.
[0006] Recently, Koizumi et al. have developed a process for
producing CMP-NeuAc from orotic acid and NeuAc as starting
materials by using, in combination, Brevibacterium ammoniagenes
cells which transform orotic acid to uridine 5'-triphosphate (UTP),
a recombinant E. coli which produces a CTP synthase that catalyzes
transformation of UTP to CTP, and a recombinant E. coli which
produces a CMP-NeuAc synthase (Appl. Microbiol. Biotechnol., 53,
257-261, (2000)). This process does not employ expensive CTP.
However, cumbersome steps and large-scale facilities must be
provided for preparing cells of a plurality of species, and NeuAc,
which is an expensive reagent, is still employed, discouraging
employment of the process in practice.
[0007] Meanwhile, regarding the method for producing NeuAc, there
has been known a process where colominic acid--a polymer of sialic
acid--is recovered from a microorganism, and NeuAc is obtained
through chemical decomposition of colominic acid. Recently, some
processes employing an enzyme have also been developed.
[0008] Examples of such enzymatic processes include
[0009] (1) a process for producing NeuAc from N-acetylmannosamine
(ManNAc) by use of NeuAc lyase or NeuAc synthase (J. Am. Chem.
Soc., 110, 6481 (1988), J. Am. Chem. Soc., 110, 7159 (1988), and
Japanese Patent Application Laid-Open (kokai) No. 10-4961);
[0010] (2) a process for producing NeuAc through transformation of
N-acetylglucosamine (GlcNAc) to N-acetylmannosamine (ManNAc) under
alkaline conditions and subsequent treatment of ManNAc with NeuAc
lyase or NeuAc synthase (Japanese Patent Application Laid-Open
(kokai) No. 5-211884, Biotechnology And Bioengineering, Vol. 66,
No. 2 (1999), and Enzyme Microb. Technol., Vol. 20 (1997)); and
[0011] (3) a process for producing NeuAc from GlcNAc by use of
N-acetylglucosamine (GlcNAc) 2-epimerase which catalyzes
transformation of GlcNAc to ManNAc, and NeuAc lyase or NeuAc
synthase (WO95/26399, Japanese Patent Application Laid-Open (kokai)
Nos. 3-180190, and 2001-136982).
[0012] However, these processes have drawbacks. The process (1)
employs ManNAc, which is an expensive starting material. Process
(2) includes a cumbersome step for isolating ManNAc from a mixture
of GlcNAc and ManNAc, although the process employs inexpensive
GlcNAc as a starting material. The problem with process (3) resides
in that, as shown in the following scheme, it employs GlcNAc
2-epimerase, which functions only in the presence of ATP. Thus,
expensive ATP must be used, or ATP must be produced from adenine--a
precursor of ATP--by use of a microorganism, making the process
unsatisfactory.
<Process (3)>
##STR00001##
[0013] DISCLOSURE OF THE INVENTION
[0014] The present inventors have studied the synthesis of NeuAc
employing GlcNAc as a substrate in the presence of an intracellular
enzyme found in E. coli, and have found that GlcNAc is transformed
to GlcNAc 6-phosphate (GlcNAc-6P) although virtually no NeuAc is
synthesized. Thus, the inventors have attempted to establish a
NeuAc synthesis system via the following pathway starting with
GlcNAc.
[0015] As a result, the present inventors have found that NeuAc can
be produced at high yield through enhancement of GlcNAc-6P
2-epimerase (EC 5.1.3.9) activity and NeuAc lyase activity or NeuAc
synthase activity, and that the synthesis system does not require
ATP, which is an expensive reagent.
##STR00002##
[0016] The inventors have further conducted research on a CTP
synthesis system including reactions for synthesizing CMP-NeuAc, in
an attempt to combine, with the aforementioned NeuAc synthesis
system, a microorganism-based transformation system which forms CTP
by use of inexpensive CMP as a starting material, and a variety of
microorganisms have been tested. The inventors have found that,
when a microorganism (e.g., E. coli) other than yeast is employed
CMP-NeuAc is synthesized only in a small amount, whereas when yeast
cells are employed, CMP-NeuAc can be synthesized at high yield. The
inventors have also found that phosphoenolpyruvate (PEP) required
for NeuAc synthase reaction can be advantageously provided by yeast
cells, eliminating the need for further addition of PEP to the
reaction system. The present invention has been accomplished on the
basis of these findings.
[0017] Accordingly, the present invention is directed to a process
for producing CMP-N-acetylneuraminic acid (CMP-NeuAc), which
comprises adding yeast cells, N-acetylglucosamine-6-phosphate
2-epimerase (GlcNAc-6P 2-epimerase), N-acetylneuraminic acid lyase
(NeuAc lyase), and CMP-N-acetylneuraminic acid synthase (CMP-NeuAc
synthase) to a reaction system containing N-acetylglucosamine
(GlcNAc), pyruvate, and cytidine 5'-monophosphate (CMP) and
inducing reaction of the mixture.
[0018] The present invention is also directed to a process for
producing CMP-N-acetylneuraminic acid (CMP-NeuAc), which comprises
adding yeast cells, N-acetylglucosamine-6-phosphate 2-epimerase
(GlcNAc-6P 2-epimerase), N-acetylneuraminic acid synthase (NeuAc
synthase), and CMP-N-acetylneuraminic acid synthase (CMP-NeuAc
synthase) to a reaction system containing N-acetylglucosamine
(GlcNAc) and cytidine 5'-monophosphate (CMP) and inducing reaction
of the mixture.
BRIEF DESCRIPTION OF FIGURE
[0019] The CMP-NeuAc synthesis routes of the present invention,
including a process (A) employing NeuAc lyase and a process (B)
employing NeuAc synthase, will next be described with reference to
the schemes shown in FIG. 1. Notably, phosphoenolpyruvate (PEP),
which is essential to the reaction system (B), is not required to
be added to the system, because PEP is synthesized from glucose
contained in culture medium through (metabolic) bioreaction of
yeast and E. coli and fed to the system.
[0020] In the schemes (A) and (B) of FIG. 1, the reference numerals
denote the following: [0021] (1): GlcNAc-6P 2-epimerase [0022] (2):
NeuAc lyase [0023] (3): CMP-NeuAc synthase [0024] (4): NeuAc
synthase
BEST MODES FOR CARRYING OUT THE INVENTION
(1) Preparation of Enzymes and Other Materials
[0025] N-acetylglucosamine-6-phosphate 2-epimerase (GlcNAc-6P
2-epimerase) ((1) above) which is added to the above reaction
system (A) or (B) refers to an enzyme exhibiting catalytic activity
on transformation of GlcNAc 6-phosphate to ManNAc 6-phosphate.
N-acetylneuraminic acid lyase (NeuAc lyase) ((2) above) which is
added to the above reaction system (A) refers to an enzyme
exhibiting catalytic activity on reaction of ManNAc and pyruvate
serving as substrates, to thereby synthesize NeuAc.
N-acetylneuraminic acid synthase (NeuAc synthase) ((4) above) which
is added to the above reaction system (B) refers to an enzyme
exhibiting catalytic activity on reaction of MaNAc and
phosphoenolpyruvate (PEP) serving as substrates, to thereby
synthesize NeuAc. CMP-N-acetylneuraminic acid synthase (CMP-NeuAc
synthase) ((3) above) which is added to the above reaction system
(A) or (B) refers to an enzyme exhibiting a catalytic activity on
reaction of NeuAc and CTP serving as substrates, to thereby
synthesize CMP-NeuAc.
[0026] Examples of enzymes exhibiting such an enzymatic activity
include cells (including transformants) and processed products
thereof. Among them, enzymes derived from a microorganism are
preferably employed, from the viewpoint of ease of preparation and
other factors. GlcNAc-6P 2-epimerase, N-acetylneuraminic acid
lyase, N-acetylneuraminic acid synthase, and CMP-NeuAc synthase
which are derived from a microorganism are known enzymes, and can
be prepared through a routine method.
[0027] In order to enhance the aforementioned enzyme activity, a
so-called recombinant DNA technique is preferably employed. In a
specific procedure, genes encoding enzymes are cloned (J.
Bacteriol., 181, 47-54, 1999; J. Bacteriol., 181, 4526-4532, 1999;
Nucleic Acids. Res., 13, 8843-8852, 1985; Agric. Biol. Chem., 50,
2155-2158, 1986; FEMS Microbiol. Lett., 75, 161-166, 1992; J. Biol.
Chem., 271, 15373-15380, 1996; J. Biol. Chem., 264, 14769-14774,
1989; J. Bacteriol., 177, 312-319, 1995; and Mol. Microbiol., 35,
1120-1134, 2000), and the cloned genes are expressed in a large
amount of cells of a microorganism.
[0028] In the present invention, cells produced through
co-expression of two or more genes or a processed product thereof
may also be employed. Although no particular limitation is imposed
on the above cells, preferred examples include transformants which
exhibit enhanced enzyme activity of both GlcNAc-6P 2-epimerase and
N-acetylneuraminic acid lyase, and transformants which exhibit
enhanced enzyme activity of both GlcNAc-6P 2-epimerase and
N-acetylneuraminic acid synthase.
[0029] Cloning of genes, preparation of expression vectors by use
of a cloned DNA fragment, preparation of enzyme proteins exhibiting
an enzyme activity of interest by use of the expression vectors,
etc. are techniques known to those skilled in the field of
molecular biology, and may be performed through a method described
in, for example, "Molecular Cloning" (compiled by Maniatis et al.,
Cold Spring Harbor Laboratories, Cold Spring Harbor, N.Y.
(1982)).
[0030] In a specific procedure, probes are synthesized on the basis
of a reported nucleotide sequence, and DNA fragments containing a
gene encoding an enzyme protein exhibiting an enzyme activity of
interest are cloned from chromosomal DNAs of a microorganism.
Although no particular limitation is imposed on a host to be
employed for cloning, E. coli is preferably employed, from the
viewpoint of ease of handling and availability.
[0031] In order to establish a high expression system of a cloned
gene, the following procedure may be employed. For example, the
nucleotide sequence of a cloned DNA fragment is analyzed through
the Maxam-Gilbert method (Methods in Enzymology, 65, 499 (1980)),
the dideoxy chain termination method (Methods in Enzymology, 101,
20 (1983)), or a similar method, whereby a coding domain of the
gene is specified. In order to enable the gene to be expressed in
cells of a corresponding host microorganism, a recombinant
expression vector containing an expression-regulating signal
(transcription initiating signal and translation initiating signal)
ligated to the upstream side of the gene is prepared.
[0032] A variety of plasmid vectors and phage vectors may be
employed. Among them, a plasmid vector which can be replicated in
E. coli cells, has an appropriate drug resistant marker and a
specific restriction enzyme cleavage site, and permits a large
multiplication number within the cells is preferably employed.
Specific examples of the plasmid vector include pBR322 (Gene, 2, 95
(1975)), pUC18, and pUC19 (Gene, 33, 103 (1985)).
[0033] By use of the thus-prepared recombinant vector, E. coli is
transformed. Examples of the E. coli employed as a host include K12
strain, C600, JM105, and JM109 (Gene, 33, 103-119 (1985)), which
are employed in recombinant DNA experiments. Alternatively, there
may also be employed, as a host, E. coli to which lip gene mutation
relating to metabolism of pyruvate has been introduced (e.g.,
W1485lip2 (ATCC25645)) so as to reduce metabolism of pyruvate other
than that which occurs in relation to NeuAc synthesis.
[0034] A variety of methods for transforming E. coli have already
been reported, and, for example, a plasmid is incorporated into
cells through treatment of the cells with calcium chloride at low
temperature (J. Mol. Biol., 53, 159 (1970)).
[0035] The thus-prepared transformants are cultured in a medium
where the corresponding microorganism can grow, and expression of
the cloned gene of enzyme protein exhibiting enzyme activity of
interest is induced. Culturing is performed until the enzyme
protein is accumulated in a large amount within the cells. The
transformants may be cultured through a routine method in a medium
containing nutrients (e.g., a carbon source and a nitrogen source)
required for the growth of the microorganism. In an exemplary
procedure, the transformants are cultured in a medium which is
generally employed for culturing E. coli (e.g., a bouillon medium,
an LB medium (1% trypton, 0.5% yeast extract, and 1% saline), or a
2.times.YT medium (1.6% trypton, 1% yeast extract, and 0.5%
saline)) at 30 to 50.degree. C. for about 10 to 50 hours with, if
necessary, aeration and stirring. When a plasmid is employed as a
vector, an appropriate antibiotic (in accordance with a drug
resistant marker of the plasmid; e.g., ampicillin or kanamycin) is
added to the culture in an appropriate amount in order to prevent
loss of the plasmid during culturing.
[0036] Examples of a mass of cells exhibiting enzyme activity of
interest include those collected, from the culture liquid obtained
through the above method, through a solid-liquid separation means
such as centrifugal separation or membrane separation.
Alternatively, there may also be employed, as a cell processed
product, a product obtained from processing the thus-collected cell
product through a generally employed treatment method such as
mechanical breaking (by use of a Waring blender, a French press, a
homogenizer, a mortar, etc.), freezing and thawing, autolysis,
drying (lyophilization, drying in air, etc.), an enzyme treatment
(with lysozyme), ultrasonication, a chemical treatment (with acid,
alkali, etc.); or crude or purified enzymes obtained by separating
a fraction exhibiting enzyme activity of interest from the cell
processed product and subjecting the fraction to a routine enzyme
purification means (e.g., salting out, isoelectric precipitation,
organic solvent precipitation, dialysis, or chromatographic
treatments).
[0037] Examples of the yeast employed for transforming CMP to CTP
include commercially available bakers' yeasts and wine yeasts.
These commercial yeasts are very advantageous, in that a step of
producing yeast cells can be omitted. Although either fresh yeast
cells or dried yeast cells may be employed, dried yeast cells are
preferably employed, from the viewpoint of yield and ease of
handling.
(2) Synthesis of CMP-NeuAc
[0038] Commercially available products of GlcNAc, pyruvate, and CMP
may be employed in CMP-NeuAc synthesis reaction. The concentration
of each reagent may be appropriately selected from a range of 1 to
5,000 mM, preferably 10 to 1,000 mM.
(Process Employing NeuAc Lyase)
[0039] The CMP-NeuAc synthesis reaction may be carried out by
adding GlcNAc-6P 2-epimerase, NeuAc lyase, and CMP-NeuAc synthase,
each in an amount of 0.2 mg or more based on 1 mL of reaction
solution, preferably 2 to 100 mg, and dry yeast in an amount of 1
to 20% (w/v) to a reaction system containing GlcNAc, CMP, and
pyruvate, followed by allowing the mixture to react at 50.degree.
C. or lower, preferably 15 to 40.degree. C., for about 1 to 150
hours with, if necessary, stirring.
[0040] Alternatively, the above reaction may be performed in two
steps so as to improve synthesis yield of CMP-NeuAc. Firstly,
GlcNAc-6P 2-epimerase and NeuAc lyase are added to a reaction
system containing GlcNAc and pyruvate, and the mixture is allowed
to react at 50.degree. C. or lower (preferably 15 to 40.degree. C.)
for about 1 to 50 hours, thereby synthesizing NeuAc. Subsequently,
CMP, yeast cells, and CMP-NeuAc synthase are added to the reaction
mixture, and the mixture is allowed to react for about 5 to 50
hours, thereby synthesizing CMP-NeuAc. Here, in the NeuAc
synthesis, CMP may be added in advance to a reaction system.
(Process Employing NeuAc Synthase)
[0041] The CMP-NeuAc synthesis reaction may be carried out by
adding GlcNAc-6P 2-epimerase, NeuAc synthase, and CMP-NeuAc
synthase, each in an amount of 0.2 mg or more based on 1 mL of
reaction solution, preferably 2 to 100 mg, and dry yeast in an
amount of 1 to 20% (w/v) to a reaction system containing GlcNAc and
CMP, followed by allowing the mixture to react at 50.degree. C. or
lower (preferably 15 to 40.degree. C.) for about 1 to 150 hours
with, if necessary, stirring.
[0042] To the aforementioned CMP-NeuAc synthesis systems, an
inorganic phosphoric acid, magnesium, and an energy source are
preferably added in accordance with needs.
[0043] An inorganic phosphoric acid such as potassium phosphate may
be used without any modification. However, an inorganic phosphoric
acid in the form of a phosphate buffer is preferably used. The
concentration of inorganic phosphoric acid may be appropriately
selected from a range of 1 to 1,000 mM, preferably 10 to 400 mM.
When a phosphate buffer is used, the pH thereof may be
appropriately selected from a range of 5 to 10.
[0044] Examples of usable magnesium species include inorganic acid
magnesium salts such as magnesium sulfate, magnesium nitrate, and
magnesium chloride; and organic acid magnesium salts such as
magnesium citrate. The concentration of magnesium species may be
appropriately selected from a range of 1 to 1,000 mM.
[0045] Examples of usable energy sources include sugars such as
glucose, fructose, and sucrose; and organic acids such as acetic
acid and citric acid. The concentration of energy source may be
appropriately selected from a range of 1 to 5,000 mM, preferably 10
to 1,000 mM.
[0046] The thus-produced CMP-NeuAc may be isolated and purified
through a conventional sugar nucleotide isolation/purification
means (e.g., ion exchange chromatography, adsorption
chromatography, salting out, or affinity chromatography).
EXAMPLES
[0047] Hereinafter, the present invention will be described in more
detail by way of Examples, which should not be construed as
limiting the invention thereto. In the Examples, preparation of DNA
samples, cleavage with restriction enzymes, DNA ligation by use of
a T4 DNA ligase, and transformation of E. coli were all performed
as described in "Molecular Cloning, A Laboratory Manual, Second
Edition" (complied by Sambrook, et al., Cold Spring Harbor
Laboratory, Cold Spring Harbor, N.Y. (1989)). Also, restriction
enzymes, AmpliTaq DNA polymerase, and T4 DNA ligase were purchased
from Takara Bio Inc.
[0048] Quantitation of CMP-NeuAc in a reaction mixture was carried
out by means of HPLC. Specifically, an ODS-HS302 column (product of
YMC) was used for separation, and 1 mM tetrabutylammonium sulfate
and 50 mM magnesium acetate solution were used to prepare an
eluant. Quantitation of sugar such as NeuAc was carried out by
means of HPLC making use of HPAE-PAD. Specifically, a CarboPac PA1
column ED40 (product of Dionex) was used for separation and
detection purposes, and solution A (0.1N NaOH) and solution B (0.1N
NaOH, 0.5M sodium acetate), with a gradient therebetween, were used
to prepare an eluant.
Example 1
(1) Cloning of nanA Gene Encoding N-acetylneuraminic acid lyase
[0049] Chromosomal DNA (ATCC 51907D) of Haemophilus influenzae (H.
influenzae) Rd strain was used as a template, and the two
below-described primer DNA sequences were synthesized according to
a method known per se. The N-acetylneuraminic acid lyase (nanA)
gene of H. influenzae was amplified through PCR.
TABLE-US-00001 Primer (A): (SEQ ID NO: 1)
5'-CACCATGGCGAAGATATTGCCGCTCAAACTA-3' Primer (B): (SEQ ID NO: 2)
5'-CCGAATTCATTTATGACAAAAATTTCGCTTTCAAG-3'
[0050] Amplification of the nanA gene through PCR was performed in
a DNA Thermal Cycler (product of Perkin-Elmer Cetus Instrument) by
adding thereto a 100 .mu.L reaction mixture containing 50 mM
potassium chloride, 10 mM Tris HCl (pH 8.3), 1.5 mM magnesium
chloride, 0.001% gelatin, 0.1 .mu.g template DNA, DNA primers (A)
and (B) (each 0.2 .mu.M), and AmpliTaq DNA polymerase (2.5 units).
The cycling protocol consisted of 25 cycles of the following three
steps: strand denaturation at 94.degree. C. for 1 minute, annealing
at 55.degree. C. for 1.5 minutes, and polymerization at 72.degree.
C. for 3 minutes.
[0051] Subsequent to gene amplification, the reaction mixture was
treated with a phenol/chloroform (1:1) mixture. To the
water-soluble fraction, ethanol was added in a volume twice that of
the fraction, to thereby precipitate DNA. The DNA collected through
precipitation was subjected to agarose gel electrophoresis as
described in literature (Molecular Cloning, see above), to thereby
purify DNA fragments having a size of 1.2 kb. The DNA was cleaved
with restriction enzymes NcoI and EcoRI, followed by ligation, by
use of T4 DNA ligase, with plasmid pTrc99A (Pharmacia Biotech.)
which had likewise been digested with restriction enzymes NcoI and
EcoRI. By use of the ligation reaction mixture, E. coli strain
JM109 (ATCC53323) was transformed, and from the resultant
ampicillin-resistant transformants, plasmid pTrcnanA was isolated.
pTrcnanA has a structure in which a DNA fragment containing a
structural gene of nanA gene of H. influenzae has been inserted to
the NcoI-EcoRI cleavage sites located downstream of the trc
promoter of pTrc99A.
(2) Cloning of nanE Gene Encoding GlcNAc-6P 2-epimerase
[0052] Chromosomal DNA of H. influenzae Rd strain was used as a
template, and the two below-described primer DNA sequences were
synthesized according to a method known per se. The GlcNAc-6P
2-epimerase (nanE) gene of H. influenzae was amplified through
PCR.
TABLE-US-00002 Primer (C): (SEQ ID NO: 3)
5'-GGTCTAGATTTAAATGAGGGGTGTTATATGT-3' Primer (D): (SEQ ID NO: 4)
5'-TCGTCGACTTATCTTGCAGATTTCACTGAATTAGCAAACCA-3'
[0053] Amplification of the nanE gene through PCR was performed in
a DNA Thermal Cycler (product of Perkin-Elmer Cetus Instrument) by
adding thereto a 100 .mu.L reaction mixture containing 50 mM
potassium chloride, 10 mM Tris HCl (pH 8.3), 1.5 mM magnesium
chloride, 0.001% gelatin, 0.1 .mu.g template DNA, DNA primers (C)
and (D) (each 0.2 .mu.M), and AmpliTaq DNA polymerase (2.5 units).
The cycling protocol consisted of 25 cycles of the following three
steps: strand denaturation at 94.degree. C. for 1 minute, annealing
at 55.degree. C. for 1.5 minutes, and polymerization at 72.degree.
C. for 3 minutes.
[0054] Subsequent to gene amplification, the reaction mixture was
treated with a phenol/chloroform (1:1) mixture. To the
water-soluble fraction, ethanol was added in a volume twice that of
the fraction, to thereby precipitate DNA. The DNA collected through
precipitation was subjected to agarose gel electrophoresis as
described in literature (Molecular Cloning, see above), to thereby
purify DNA fragments having a size of 720 b. The DNA was cleaved
with restriction enzymes XbaI and SalI, followed by ligation, by
use of T4 DNA ligase, with plasmid pTrc99A which had likewise been
digested with restriction enzymes XbaI and SalI. By use of the
ligation reaction mixture, E. coli strain JM109 was transformed,
and from the resultant ampicillin-resistant transformants, plasmid
pTrc-nanE was isolated. pTrc-nanE has a structure in which a DNA
fragment containing a structural gene of nanE gene of H. influenzae
has been inserted to the XbaI-SalI cleavage sites located
downstream of the trc promoter of pTrc99A.
(3) Construction of a Plasmid for Coexpression of nanA and nanE
Genes
[0055] The pTrcnanA plasmid obtained in above (1) was cleaved with
restriction enzymes NcoI and EcoRI, and NcoI-EcoRI fragments
containing nanA gene were recovered through agarose gel
electrophoresis. The recovered fragments were ligated to the
pTrc-nanE plasmid obtained in (2) above through digestion with NcoI
and EcoRI, using a T4 DNA ligase. By use of the ligation reaction
mixture, E. coli strain JM109 was transformed, and from the
resultant ampicillin-resistant transformants, plasmid pTrcAE was
isolated. pTrcAE has a structure in which a DNA fragment containing
structural genes of nanE and nanA of H. influenzae has been
inserted to the NcoI-SalI cleavage sites located downstream of the
trc promoter of pTrc99A.
(4) Synthesis of NeuAc
[0056] E. coli W1485lip2 (ATCC25645) was engineered so as to harbor
the plasmid pTrcAE constructed in (3) above, and inoculated in a
2.times.YT medium (500 mL) supplemented with 100 .mu.g/mL
ampicillin. Shaking culture was performed at 37.degree. C. When the
cell count had reached 1.times.10.sup.8 cells/mL, isopropyl
.beta.-D-thiogalactoside (IPTG) was added to the culture system so
as to attain a final concentration of 0.2 mM. Shaking culture was
continued at 37.degree. C. for 26 hours. After completion of
culturing, the culture was subjected to centrifugal separation
(9,000.times.g, 10 minutes), whereby a 25-mL culture broth (which
equals to 50 mg cells) was recovered. To the recovered culture
cells was added a potassium phosphate buffer (200 mM, pH 8.0, 5 mL)
containing 100 mM GlcNAc, 20 mM magnesium chloride, 50 mM glucose,
300 mM sodium pyruvate, and 0.5% (v/v) xylene, and the mixture was
allowed to react at 28.degree. C. under stirring. At points in time
14 and 24 hours after the start of reaction, sodium pyruvate (110
mg) was added, and at 48 hours, the reaction mixture was
heat-treated at 100.degree. C. for 5 minutes, whereby reaction was
stopped. Analysis of the resultant reaction mixture by means of
HPLC (HPAE-PAD, Dionex) designed for sugar analysis confirmed
production of 43.7 mM NeuAc.
[0057] A control microorganism (E. coli W1485lip2 harboring plasmid
pTrc99A) was subjected to similar reactions. However, production of
NeuAc was not detected (which means production was 0.5 mM or
less).
(5) Cloning of neuA Gene Encoding CMP-NeuAc Synthase
[0058] Chromosomal DNA of H. influenzae Rd strain was used as a
template, and the two below-described primer DNA sequences were
synthesized according to a method known per se. The CMP-NeuAc
synthase (neuA) gene of H. influenzae was amplified through
PCR.
TABLE-US-00003 Primer (E): (SEQ ID NO: 5)
5'-TGCCATGGTGAAAATAATAATGACAAGAA-3' Primer (F): (SEQ ID NO: 6)
5'-AACTGCAGTGCAGATCAAAAGTGCGGCC-3'
[0059] Amplification of the neuA gene through PCR was performed in
a DNA Thermal Cycler (product of Perkin-Elmer Cetus Instrument) by
adding thereto a 100 .mu.L reaction mixture containing 50 mM
potassium chloride, 10 mM Tris HCl (pH 8.3), 1.5 mM magnesium
chloride, 0.001% gelatin, 0.1 .mu.g template DNA, DNA primers (E)
and (F) (each 0.2 .mu.M), and AmpliTaq DNA polymerase (2.5 units).
The cycling protocol consisted of 25 cycles of the following three
steps: strand denaturation at 94.degree. C. for 1 minute, annealing
at 55.degree. C. for 1.5 minutes, and polymerization at 72.degree.
C. for 3 minutes.
[0060] Subsequent to gene amplification, the reaction mixture was
treated with a phenol/chloroform (1:1) mixture. To the
water-soluble fraction, ethanol was added in a volume twice that of
the fraction, to thereby precipitate DNA. The DNA collected through
precipitation was subjected to agarose gel electrophoresis as
described in literature (Molecular Cloning, see above), to thereby
purify DNA fragments having a size of 720 b. The DNA was cleaved
with restriction enzymes NcoI and PstI, followed by ligation, by
use of T4 DNA ligase, with plasmid pTrc99A which had likewise been
digested with restriction enzymes NcoII and PstI. By use of the
ligation reaction mixture, E. coli strain JM109 was transformed,
and from the resultant ampicillin-resistant transformants, plasmid
pTrcsiaBNP was isolated. pTrcsiaBNP has a structure in which a DNA
fragment containing a structural gene of neuA gene of H. influenzae
has been inserted to the NcoI-PstI cleavage sites located
downstream of the trc promoter of pTrc99A.
(6) Preparation of CMP-NeuAc Synthase
[0061] E. coli JM109 harboring the plasmid pTrcsiaBNP was
inoculated in a 2.times.YT medium (100 mL) supplemented with 100
.mu.g/mL ampicillin. Shaking culture was performed at 37.degree. C.
When the cell count had reached 4.times.10.sup.8 cells/mL, IPTG was
added to the culture system so as to attain a final concentration
of 0.25 mM. Shaking culture was continued at 37.degree. C. for 6
hours. After completion of culturing, the culture was subjected to
centrifugal separation (9,000.times.g, 10 minutes), whereby the
cells were recovered. The cells were suspended in a buffer (5 mL)
(100 mM Tris-HCl (pH 7.8), 10 mM MgCl.sub.2). The cells were
ultrasonically disrupted, and the resultant cell residues were
removed through centrifugation (20,000.times.g, 10 minutes).
[0062] The thus-obtained supernatant fraction was employed as an
enzyme solution, and CMP-NeuAc synthase activity as measured with
this enzyme solution is shown in Table 1 together with the data
from a control microorganism (E. coli K-12 JM109 harboring
pTrc99A). In the present invention, CMP-NeuAc synthase activity
units were determined by measuring and calculating activity in
relation to the synthesis of CMP-NeuAc from 5'-CMP and
N-acetylneuraminic acid through the below-described method.
(Measurement of CMP-NeuAc Synthase Activity and Calculation of
Units)
[0063] The CMP-NeuAc synthase was added to 50 mM Tris-HCl buffer
(pH 8.0) containing 20 mM magnesium chloride, 5 mM CTP, and 10 mM
N-acetylneuraminic acid, to thereby initiate reaction for five
minutes at 37.degree. C. As a control, a cell lysate of E. coli
JM109 harboring pTrc99A was employed in stead of CMP-NeuAc synthase
and similar reaction was performed.
[0064] To the reaction mixture, 70% ethanol (twice the volume of
the mixture) was added to thereby stop the reaction, and the
mixture was diluted and then analyzed through HPLC. The separation
process was performed through use of an HS-302 column (product of
YMC) and, as an eluent, a mixture of 50 mM magnesium acetate and an
aqueous 1 mM tetrabutylammonium solution. From the results of the
HPLC analysis, amount of CMP-NeuAc contained in the reaction
mixture was calculated. The activity of the synthase capable of
synthesizing 1 .mu.mole CMP-NeuAc in one minute at 37.degree. C.
was regarded as one unit, and the CMP-NeuAc synthase activity was
calculated.
TABLE-US-00004 TABLE 1 CMP-NeuAc synthase Activity
Microorganism/Plasmid (units/mg protein) JM109/pTrc99A <0.01
JM109/pTrcsiaBNP 2.45
(7) Synthesis of CMP-NeuAc
[0065] E. coli K-12 ME8417 (FERM BP-6847: Aug. 18, 1999, National
Institute of Advanced Industrial Science and Technology, Patent
Microorganisms Depositary (Chuo 6, 1-1-1 Higashi, Tsukuba-shi,
Ibaraki-ken, Japan (postal code: 305-8566)) was engineered so as to
harbor the plasmid pTrcAE constructed in (3) above, and inoculated
in a 2.times.YT medium (500 mL) supplemented with 100 .mu.g/mL
ampicillin. Shaking culture was performed at 37.degree. C. When the
cell count had reached 4.times.10.sup.8 cells/mL, IPTG was added to
the culture system so as to attain a final concentration of 0.2 mM.
Shaking culture was continued at 37.degree. C. for 8.5 hours. After
completion of culturing, the culture was subjected to centrifugal
separation (9,000.times.g, 10 minutes), whereby a 25-mL culture
broth (which equals to 50 mg of cells) were recovered. To the
recovered culture cells was added a potassium phosphate buffer (200
mM, pH 8.0, 5 mL) containing 50 mM CMP, 100 mM GlcNAc, 20 mM
magnesium chloride, 50 mM glucose, and 250 mM sodium pyruvate, and
0.5% (v/v) xylene, and the mixture was allowed to react at
28.degree. C. under stirring.
[0066] Twenty-four hours after the reaction started, dry baker's
yeast (product of Oriental Yeast) (250 mg), CMP-NeuAc synthase (3.4
units/mL reaction mixture) prepared in (6) above, and 1M magnesium
chloride solution (100 .mu.L) were added to the reaction mixture,
and reaction was allowed to proceed for a total of 62 hours. At a
point in time 14 hours after the start of reaction, sodium pyruvate
(110 mg) was added, at 24 and 38 hours, sodium pyruvate (110 mg)
and glucose (180 mg) was added, and, at 48 hours, sodium pyruvate
(55 mg) and glucose (180 mg) was added to the reaction mixture.
[0067] Analysis of the supernatant of the reaction mixture through
HPLC reveals that 21.4 mM CMP-NeuAc was produced.
Comparative Example 1
(1) Cloning of cmk Gene Encoding CMP Kinase
[0068] A chromosomal DNA prepared from E. coli JM109 through a
method described by Saito and Miura (Biochim. Biopys. Acta., 72,
619 (1963)) was used as a template, and the two below-described
primer DNA sequences were synthesized according to a method known
per se. The CMP kinase (cmk) gene of E. coli was amplified through
PCR.
TABLE-US-00005 Primer (G): (SEQ ID NO: 7)
5'-TTGAATTCTAAGGAGATAAAGATGACGGCAATT-3' Primer (H): (SEQ ID NO: 8)
5'-TTGAGCTCTGCAAATTCGGTCGCTTATGCG-3'
[0069] Amplification of the cmk gene through PCR was performed in a
DNA Thermal Cycler (product of Perkin-Elmer Cetus Instrument) by
adding thereto a 100 .mu.L reaction mixture containing 50 mM
potassium chloride, 10 mM Tris HCl (pH 8.3), 1.5 mM magnesium
chloride, 0.001% gelatin, 0.1 .mu.g template DNA, DNA primers (G)
and (H) (each 0.2 .mu.M), and AmpliTaq DNA polymerase (2.5 units).
The cycling protocol consisted of 25 cycles of the following three
steps: strand denaturation at 94.degree. C. for 1 minute, annealing
at 55.degree. C. for 1.5 minutes, and polymerization at 72.degree.
C. for 3 minutes.
[0070] Subsequent to gene amplification, the reaction mixture was
treated with a phenol/chloroform (1:1) mixture. To the
water-soluble fraction, ethanol was added in a volume twice that of
the fraction, to thereby precipitate DNA. The DNA collected through
precipitation was subjected to agarose gel electrophoresis as
described in literature (Molecular Cloning, see above), to thereby
purify DNA fragments having a size of 720 b. The DNA was cleaved
with restriction enzymes EcoRI and SacI, followed by ligation, by
use of T4 DNA ligase, with plasmid pTrc99A which had likewise been
digested with restriction enzymes EcoRI and SacI. By use of the
ligation reaction mixture, E. coli strain JM109 was transformed,
and from the resultant ampicillin-resistant transformants, plasmid
pTrcCMKAB was isolated. pTrcCMKAB has a structure in which a DNA
fragment containing a structural gene of cmk gene of E. coli has
been inserted to the EcoR-SacI cleavage sites located downstream of
the trc promoter of pTrc99A.
(2) Construction of a Plasmid for Coexpression of cmk and neuA
Genes
[0071] The pTrcsiaBNP plasmid obtained in Example 1 was cleaved
with restriction enzymes NcoI and EcoRI, and NcoI-EcoRI fragments
containing neuA gene were recovered through agarose gel
electrophoresis. The recovered fragments were ligated to the
pTrcCMKAB plasmid obtained in Comparative Example (1) above through
digestion with NcoI and EcoRI, using a T4 ligase. By use of the
ligation reaction mixture, E. coli strain JM109 was transformed,
and from the resultant ampicillin-resistant transformants, plasmid
pTrcSBCK was isolated. pTrcSBCK has a structure in which a DNA
fragment containing structural genes of neuA of H. influenzae and
cmk of E. Coli has been inserted to the NcoI-SalI cleavage sites
located downstream of the trc promoter of pTrc99A.
(3) Synthesis of CMP-NeuAc
[0072] A 25-mL culture broth (equivalent to 50-mg cells) of E. coli
ME8417/pTrcAE prepared in Example 1 was added to 200 mM potassium
phosphate buffer (pH 8.0, 2.5 mL) containing 100 mM GlcNAc, 20 mM
magnesium chloride, 50 mM glucose, 250 mM sodium pyruvate, and 0.5%
(v/v) xylene. The mixture was allowed to react under stirring for
24 hours at 28.degree. C.
[0073] A 25-mL culture broth (equivalent to 50-mg cells) of E. coli
ME8417 harboring the plasmid pTrcSBCK constructed in (2) above was
added to 200 mM potassium phosphate buffer (pH 8.0, 2.5 mL)
containing 100 mM CMP, 20 mM magnesium chloride, and 250 mM sodium
pyruvate, followed by ultrasonic treatment.
[0074] The ultrasonic-treated solution (2.5 mL) was added to the
reaction mixture 24 hours after the reaction started, and the
resultant mixture was further allowed to react under stirring at
28.degree. C. At points in time 14 and 24 hours after the start of
reaction, 55 mg of sodium pyruvate was added, and, at 38 hours, 110
mg of sodium pyruvate was added thereto.
[0075] After reaction was allowed to proceed for a total of 48
hours, the supernatant of the reaction mixture was analyzed through
HPLC. The results indicate that 6.28 mM CMP-NeuAc was produced.
Example 2
(1) Cloning of neuB1 Gene Encoding N-acetylneuraminic acid
Synthase
[0076] Chromosomal DNA of Campylobacter jejuni 1652 strain was used
as a template, and the two below-described primer DNA sequences
were synthesized according to a method known per se. The
acetylneuraminic acid synthase (neuB1) gene was amplified through
PCR.
TABLE-US-00006 Primer (I): (SEQ ID NO: 9)
5'-TACGATTATTTTCCTGATGCTC-3' Primer (J): (SEQ ID NO: 10)
5'-TCTCCAAGCTGCATTAAACGCC-3'
[0077] Amplification of the neuB1 gene through PCR was performed in
a DNA Thermal Cycler (product of Perkin-Elmer Cetus Instrument) by
adding thereto a 100 .mu.L reaction mixture containing 50 mM
potassium chloride, 10 mM Tris HCl (pH 8.3), 1.5 mM magnesium
chloride, 0.001% gelatin, 0.1 .mu.g template DNA, DNA primers (A)
and (B) (each 0.2 .mu.M), and AmpliTaq DNA polymerase (2.5 units).
The cycling protocol consisted of 30 cycles of the following three
steps: strand denaturation at 94.degree. C. for 1 minute, annealing
at 55.degree. C. for 1.5 minutes, and polymerization at 72.degree.
C. for 3 minutes.
[0078] Subsequent to gene amplification, the reaction mixture was
treated with a phenol/chloroform (1:1) mixture. To the
water-soluble fraction, ethanol was added in a volume twice that of
the fraction, to thereby precipitate DNA. The DNA collected through
precipitation was subjected to agarose gel electrophoresis as
described in literature (Molecular Cloning, see above), to thereby
purify DNA fragments having a size of 2.2 kb. The DNA fragments
were used as a template, and the two below-described primer DNA
sequences were synthesized according to a method known per se. The
neuB1 gene of C. jejuni was again amplified through PCR.
TABLE-US-00007 Primer (K): (SEQ ID NO: 11)
5'-AAGGATCCTCTAGTGAGGCTTATGGAA-3' Primer (L): (SEQ ID NO: 12)
5'-GTCTGCAGATTTAATCTTAGAATAATCAGCCC-3'
[0079] Amplification of the neuB1 gene through PCR was performed in
a DNA Thermal Cycler (product of Perkin-Elmer Cetus Instrument) by
adding thereto a 100 .mu.L reaction mixture containing 50 mM
potassium chloride, 10 mM Tris HCl (pH 8.3), 1.5 mM magnesium
chloride, 0.001% gelatin, 0.1 .mu.g template DNA, DNA primers (A)
and (B) (each 0.2 .mu.M), and AmpliTaq DNA polymerase (2.5 units).
The cycling protocol consisted of 25 cycles of the following three
steps: strand denaturation at 94.degree. C. for 1 minute, annealing
at 55.degree. C. for 1.5 minutes, and polymerization at 72.degree.
C. for 3 minutes.
[0080] Subsequent to gene amplification, the reaction mixture was
treated with a phenol/chloroform (1:1) mixture. To the
water-soluble fraction, ethanol was added in a volume twice that of
the fraction, to thereby precipitate DNA. The DNA collected through
precipitation was subjected to agarose gel electrophoresis, to
thereby purify DNA fragments having a size of 1.2 kb. The DNA was
cleaved with restriction enzymes BamHI and PstI, followed by
ligation, by use of T4 DNA ligase, with plasmid pTrc99A (Pharmacia
Biotech.) which had likewise been digested with restriction enzymes
BamHI and PstI. By use of the ligation reaction mixture, E. coli
strain JM109 was transformed, and from the resultant
ampicillin-resistant transformants, plasmid pTrcneuB1 was isolated.
pTrcneuB1 has a structure in which a DNA fragment containing a
structural gene of neuB1 gene of C. jejuni has been inserted to the
BamHI-PstI cleavage sites located downstream of the trc promoter of
pTrc99A (FERM BP-8248: Jun. 25, 2002, National Institute of
Advanced Industrial Science and Technology, Patent Microorganisms
Depositary (Chuo 6, 1-1-1 Higashi, Tsukuba-shi, Ibaraki-ken, Japan
(postal code: 305-8566)).
(2) Construction of a Plasmid for Coexpression of nanE and neuB1
Genes
[0081] The pTrcneuB1 plasmid prepared in (1) above was cleaved with
a restriction enzyme BamHI and blunted with a T4 DNA polymerase.
The product was cleaved with a restriction enzyme PstI, and
(BamHI)-PstI fragments containing neuB1 gene were collected through
agarose gel electrophoresis. Subsequently, the pTrcnanE plasmid
prepared in Example 1 (2) was cleaved with a restriction enzyme
SalI and blunted with a T4 DNA polymerase, and then cleaved with a
restriction enzyme PstI. The resultant fragments were ligated to
the (BamHI)-PstI fragments containing neuB1 gene using a T4 DNA
ligase. By use of the ligation reaction mixture, E. coli strain
JM109 was transformed, and from the resultant ampicillin-resistant
transformants, plasmid pTrcNENB was isolated. pTrcNENB has a
structure in which a DNA fragment containing structural genes of
nanE of H. influenzae and neuB1 of C. jejuni has been inserted to
the XbaI-PstI cleavage sites located downstream of the trc promoter
of pTrc99A.
(3) Synthesis of CMP-NeuAc
[0082] E. coli MC1061 (ATCC53338) was engineered so as to harbor
the plasmid pTrcNENB constructed in (2) above. To the cultured cell
(50 mg) were added 175 mM potassium phosphate buffer (pH 8.0) (5
mL) containing 50 mM CMP, 100 mM GlcNAc, 30 mM magnesium chloride,
200 mM glucose, 100 mM sodium pyruvate, 0.5% (v/v) xylene, 4% (w/v)
dry baker's yeast (product of Oriental yeast), and CMP-NeuAc
synthase (1.7 units/mL reaction mixture) prepared in Example 1 (6).
Reaction was allowed to proceed under stirring for 72 hours at
28.degree. C. At points in time 14, 24, 38, 48, and 62 hours after
the start of reaction, glucose (180 mg) was added to the
mixture.
[0083] Analysis of the supernatant of the reaction mixture through
HPLC reveals that 25.6 mM CMP-NeuAc was produced.
INDUSTRIAL APPLICABILITY
[0084] The process of the present invention employing NeuAc lyase
requires no expensive ATP and enables, for the first time,
efficient production of CMP-NeuAc from inexpensive GlcNAc, CMP, and
pyruvate. Therefore, the process of the present invention is
considerably useful as a process for mass-production of
CMP-NeuAc.
[0085] The process of the present invention employing NeuAc
synthase also requires no expensive ATP and enables, for the first
time, efficient production of CMP-NeuAc from inexpensive GlcNAc,
CMP, and pyruvate, since phosphoenolpyruvate (PEP), which is
essential to the NeuAc synthase reaction, is synthesized and
supplied from glucose through (metabolic) bioreaction of yeast and
E. coli, omitting the need of addition of phosphoenolpyruvate (PEP)
to the reaction system. Therefore, the process of the present
invention is considerably useful as a process for mass-production
of CMP-NeuAc.
[0086] In particular, the process of the present invention
employing NeuAc synthase is simple and excellent as compared with
the process of the present invention employing NeuAc lyase, which
requires two steps of reaction.
Sequence CWU 1
1
12131DNAArtificial Sequenceprimer for amplification of nanA gene
1caccatggcg aagatattgc cgctcaaact a 31235DNAArtificial
Sequenceprimer for amplification of nanA gene 2ccgaattcat
ttatgacaaa aatttcgctt tcaag 35331DNAArtificial Sequenceprimer for
amplification of nanE gene 3ggtctagatt taaatgaggg gtgttatatg t
31441DNAArtificial Sequenceprimer for amplification of nanE gene
4tcgtcgactt atcttgcaga tttcactgaa ttagcaaacc a 41529DNAArtificial
Sequenceprimer for amplification of neuA gene 5tgccatggtg
aaaataataa tgacaagaa 29628DNAArtificial Sequenceprimer for
amplification of neuA gene 6aactgcagtg cagatcaaaa gtgcggcc
28733DNAArtificial Sequenceprimer for amplification of cmk gene
7ttgaattcta aggagataaa gatgacggca att 33830DNAArtificial
Sequenceprimer for amplification of cmk gene 8ttgagctctg caaattcggt
cgcttatgcg 30922DNAArtificial Sequenceprimer for amplification of
neuB1 gene 9tacgattatt ttcctgatgc tc 221022DNAArtificial
Sequenceprimer for amplification of neuB1 gene 10tctccaagct
gcattaaacg cc 221127DNAArtificial Sequenceprimer for amplification
of neuB1 gene 11aaggatcctc tagtgaggct tatggaa 271232DNAArtificial
Sequenceprimer for amplification of neuB1 gene 12gtctgcagat
ttaatcttag aataatcagc cc 32
* * * * *