U.S. patent application number 09/891287 was filed with the patent office on 2002-07-25 for method for producing nucleotide by fermentation.
This patent application is currently assigned to AJINOMOTO CO., INC.. Invention is credited to Kakehi, Masahiro, Sugimoto, Shinichi, Tabira, Yukiko, Usuda, Yoshihiro.
Application Number | 20020098494 09/891287 |
Document ID | / |
Family ID | 18701553 |
Filed Date | 2002-07-25 |
United States Patent
Application |
20020098494 |
Kind Code |
A1 |
Kakehi, Masahiro ; et
al. |
July 25, 2002 |
Method for producing nucleotide by fermentation
Abstract
Nucleoside 5'-phosphate ester is produced by culturing a
bacterium belonging to the genus Escherichia having an ability to
produce nucleoside 5'-phosphate ester, in which ushA gene and aphA
gene do not function normally, in a medium to produce and
accumulate nucleoside 5'-phosphate ester in the medium, and
collecting the nucleoside 5'-phosphate ester from the medium.
Inventors: |
Kakehi, Masahiro;
(Kawasaki-shi, JP) ; Usuda, Yoshihiro;
(Kawasaki-shi, JP) ; Tabira, Yukiko;
(Kawasaki-shi, JP) ; Sugimoto, Shinichi;
(Kawasaki-shi, JP) |
Correspondence
Address: |
OBLON SPIVAK MCCLELLAND MAIER & NEUSTADT PC
FOURTH FLOOR
1755 JEFFERSON DAVIS HIGHWAY
ARLINGTON
VA
22202
US
|
Assignee: |
AJINOMOTO CO., INC.
15-1, Kyobashi 1-chome, Tokyo
Chuo-ku
JP
|
Family ID: |
18701553 |
Appl. No.: |
09/891287 |
Filed: |
June 27, 2001 |
Current U.S.
Class: |
435/6.12 ;
435/252.33; 435/6.15; 435/89 |
Current CPC
Class: |
C12P 19/30 20130101;
C12N 9/16 20130101; C12P 19/32 20130101 |
Class at
Publication: |
435/6 ;
435/252.33; 435/89 |
International
Class: |
C12Q 001/68; C12P
019/30; C12N 001/21 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 5, 2000 |
JP |
2000-204260 |
Claims
What is claimed is:
1. A method for producing nucleoside 5'-phosphate ester, comprising
the steps of culturing a bacterium belonging to the genus
Escherichia having an ability to produce nucleoside 5'-phosphate
ester, in which ushA gene and aphA gene do not function normally,
in a medium to produce and accumulate nucleoside 5'-phosphate ester
in the medium, and collecting the nucleoside 5'-phosphate ester
from the medium.
2. The method for producing nucleoside 5'-phosphate ester according
to claim 1, wherein mutations are introduced into the ushA gene and
the aphA gene or these genes are disrupted so that they do not
function normally.
3. The method for producing nucleoside 5'-phosphate ester according
to claim 1 or 2, wherein the nucleoside 5'-phosphate ester is
selected from the group consisting of 5'-inosinic acid or
5'-guanylic acid.
4. A bacterium belonging to the genus Escherichia having an ability
to produce nucleoside 5'-phosphate ester, in which ushA gene and
aphA gene are disrupted.
5. The bacterium belonging to the genus Escherichia according to
claim 4, wherein the nucleoside 5'-phosphate ester is selected from
the group consisting of 5'-inosinic acid or 5'-guanylic acid.
6. A method for searching for a 5'-nucleotidase gene affecting
accumulation of nucleoside 5'-phosphate ester, comprising the steps
of: culturing a parent strain of microorganism and a derivative
strain thereof in which a known 5'-nucleotidase is deleted in a
minimal medium containing a first nucleoside 5'-phosphate ester as
a sole carbon source and a minimal medium containing a second
nucleoside 5'-phosphate ester as a sole carbon source to examine
expression profiles of genes in the parent strain and the
derivative strain, calculating a product of a ratio of expression
amounts of each gene in the parent strain and the derivative strain
when they are cultured in the medium containing the first
nucleoside 5'-phosphate ester as a carbon source and a ratio of
expression amounts of each gene in the parent strain and the
derivative strain when they are cultured in the medium containing
the second nucleoside 5'-phosphate ester as a carbon source, and
selecting one or more genes showing a larger value of the
product.
7. The method for searching for a 5'-nucleotidase gene according to
claim 6, wherein the first and second nucleoside 5'-phosphate
esters are 5'-inosinic acid and 5'-guanylic acid.
8. The method for searching for a 5'-nucleotidase gene according to
claim 6 or 7, further comprising the step of selecting a gene that
can code for a signal sequence required for transition of a protein
into periplasm from the selected genes.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] The present invention relates to a method for producing
nucleotides by fermentation. Nucleotides such as nucleoside
5'-phosphate esters are useful as seasonings, drugs, raw materials
thereof and so forth.
[0003] 2. Description of the Related Art
[0004] As methods for industrial production of nucleoside
5'-phosphate esters, there are known methods comprising producing
nucleoside by fermentation and enzymatically phosphorylating the
obtained nucleoside to obtain nucleoside 5'-phosphate ester.
[0005] On the other hand, methods of directly producing nucleoside
5'-phosphate esters by fermentation have also been proposed. For
example, Japanese Patent Publication (Kokoku) No. 56-12438
discloses a method for producing 5'-guanylic acid, which comprises
culturing a mutant strain of a bacterium belonging to the genus
Bacillus showing adenine auxotrophy and resistance to decoyinine or
methionine sulfoxide and having an ability to produce 5'-guanylic
acid (guanosine 5'-monophosphate, also abbreviated as "GMP"
hereinafter) and collecting GMP produced and accumulated in the
medium. Further, there are several reports on deriving strains
which produce 5'-inosinic acid (inosine 5'-monophosphate, also
abbreviated as "IMP" hereinafter) from inosine producing strains of
Bacillus subtilis (Magasanik, B. et al., J. Biol. Chem., 226, 339
(1957); Fujimoto, M., et al., Agr. Biol. Chem., 30, 605 (1966)).
However, the production of nucleoside 5'-phosphate esters by direct
fermentation generally suffers from insufficient yield, and it is
not so practical compared with the aforementioned enzymatic
methods.
[0006] As the reasons for the difficulty of IMP production by
direct fermentation, there are mentioned bad cell permeability of
IMP and quite ubiquitous distribution of degradative enzymes that
decompose IMP (Nucleic Acid Fermentation, Edited by Aminosan
Kakusan Shudankai, Kodansha Scientific, Japan). To overcome these
obstacles, there has been attempted to delete nucleotide
degradative activity. As degradative enzymes that decompose IMP
into inosine, 5'-nucleotidase, acid phosphatase, alkaline
phosphatase and so forth are conceived (Nucleic Acid Fermentation,
supra). Further, the aforementioned Japanese Patent Publication No.
56-12438 also suggests that a bacterial strain showing high GMP
yield can be obtained from a mutant strain showing reduced
nucleotidase activity.
[0007] As a technique for producing nucleoside 5'-phosphate ester
on an industrial level, a method of producing IMP by using a mutant
strain of Brevibacterium ammoniagenes has been developed (Furuya et
al., Appl. Microbiol., 16, 981 (1968)).
[0008] As described above, various studies have been made on the
production of nucleoside 5'-phosphate esters by direct
fermentation, and some successful examples are also known. However,
there are many unknown points about nucleotide degradative enzymes,
and it cannot be said that improvement of yield has been studied
sufficiently. In particular, no example of production of nucleoside
5'-phosphate esters on a practical level has been known for
bacteria belonging to the genus Escherichia.
SUMMARY OF THE INVENTION
[0009] The present invention was accomplished in view of the
technical situation described above, and an object of the invention
is to provide a method for producing nucleoside 5'-phosphate ester
such as IMP using a bacterium belonging to the genus
Escherichia.
[0010] The inventors of the present invention assiduously studied
in order to achieve the aforementioned object. As a result, they
found that a gene coding for 5'-nucleotidase other than the known
gene existed in Escherichia coli, and successfully identified the
gene. Further, they found that Escherichia coli having inosine
producing ability or guanosine producing ability became to produce
IMP or GMP, if the novel gene was disrupted in addition to the
known 5'-nucleotidase gene. Thus, they accomplished the present
invention.
[0011] That is, the present invention provides the followings.
[0012] (1) A method for producing nucleoside 5'-phosphate ester,
comprising the steps of culturing a bacterium belonging to the
genus Escherichia having an ability to produce nucleoside
5'-phosphate ester, in which ushA gene and aphA gene do not
function normally, in a medium to produce and accumulate nucleoside
5'-phosphate ester in the medium, and collecting the nucleoside
5'-phosphate ester from the medium.
[0013] (2) The method for producing nucleoside 5'-phosphate ester
according to (1), wherein mutations are introduced into the ushA
gene and the aphA gene or these genes are disrupted so that they do
not function normally.
[0014] (3) The method for producing nucleoside 5'-phosphate ester
according to (1) or (2), wherein the nucleoside 5'-phosphate ester
is selected from the group consisting of 5'-inosinic acid or
5'-guanylic acid.
[0015] (4) A bacterium belonging to the genus Escherichia having an
ability to produce nucleoside 5'-phosphate ester, in which ushA
gene and aphA gene are disrupted.
[0016] (5) The bacterium belonging to the genus Escherichia
according to (4), wherein the nucleoside 5'-phosphate ester is
selected from the group consisting of 5'-inosinic acid or
5'-guanylic acid.
[0017] (6) A method for searching for a 5'-nucleotidase gene
affecting accumulation of nucleoside 5'-phosphate ester, comprising
the steps of culturing a parent strain of microorganism and a
derivative strain thereof in which a known 5'-nucleotidase is
deleted in a minimal medium containing a first nucleoside
5'-phosphate ester as a sole carbon source and a minimal medium
containing a second nucleoside 5'-phosphate ester as a sole carbon
source to examine expression profiles of genes in the parent strain
and the derivative strain,
[0018] calculating a product of a ratio of expression amounts of
each gene in the parent strain and the derivative strain when they
are cultured in a medium containing the first nucleoside
5'-phosphate ester as a carbon source and a ratio of expression
amounts of each gene in the parent strain and the derivative strain
when they are cultured in a medium containing the second nucleoside
5'-phosphate ester as a carbon source, and selecting one or more
genes showing a larger value of the product.
[0019] (7) The method for searching for a 5'-nucleotidase gene
according to (6), wherein the first and second nucleoside
5'-phosphate esters are 5'-inosinic acid and 5'-guanylic acid.
[0020] (8) The method for searching for a 5'-nucleotidase gene
according to (6) or (7), further comprising the step of selecting a
gene that can code for a signal sequence required for transition of
a protein into periplasm from the selected genes.
[0021] According to the present invention, nucleoside 5'-phosphate
ester such as IMP and GMP can be produced by direct fermentation
using a bacterium belonging to the genus Escherichia.
PREFERRED EMBODIMENTS OF THE INVENTION
[0022] Hereafter, the present invention will be explained in
detail.
[0023] <1> Search of an Unknown 5'-nucleotidase Gene
[0024] As a known 5'-nucleotidase of Escherichia coli, UDP-sugar
hydrolase (UshA), which is a product of the ushA gene (GenBank
accession X03895), is known. It has been known that the enzyme has
5'-nucleotidase activity that catalyzes dephosphorylation of
nucleoside 5'-phosphate such as AMP, GMP, IMP and XMP to produce a
corresponding nucleoside (H. C. Neu, (1967) Journal of Biological
Chemistry, 242, 3896-3904; A. Cowman, I. R. Beacham, (1980) Gene,
12, 281-286).
[0025] The inventors of the present invention disrupted the ushA
gene of Escherichia coli W3110 strain, and examined its influence
on the nucleotide decomposing ability. The 5'-nucleotidase activity
in periplasm of the ushA gene-disrupted W3110 strain (W.DELTA.ushA)
was markedly reduced compared with the W3110 strain. However, when
growth of the W.DELTA.ushA strain was investigated in a minimal
medium containing nucleoside-5'-phosphate as a sole carbon source,
this strain could grow. Therefore, it was considered that the
nucleotide decomposing ability is not completely lost by the
disruption of only ushA. Furthermore, when nucleoside-5'-phosphate
was used as a sole carbon source, start of the growth was retarded.
Therefore, it was expected that there existed another
5'-nucleotidase that was induced when UshA did not function.
[0026] The inventor of the present invention attempted to search
for an unknown 5'-nucleotidase gene based on the aforementioned
findings, and found that a product of a gene reported as an acid
phosphatase gene (aphA) (M. C. Thaller, S. Schippa, A. Bonci, S.
Cresti, G. M. Rossolini, (1997) FEMS Microbilogy Letters, 146,
191-198, GenBank accession X86971) or yjbP (GenBank accession
AAC77025) had the 5'-nucleotidase activity.
[0027] A gene coding for such a 5'-nucleotidase that affects the
accumulation of nucleoside 5'-phosphate as described above can be
searched for as follows.
[0028] First, a microbial parent strain and a derivative strain
thereof in which a known 5'-nucleotidase is deleted are cultured in
a minimal medium containing a first nucleoside 5'-phosphate ester
or a second nucleoside 5'-phosphate ester such as IMP or GMP as a
sole carbon source. When the microorganism is Escherichia coli, the
known 5'-nucleotidase may be the aforementioned UshA.
[0029] Subsequently, gene expression profiles of these strains are
investigated. Specifically, a ratio of expression amounts in the
wild strain and the derivative strain is investigated for each
gene.
[0030] Then, a product of a ratio of expression amounts of a gene
in the parent strain and the derivative strain when they are
cultured in a medium containing the first nucleoside 5'-phosphate
as a carbon source and a ratio of expression amounts of the gene in
the parent strain and the derivative strain when they are cultured
in a medium containing the second nucleoside 5'-phosphate as a
carbon source is calculated for each gene, and one or more genes
showing a larger value of the product are selected.
[0031] Although the method for gene expression profiling is not
particularly limited, the DNA array method (H. Tao, C. Bausch, C.
Richmond, F. R. Blattner, T. Conway, (1999) Journal of
Bacteriology, 181, 6425-6440) can be mentioned, for example.
[0032] From the aforementioned selected genes, target genes can be
further narrowed down by selecting genes that may code a signal
sequence required for transition of protein to periplasm. This is
because it is expected that the target 5'-nucleotidase transits to
periplasm and function therein.
[0033] As for Escherichia coli, as shown in the examples mentioned
later, two kinds of genes, b0220 (also referred to as o157) and
yjbP, were selected. Among these genes, yjbp was an acid
phosphatase gene (aphA). On the other hand, b0220 was a gene of
which function was unidentified, which was designated as ykfE. When
these genes were amplified in Escherichia coli, remarkable increase
of 5'-nucleotidase activity was not observed in the ykfE
gene-amplified strain, whereas remarkable increase of
5'-nucleotidase activity was observed in the aphA gene-amplified
strain. Thus, it was confirmed that the aphA gene product (AphA)
had the 5'-nucleotidase activity. In this way, apha was found as a
gene coding for 5'-nucleotidase that affected the accumulation of
nucleoside 5'-phosphate.
[0034] <2> Bacterium Belonging to the Genus Escherichia of
the Present Invention
[0035] The Bacterium belonging to the genus Escherichia of the
present invention is a bacterium belonging to the genus Escherichia
having an ability to produce nucleoside 5'-phosphate, in which the
ushA gene and the apha gene do not function normally. The Bacterium
belonging to the genus Escherichia itself is not particularly
limited so long as it is a microorganism belonging to the genus
Escherichia such as Escherichia coli. However, specifically, those
mentioned in the reference of Neidhardt et al. (Neidhardt, F. C. et
al., Escherichia coli and Salmonella Typhimurium, American Society
for Microbiology, Washington D.C., 1208, Table 1) can be used.
[0036] The Bacterium belonging to the genus Escherichia of the
present invention can be obtained by, for example, breeding a
mutant strain or genetic recombinant strain in which the ushA gene
and the aphA gene do not normally function using a Bacterium
belonging to the genus Escherichia having purine nucleoside
producing ability as a parent strain. Further, the Bacterium
belonging to the genus Escherichia of the present invention can
also be obtained by breeding similar to the breeding of purine
nucleoside producing strain using a strain in which the ushA gene
and the aphA gene do not normally function as a parent strain.
[0037] Examples of bacteria belonging to the genus Escherichia
having purine nucleoside producing ability include bacteria
belonging to the genus Escherichia having an ability to produce
inosine, guanosine, adenosine, xanthosine, purine riboside,
6-methoxypurine riboside, 2,6-diaminopurine riboside,
6-fluoropurine riboside, 6-thiopurine riboside,
2-amino-6-thiopurine riboside, mercaptoguanosine or the like. By
breeding a mutant strain or genetic recombinant strain in which the
ushA gene and the aphA gene do not normally function using these
Escherichia bacteria having purine nucleoside producing ability as
a parent strain, bacteria belonging to the genus Escherichia having
an ability to produce nucleoside 5'-phosphate ester corresponding
to each purine nucleoside can be obtained.
[0038] The purine nucleoside producing ability referred to in the
present invention means an ability to produce and accumulate a
purine nucleoside in a medium. Further, the expression of "having
purine nucleoside producing ability" means that the microorganism
belonging to the genus Escherichia produces and accumulates a
purine nucleoside in a medium in an amount larger than that
obtained with a wild strain of E. coli, for example, the W3110
strain.
[0039] Further, the ability to produce nucleoside 5'-phosphate
ester means an ability to produce and accumulate nucleoside
5'-phosphate ester in a medium. Furthermore, the expression of
"having purine nucleoside producing ability" means that the
microorganism belonging to the genus Escherichia produces and
accumulates a purine nucleoside in a medium in an amount larger
than that obtained with a wild strain of E. coli, for example, the
W3110 strain, and it preferably means that the microorganism
produces and accumulates nucleoside 5'-phosphate ester in an amount
of 100 mg/L or more, more preferably 500 mg/L or more, further
preferably 1000 mg/L or more, when it is cultured under the
conditions mentioned in Example 6 described later.
[0040] Bacteria belonging to the genus Escherichia having purine
nucleoside producing ability are detailed in International Patent
Publication WO99/03988, for example. More specifically, there can
be mentioned the Escherichia coli FADRaddG-8-3::KQ strain (purFKQ,
purA.sup.-, deoD.sup.-, purR.sup.-, add.sup.-, gsk.sup.-) described
in the above international patent publication. This strain harbors
a mutant purF coding for PRPP amidotransferase of which feedback
inhibition by AMP and GMP is desensityzed, and in which the lysine
residue at a position of 326 is replaced with a glutamine residue,
and a succinyl-AMP synthase gene (purA), purine nucleoside
phosphorylase gene (deoD), purine repressor gene (purR), adenosine
deaminase gene (add), and inosine/guanosine kinase gene (gsk) are
disrupted. This strain given with a private number of AJ13334 was
deposited on Jun. 24, 1997 at the National Institute of Bioscience
and Human-Technology, Agency of Industrial Science and Technology,
Ministry of International Trade and Industry (currently, the
independent administrative corporation, National Institute of
Advanced Industrial Science and Technology, International Patent
Organism Depositary)(Chuo Dai-6, 1-1 Higashi 1-Chome, Tsukuba-shi,
Ibaraki-ken, Japan, postal code: 305-5466) as an international
deposit under the provisions of the Budapest treaty, and received
an accession number of FERM BP-5993. This strain has an ability to
produce inosine and guanosine. Further, the strain obtained by
introducing a plasmid containing a mutant purF gene into the
FADRaddeddyicPpgixapA strain, which was constructed as described in
the Example to be mentioned later, can also be suitably used as an
inosine producing bacterium. Guanosine producing ability can be
enhanced by introducing the guaA and guaB genes that encode IMP
dehydrogenase and GMP synthetase, respectively, into an inosine
producing bacterium. In the present invention, the bacterial strain
is not limited to the aforementioned strains, and any strains
having purine nucleoside producing ability can be used without any
particular limitation.
[0041] A mutant strain or genetic recombinant strain in which the
ushA gene and the aphA gene do not function normally can be
obtained by modifying the genes so that the activities of
5'-nucleotidases that are the products of the genes should be
decreased or deleted, or transcription of these genes should be
decreased or eliminated. Such a microorganism can be obtained by,
for example, replacing the ushA gene and the aphA gene on the
chromosome with an ushA gene and aphA gene that do not function
normally (also referred to as "disrupted ushA gene" and "disrupted
aphA gene" hereinafter) by homologous recombination utilizing a
genetic recombination method (Experiments in Molecular Genetics,
Cold Spring Harbor Laboratory press (1972); Matsuyama, S. and
Mizushima, S., J. Bacteriol., 162, 1196 (1985)).
[0042] In homologous recombination, a plasmid or the like having a
sequence showing homology to a sequence on a chromosome is
introduced into a bacterial cell. Then, recombination occurs at a
certain frequency at a position of the homologous sequence so that
the whole introduced plasmid is incorporated into the chromosome.
When recombination is further caused thereafter at the position of
the homologous sequence, the plasmid is again removed from the
chromosome. At this time, depending on the position of the
recombination, the disrupted gene may remain on the chromosome, and
the original normal gene may be removed together with the plasmid.
By selecting such a bacterial strain, a strain in which the normal
ushA gene or aphA gene on the chromosome is replaced with the
disrupted ushA gene or the disrupted aphA gene can be obtained.
[0043] A gene disruption technique based on such homologous
recombination has already been established, and a method utilizing
a linear DNA, a method utilizing a temperature sensitive plasmid
and so forth can be used. The disruption of the ushA gene and the
aphA gene can also be performed by using a plasmid containing an
ushA gene or aphA gene internally inserted with a marker gene such
as a drug resistance gene, which cannot replicate in a target
microbial cell. That is, in a transformant that was transformed
with the aforementioned plasmid and hence acquired drug resistance,
the marker gene is incorporated into the chromosomal DNA. Since it
is highly probable that this marker gene is incorporated into the
chromosome by homologous recombination of the ushA gene or aphA
gene sequences located on the both ends of the marker gene with
those genes on the chromosome, a gene-disrupted strain can be
selected efficiently.
[0044] The disrupted ushA gene and the disrupted aphA gene used for
the gene disruption can be obtained by, specifically, deleting a
certain region of these genes by digestion with a restriction
enzyme and ligation, inserting another DNA fragment (marker gene
etc.) into these genes, or introducing substitution, deletion,
insertion, addition or inversion of one or more nucleotides into a
nucleotide sequence of coding region, promoter region or the like
of the ushA gene or the aphA gene by the site-specific mutagenesis
(Kramer, W. and Frits, H. J., Methods in Enzymology, 154, 350
(1987)) or treatment with a chemical agent such as sodium
hyposulfite or hydroxylamine (Shortle, D. and Nathans, D., Proc.
Natl. Acad. Sci. U.S.A., 75, 270 (1978)) so that activity of the
encoded repressor should be decreased or deleted, or transcription
of the ushA gene or the aphA gene should be decreased or
eliminated. Among these embodiments, the method of deleting a
certain region of the ushA gene or aphA by digestion with a
restriction enzyme and ligation and the method of inserting another
DNA fragment into these genes are preferred in view of certainty
and stability of the methods. The order of the gene disruption of
the ushA gene and the aphA gene is not particularly limited, and
either one may be disrupted first.
[0045] The nucleotide sequences of the ushA gene and the aphA genes
themselves are known, and hence they can be easily obtained by PCR
or hybridization based on such nucleotide sequences. For example,
the ushA gene can be obtained from chromosome DNA of Escherichia
coli by PCR using the primers shown in SEQ ID NOS: 1 and 2, for
example. Further, the N-terminal region of the aphA gene can be
obtained by PCR using the primers shown in SEQ ID NOS: 3 and 7, and
the C-terminal region of the same can be obtained by PCR using the
primers shown in SEQ ID NOS: 4 and 8.
[0046] Whether the target gene has been disrupted or not can be
confirmed by analyzing the gene on a chromosome by Southern
blotting or PCR.
[0047] <3> Method for Producing Nucleoside 5'-phosphate
Ester
[0048] Nucleoside 5'-phosphate ester can be produced by culturing a
bacterium belonging to the genus Escherichia having an ability to
produce nucleoside 5'-phosphate ester, in which the ushA gene and
the aphA gene do not function normally, in a medium to produce and
accumulate nucleoside 5'-phosphate ester in the medium, and
collecting the nucleoside 5'-phosphate ester from the medium.
[0049] The medium may be a usual medium containing a carbon source,
nitrogen source, inorganic ions, and other organic components, if
needed. As the carbon source, there can be used saccharides such as
glucose, lactose, galactose, fructose, arabinose, maltose, xylose,
trehalose, ribose and starch hydrolysate, alcohols such as
glycerol, mannitol and sorbitol, organic acids such as gluconic
acid, fumaric acid, citric acid and succinic acid and so forth.
[0050] As the nitrogen source, there can be used inorganic ammonium
salts such as ammonium sulfate, ammonium chloride, and ammonium
phosphate, organic nitrogen such as soybean hydrolysate, ammonia
gas, aqueous ammonia and so forth.
[0051] As the organic trace nutrients, it is desirable to add
required substances including vitamins such as vitamin B1, nucleic
acids such as adenine and RNA or yeast extract in a suitable
amount. In addition to these, a small amount of potassium
phosphate, magnesium sulfate, iron ions, manganese ions and so
forth are added as required.
[0052] Culture is preferably carried out under an aerobic condition
for 16-72 hours. The culture temperature is controlled to be
30.degree. C. to 45.degree. C., and pH is controlled to be 5 to 8
during the culture. Inorganic or organic, acidic or alkaline
substances as well as ammonia gas and so forth can be used for pH
adjustment.
[0053] Collection of nucleoside 5'-phosphate ester from fermented
liquor is usually carried out by a combination of an ion exchange
resin method, a precipitation method and other known
techniques.
BEST MODE FOR CARRYING OUT THE INVENTION
[0054] The present invention will be further specifically explained
hereinafter with reference to the following examples.
EXAMPLE 1
Effect of ushA Disruption on Nucleotide Production of Escherichia
coli.
[0055] <1> Construction of ushA-disrupted Strain
[0056] From genomic DNA of the Escherichia coli W3110 strain, a
ushA gene fragment was amplified by PCR. The genomic DNA was
extracted by using RNA/DNA maxi Kit (produced by Qiagen). PCR was
performed by using the primers shown in SEQ ID NOS: 1 and 2 and
Pyrobest DNA Polymerase (produced by Takara Shuzo) according to the
instruction appended to the polymerase. After PCR, the amplified
DNA fragments were purified by using Wizard PCR Preps (produced by
Promega). After digestion with restriction enzymes SphI and SalI
(produced by Takara Shuzo), the purified DNA fragments were
subjected to a phenol/chloroform treatment and ethanol
precipitation. pHSG397 (produced by Takara Shuzo) similarly
digested with SphI and SalI was ligated by using DNA ligation Kit
Ver.2 (produced by Takara Shuzo). Competent cells of JM109
(produced by Takara Shuzo) were transformed with the above ligation
mixture, and plated on an LB agar plate containing 30 .mu.g/mL of
chloramphenicol (produced by Sigma) (LB+chloramphenicol plate).
After culturing at 37.degree. C. overnight, grown colonies were
cultured in LB medium containing 30 .mu.g/mL of chloramphenicol at
37.degree. C. in a test tube, and a plasmid was extracted using an
automatic plasmid extractor, PI-50 (produced by Kurabo Industries).
The obtained plasmid was designated as pHSGushA.
[0057] Then, an HpaI fragment was removed from the ushA gene
contained in pHSGushA as follows. pHSGushA was digested with a
restriction enzyme HpaI (produced by Takara Shuzo), subjected to a
phenol/chloroform treatment and ethanol precipitation, and ligated
by using DNA Ligation Kit Ver.2. JM109 was transformed with this
ligation solution, and a plasmid was extracted from emerged
colonies. The obtained plasmid was digested with SphI and SalI, and
subjected to agarose gel electrophoresis to select a plasmid
containing an inserted target fragment in which the HpaI digestion
fragment was deleted from the ushA gene region.
[0058] The obtained plasmid fragment and a fragment obtained by
digesting the temperature sensitive plasmid pMAN997 described in
International Patent Publication WO99/03988 with SphI and SalI were
ligated. JM109 was transformed with the ligation solution, and
colonies were selected at 30.degree. C. on an LB agar plate
containing 50 .mu.g/mL of ampicillin (produced by Meiji Seika
Kaisha) (LB+ampicillin plate). The colonies were cultured in LB
medium containing 50 .mu.g/mL of ampicillin at 30.degree. C. in a
test tube, and plasmids were extracted. A plasmid from which a
fragment of a desired length could be obtained by digestion with
SphI and SalI was used as a plasmid for ushA disruption,
pMAN.DELTA.ushA. The above pMAN997 was obtained by exchanging
VspI-HindIII fragments of pMAN031 (J. Bacteriol., 162, 1196 (1985))
and pUC19 (produced by Takara Shuzo).
[0059] The W3110 strain was transformed with pMAN.DELTA.ushA, and
colonies were selected on an LB+ampicillin plate at 30.degree. C.
The selected clones were cultured at 30.degree. C. overnight as
liquid culture. The culture broth was diluted 10.sup.-3 times, and
inoculated on an LB+ampicillin plate, and colonies were selected at
42.degree. C. The selected clones were applied and spread on an
LB+ampicillin plate, and cultured at 30.degree. C. Then, 1/8 of the
cells on the plate were suspended in 2 mL of LB medium, and
cultured at 42.degree. C. for 4 to 5 hours with shaking. The cells
diluted 10.sup.-5 times were seeded on an LB plate, and several
hundreds of colonies among the obtained colonies were inoculated on
an LB plate and LB+ampicillin plate, and growth was confirmed to
select ampicillin sensitive strains. Colony PCR was performed for
several strains among the ampicillin sensitive strains to confirm
the deletion of ushA gene. In this way, an ushA-disrupted strain
derived from E. coli W3110, W.DELTA.ushA, was obtained.
[0060] <2> Measurement of 5'-nucleotidase and Nucleotide
Assimilation Culture
[0061] W3110 and W.DELTA.ushA were cultured at 37.degree. C. in LB
medium, and periplasm was extracted from cells in a proliferation
phase according to the method of Edwards et al. (C. J. Edwards, D.
J. Innes, D. M. Burns, I. R. Beacham, (1993) FEMS Microbiology
Letters, 114, 293-298). By using the procedure described in the
above reference, 5'-nucleotidase activity of periplasmic proteins
for IMP, GMP and AMP was measured. Activity producing 1 .mu.mol of
phosphoric acid per minute was defined as 1 unit. As a result, the
periplasmic 5'-nucleotidase activity of W.DELTA.ushA was markedly
decreased compared with W3110 as shown in Table 1.
1TABLE 1 Periplasmic 5'-nucleotidase activity (Unit/mg of protein)
Substrate Strain IMP GMP AMP W3110 14.0 10.8 14.2 W.DELTA.ushA 0.21
0.16 0.03
[0062] In order to confirm whether W.DELTA.ushA had completely lost
the nucleotide decomposition ability, its growth was investigated
in a minimal medium containing a nucleotide as a sole carbon
source. W3110 and W.DELTA.ushA were cultured overnight at
37.degree. C. in LB medium, then washed with physiological saline,
added to 50 mL of M9 minimal medium (J. H. Miller, "A SHORT COURSE
IN BACTERIAL GENETICS", Cold Spring Harbor Laboratory Press, New
York, 1992) containing 5.8 g/L of IMP or 6.7 g/L of GMP, and
cultured at 37.degree. C. After a suitable time had passed, the
culture broth was collected and its absorbance at 600 nm was
measured by using a spectrophotometer DU640 (produced by Beckman).
Although the growth of W.DELTA.ushA degraded in M9 medium
containing IMP or GMP as a carbon source, it could grow in such a
medium. This suggested that the nucleotide degradative ability was
not completely lost by the disruption of only ushA. Further, since
the start of growth was retarded, existence of another
5'-nucleotidase was expected, which was induced when UshA did not
function.
EXAMPLE 2
Search of Novel 5'-nucleotidase Gene
[0063] It was considered that the 5'-nucleotidase gene predicted in
Example 1 was more strongly expressed in W.DELTA.ushA compared with
W3110 when they were cultured in M9 medium containing IMP or GMP as
a carbon source. In order to identify the 5'-nucleotidase
considered to function in W.DELTA.ushA, gene expression profiles of
W3110 and W.DELTA.ushA cultured in M9 medium containing IMP or GMP
as a carbon source were compared.
[0064] For comparison of gene expression profiles, the DNA array
method (H. Tao, C. Bausch, C. Richmond, F. R. Blattner, T. Conway,
(1999) Journal of Bacteriology, 181, 6425-6440) was used. Panorama
E. coli Gene Arrays (produced by Sigma Genosis) is a DNA array
composed of a nylon membrane spotted with amplified DNA fragment of
4290 genes of E. coli, and mRNA expression amounts of the total
genes of E. coli can be comprehensively analyzed at once by using
it.
[0065] W3110 and W.DELTA.ushA were cultured in M9 medium containing
IMP or GMP as a sole carbon source, and RNA was extracted from the
cells at a proliferation phase by using RNeasy mini Kit (produced
by Qiagen). The extracted RNA solution was added with MgCl.sub.2
and DNaseI (Boeringer Mannheim) at final concentrations of 10 mM
and 0.25 U/ml, respectively, to decompose contaminated genomic DNA,
and the total RNA were then purified by phenol/chloroform
extraction and ethanol precipitation. A reverse transcription
reaction was performed by using AMV reverse transcriptase (produced
by Promega), dATP, dGTP, dTTP, [.alpha.-.sup.33P]-dCTP (all
produced by Amersham Pharmacia), and random primer pd(N).sub.6
(produced by Amersham Pharmacia) according to the instructions
appended to Panorama E. coli Gene Arrays to prepare a cDNA probe.
The obtained cDNA probe was purified by using ProbeQuant (produced
by Amersham Pharmacia).
[0066] By using the cDNA probe obtained above, hybridization and
washing were performed according to the instruction appended to
Panorama E. coli Gene Arrays. The membrane was enclosed in a
hybridization bag, and brought into contact with an imaging plate
(produced by Fuji Photo Film) for 48 hours, and an image was
captured by using FLA3000G (produced by Fuji Photo Film).
Concentration of each spot was quantified by using image analysis
software, AIS (produced by Imaging Research), and ratio of each
spot concentration with respect to the sum of the total spot
concentrations on the same membrane was represented for every
membrane. Increase and decrease of gene expression was investigated
by comparing values of this ratio for each gene.
[0067] In this way, genes of which expression amount were larger in
W.DELTA.ushA compared with W3110 when they were cultured in M9
medium containing IMP as a carbon source, and genes of which
expression amount were larger in W.DELTA.ushA compared with W3110
when they were cultured in M9 medium containing GMP as a carbon
source were selected, respectively. However, since the change of
the carbon source for the culture might cause variation of
expression amounts of many genes, the number of selected genes was
large, and it was difficult to confirm function of each gene.
Therefore, as means for narrowing down the candidate genes, the
following screening method was employed.
[0068] Since it was considered that the target 51-nucleotidase gene
showed increased expression amount in both of the cultures
utilizing IMP and GMP as the carbon source, a product of a ratio of
expression amounts in W.DELTA.ushA and W3110 (W.DELTA.ushA/W3110)
obtained when they were cultured with IMP as the carbon source and
a ratio of expression amounts in W.DELTA.ushA and W3110
(W.DELTA.ushA/W3110) obtained when they were cultured with GMP as
the carbon source was calculated, and a gene showing a large value
for the product was searched for. The genes that showed larger
values of top 50 are shown in Table 2 (1-25th places) and Table 3
(26-50th places). Among these, genes of which functions were
unknown were selected as candidates that might have the
5'-nucleotidase activity. Since W.DELTA.ushA could grow by
decomposing extracellular nucleotides, it was expected that the
target 5'-nucleotidase should migrate to periplasm and function
therein. Therefore, from those genes of which functions were
unknown, only those having a signal sequence required for
transition of protein to periplasm were selected. By these
screenings, the candidate genes were narrowed down to two kinds,
b0220 (or o157) and yjbp.
[0069] When these genes were investigated, it was found that b0220
was a gene reported as a gene of unidentified function designated
as ykfE, and yjbP was a gene reported as an acid phosphatase gene
(aphA) (M. C. Thaller, S. Schippa, A. Bonci, S. Cresti, G. M.
Rossolini, (1997) FEMS Micorobilogy Letters, 146, 191-198).
2TABLE 2 Gene expression profiles observed in W3110 and
W.DELTA.ushA when they were cultured in M9 medium containing IMP or
GMP as carbon source (1-25th places) IMP expression GMP expression
Ratio (I) ratio (G) I .times. G Gene 11.3 5.5 61.7 pyrE 3.5 7.3
25.2 malE 4.5 2.0 9.1 pyrI 3.6 2.2 8.0 udp 3.9 2.0 7.9 deoD 2.8 2.6
7.2 yeiN 1.9 3.7 7.2 lamB 5.1 1.2 6.0 b0220 (o157) 3.5 1.7 5.9 DeoA
2.1 2.7 5.5 YeiC 2.1 2.6 5.4 tsx 3.0 1.8 5.3 b1036 (o173) 4.2 1.2
4.9 DeoC 2.3 2.1 4.8 NupC 2.4 2.0 4.8 FadB 2.1 2.3 4.8 YejD 1.5 3.2
4.8 MalF 1.9 2.3 4.4 CirA 2.6 1.7 4.3 CarA 1.5 2.9 4.2 LivJ 3.2 1.3
4.0 TalB 0.9 4.5 4.0 FliD 1.5 2.6 4.0 MalM 1.6 2.4 3.9 DppA 1.0 4.0
3.8 FliC
[0070]
3TABLE 3 Gene expression profiles observed in W3110 and
W.DELTA.ushA when they were cultured in M9 medium containing IMP or
GMP as carbon source (26-50th places) IMP expression GMP expression
Ratio (I) Ratio (G) I .times. G Gene 0.8 4.4 3.7 CheA 2.8 1.3 3.7
DeoB 1.3 2.7 3.6 G1pK 2.1 1.7 3.5 b2341 (f714) 1.8 1.8 3.3 YeiK 2.8
1.2 3.3 Cdd 2.0 1.6 3.2 b2673 (o81) 1.8 1.7 3.1 YelP 1.9 1.7 3.1
YeiR 0.9 3.3 3.0 MotB 3.1 1.0 3.0 YafP 2.0 1.5 3.0 b0221 (f826) 1.6
1.8 2.9 yjbP 0.7 4.0 2.9 tap 1.9 1.5 2.9 pyrH 1.5 1.9 2.8 sseA 1.8
1.6 2.8 ybeK 0.8 3.3 2.7 flgN 1.9 1.4 2.7 glnA 2.0 1.3 2.7 ygaD 2.3
1.2 2.7 entE 1.7 1.6 2.6 yafY 1.9 1.4 2.6 nupG 1.8 1.7 2.6 fepA 1.2
2.2 2.6 b3524 (hypothetical)
EXAMPLE 3
Evaluation of Candidate Genes by Gene Amplification
[0071] Strains in which the candidate genes obtained in Example 2,
ykfE and aphA, were each amplified were prepared to investigate the
influence of the gene amplification on the 5'-nucleotidase
activity. The gene fragments of ykfE and aphA were amplified by
using the primers shown in SEQ ID NOS: 3 and 4, and the primers
shown in SEQ ID NOS: 5 and 6, respectively. The ykfE fragment was
cloned into a vector pSTV28 (produced by Takara Shuzo) at a
cleavage site obtained with restriction enzymes SalI and PstI
(produced by Takara Shuzo) to obtain pSTVykfE. Further, the aphA
fragment was cloned into pSTV28 at a cleavage site obtained with
SalI and SphI to obtain pSTVaphA. W.DELTA.ushA was transformed with
each of the plasmids prepared as described above, and cultured at
37.degree. C. in LB medium containing 30 .mu.g/mL of
chloramphenicol. The 5'-nucleotidase activity for IMP, GMP and AMP
as a substrate in periplasm of cells in a proliferation phase was
measured. As a result, the aphA gene amplification provided marked
increase of the 5'-nucleotidase activity compared with a strain
harboring only the vector as shown in Table 4, and thus it was
confirmed that the AphA protein had the activity. On the other
hand, the ykfE-amplified strain did not show significant increase
of the activity, and thus it was determined that it did not have
the 5'-nucleotidase activity.
4TABLE 4 5'-Nucleotidase activity in periplasm of aphA- and
ykfE-amplified strains (U/mg of protein) Substrate Strain IMP GMP
AMP W.DELTA.ushA/pSTV 0.074 0.067 0.024 W.DELTA.ushA/pSTVykfE 0.15
0.15 0.067 W.DELTA.ushA/pSTVaphA 3.2 3.5 1.8
EXAMPLE 4
Introduction of aphA Disruption into W.DELTA.ushA
[0072] Gene disruption was performed in W.DELTA.ushA strain for
aphA, which was expected to be a gene for the 51-nucleotidase
activity. A fragment of the N-terminus region and fragment of the
C-terminus region of aphA were amplified by PCR using the primers
shown in SEQ ID NOS: 3 and 7 and the primers shown in SEQ ID NOS: 4
and 8, respectively, and purified by using Wizard PCR Preps. The
amplification reaction solutions in an amount of 1 .mu.L each were
mixed, added to a PCR reaction solution and subjected to crossover
PCR (A. J. Link, D. Phillips, G. M. Church (1997) Journal of
Bacteriology, 179, 6228-6237) using the primers shown in SEQ ID
NOS: 3 and 4 to obtain an aphA gene fragment including deletion of
its center portion of about 300 nucleotides. This fragment was
inserted into an SalI-SphI cleavage site of temperature sensitive
plasmid pMAN997 to obtain a plasmid pMAN.DELTA.aphA for gene
disruption. By using this plasmid for gene disruption, each aphA of
W3110 and W.DELTA.ushA was disrupted to obtain an aphA-deficient
strain (W.DELTA.aphA) and ushA- and aphA-double deficient strain
(W.DELTA.ushA.DELTA.aphA).
EXAMPLE 5
Measurement of 5'-nucleotidase Activity and Nucleotide Assimilation
Culture of W.DELTA.ushA.DELTA.aphA
[0073] W3110, W.DELTA.ushA, W.DELTA.aphA and
W.DELTA.ushA.DELTA.aphA were each cultured at 37.degree. C. in LB
medium, and 5'-nucleotidase activity in periplasm of cells in a
proliferation phase was measured. The results are shown in Table 5.
Although the activity in W.DELTA.aphA was reduced about by half
compared with W3110, it still strongly remained, and it was
considered that ushA contributed to it. On the other hand, the
5'-nucleotidase activity in the periplasm of
W.DELTA.ushA.DELTA.aphA, which was a double-deficient strain, was
further reduced and substantially eliminated.
5TABLE 5 5'-Nucleotidase activity of W3110, W.DELTA.ushA,
W.DELTA.aphA, and W.DELTA.ushA.DELTA.aphA (U/mg of protein)
Substrate Strain IMP GMP AMP XMP W3110 14.0 10.9 14.2 8.7
W.DELTA.aphA 5.8 4.1 6.0 3.9 W.DELTA.ushA 0.21 0.16 0.03 0.10
W.DELTA.ushA.DELTA.aphA 0.010 0.009 0.012 0.019
[0074] Furthermore, in order to investigate the nucleotide
degradative ability of each strain, these strains were cultured in
M9 medium containing IMP or GMP as a carbon source in flasks. While
growth was observed for W3110, W.DELTA.aphA and W.DELTA.ushA with
both of the carbon sources with growth intensities in that order,
growth was not observed for W.DELTA.ushA.DELTA.aphA even though it
was cultured for 300 hours, and thus it was revealed that it could
not grow in M9 medium containing IMP or GMP as a sole carbon
source. In this way, the ability to decompose extracellular
nucleotide of E. coli W3110 was successfully deleted by double
deficiency of ushA and aphA.
EXAMPLE 6
Gene Disruption for ushA and aphA in Inosine Producing
Bacterium
[0075] In order to investigate the possibility of direct
fermentation of IMP, the gene disruption was performed for ushA and
aphA in an inosine producing strain of Escherichia coli. As the
inosine producing bacterium, FADRaddeddyicPpgixapA (referred to as
"I" hereinafter) described in International Patent Publication
WO99/03988 was used. The mutant purF gene fragment contained in the
plasmid pKFpurFKQ mentioned in w099/03988 was digested with BamHI
and HindIII, then purified and ligated to pMW218 (produced by
Nippon Gene) digested with the same enzymes. The obtained plasmid
pMWpurFKQ was introduced into the I strain. The obtained strain,
I/pMWpurFKQ, became a strain having ability to accumulate about 2-3
g/L of inosine in culture broth.
[0076] The aforementioned strain FADRaddeddyicPpgixapA was a strain
in which PRPP amidotransferase gene (purF), succinyl-AMP synthase
gene (purA), purine nucleoside phosphorylase gene (deoD), purine
repressor gene (purR), adenosine deaminase gene (add),
6-phosphogluconate dehydrase gene (edd), adenine deaminase gene
(yicP), phosphoglucose isomerase gene (pgi) and xanthosine
phosphorylase gene (xapA) were disrupted. Further, pKFpurFKQ
contained a mutant purF coding for PRPP amidotransferase in which
the 326th lysine residue was replaced with a glutamine residue, and
of which feedback inhibition by AMP and GMP was canceled (see
International Patent Publication w099/03988).
[0077] By using the aforementioned plasmid pMAN.DELTA.ushA for ushA
gene disruption and the plasmid pMAN.DELTA.aphA for aphA gene
disruption, a ushA-single deficient strain
(I.DELTA.ushA/pMWpurFKQ), aphA-single deficient strain
(I.DELTA.aphA/pMWpurFKQ) and ushA- and aphA-double deficient strain
(I.DELTA.ushA.DELTA.aphA/pMWpurFKQ) were obtained.
[0078] Each of the aforementioned strains was evaluated for IMP
producing ability. Medium, culture methods and analysis method for
the evaluation of IMP producing ability are shown below.
[0079] [Base Medium: MS Medium]
6 Final concentration Glucose 40 g/L (separately sterilized)
(NH.sub.4).sub.2SO.sub.4 16 g/L KH.sub.2PO.sub.4 1 g/L MgSO.sub.4
7H.sub.2O 1 g/L FeSO.sub.4 7H.sub.2O 0.01 g/L MnSO.sub.4 4H.sub.2O
0.01 g/L Yeast extract 8 g/L CaCO.sub.3 30 g/L (separately
sterilized)
[0080] [Culture Method]
[0081] Refresh culture: stored cells were inoculated, LB agar
medium (added with necessary agents), 37.degree. C., overnight.
[0082] Seed culture: refreshed cells were inoculated, LB broth
(added with necessary agents), 37.degree. C., overnight.
[0083] Main culture: seed culture broth was inoculated in an amount
of 2%, MS medium (added with adenine and other agents as required),
37.degree. C., 20 ml, in 500-ml volume Sakaguchi flask.
[0084] [Analysis Method]
[0085] In an amount of 500 .mu.l of the culture broth was sampled
in a time course, and centrifuged at 15,000 rpm for 5 minutes, and
the supernatant was diluted 4 times with H.sub.2O and analyzed by
HPLC.
[0086] Analysis Conditions:
[0087] Column: Asahipak GS-220 (7.6 mm ID.times.500 mm L)
[0088] Buffer: 0.2 M NaH.sub.4PO.sub.4 (adjusted to pH 3.98 with
phosphoric acid)
[0089] Temperature: 55.degree. C.
[0090] Flow rate: 1.5 ml/min
[0091] Detection: UV 254 nm
[0092] Retention time (min)
7 Inosine 16.40 IMP 11.50 Guanosine 19.67 GMP 13.04
[0093] The results are shown in Table 6. In Table 6, results of two
parallel experiments are indicated, respectively. It was
demonstrated that I.DELTA.ushA.DELTA.aphA accumulated about 1.0 g/L
at most of IMP in the culture broth.
8TABLE 6 Evaluation of ushA- and aphA-deficient strains of inosine
producing bacterium by culture in flask culture time Inosine IMP
Strain (h) (g/L) (g/L) I/pMWpurFKQ 48 2.3 0 48 2.3 0
I.DELTA.ushA/pMWpurFKQ 51 3.1 0 51 2.9 0 I.DELTA.aphA/pMWpurFKQ 51
3.6 0 51 3.2 0 I.DELTA.ushA.DELTA.aphA/pMWpurFKQ 54 2.4 1.0 54 2.6
0.6
EXAMPLE 7
Production of GMP by ushA- and aphA-double Deficient Strain
[0094] In order to examine the possibility of GMP production by the
present invention, guanosine producing ability was imparted to the
ushA- and aphA-double deficient strain obtained in Example 6,
I.DELTA.ushA.DELTA.aphA/pMWpurFKQ. Impartation or enhancement of
guanosine producing ability was attained by enhancing genes of
enzymes catalyzing reactions from IMP to GMP. The reaction
converting IMP to XMP is catalyzed by IMP dehydrogenase encoded by
guaA, and the reaction converting XMP to GMP is catalyzed by GMP
synthetase encoded by guaB, and it is known that these genes
constitute an operon (guaBA) in Escherichia coli. Therefore, PCR
was performed by using the primer shown in SEQ ID NOS: 9 and 10 to
amplify guaBA operon of Escherichia coli. The amplified fragment
was purified, and the restriction enzyme sites formed on the both
ends were digested with SacI and KpnI. The digested fragment was
ligated to pSTV28 similarly digested with SacI and KpnI, and a
plasmid pSTVguaBA into which the guaBA gene was incorporated was
selected. This plasmid can coexist with the plasmid pMWpurFKQ
harbored by I.DELTA.ushA.DELTA.aphA/pMWpurFKQ.
[0095] The aforementioned pSTVguaBA was introduced into the
I.DELTA.ushA.DELTA.aphA/pMWpurFKQ strain to obtain
I.DELTA.ushA.DELTA.aphA/pMWpurFKQ/pSTVguaBA strain. Further, as a
control, I.DELTA.ushA.DELTA.aphA/pMWpurFKQ/pSTV28 strain was
prepared, which was introduced with the vector pSTV28.
[0096] According to the same culture methods and analysis method as
in Example 6, inosine, IMP, guanosine and GMP accumulated in the
culture broth were quantified for the
I.DELTA.ushA.DELTA.aphA/pMWpurFKQ/pSTVguaBA strain and
I.DELTA.ushA.DELTA.aphA/pMWpurFKQ/pSTV28 strain. The results are
shown in Table 7. In the I.DELTA.ushA.DELTA.aphA/pMWpurFKQ/pSTV28
strain used as a control, the culture time was prolonged due to the
influence of the introduction of pSTV28, and it provided a result
different from that of the
I.DELTA.ushA.DELTA.aphA/pMWpurFKQ/pSTVguaBA strain. Guanosine could
not be quantified, since its peaks overlapped with other peaks. On
the other hand, it was demonstrated that the
I.DELTA.ushA.DELTA.aphA/pMWpurFKQ/pSTVguaBA strain accumulated
about 0.1 g/L of GMP in the culture broth thanks to the
introduction of guaBA.
9TABLE 7 Culture of ushA- and aphA-deficient strain of inosine
producing bacteria in flask Culture time Inosine IMP Guanosine GMP
Strain (h) (g/L) (g/L) (g/L) (g/L) I.DELTA.ushA.DELTA.aphA/ 78 9.7
0.4 --* 0.0 pMWpurFKQ/ pSTV28 I.DELTA.ushA.DELTA.aphA/ 78 3.4 0.2
1.1 0.1 PMWpurFKQ/ PSTVguaBA *indicates that quantification was not
possible.
[0097]
Sequence CWU 1
1
10 1 27 DNA Artificial Sequence Synthetic DNA 1 cgcgcatgct
cgtcgctttg ggttttc 27 2 27 DNA Artificial Sequence Synthetic DNA 2
cgcgtcgacc acgatccggc tgaaacc 27 3 27 DNA Artificial Sequence
Synthetic DNA 3 cccgtcgaca ctgctgcgcc ttagctg 27 4 27 DNA
Artificial Sequence Synthetic DNA 4 cccctgcagg cagtattaac gttgatg
27 5 27 DNA Artificial Sequence Synthetic DNA 5 cgcgtcgaca
tcaccattgt agggtag 27 6 27 DNA Artificial Sequence Synthetic DNA 6
cgcgcatgcc agcaagacag cgaaagg 27 7 36 DNA Artificial Sequence
Synthetic DNA 7 gcatatcaat cagctggccg aacaataagc aaacgg 36 8 18 DNA
Artificial Sequence Synthetic DNA 8 gccagctgat tgatatgc 18 9 27 DNA
Artificial Sequence Synthetic DNA 9 cgcgagctca ttcagtcgat agtaacc
27 10 27 DNA Artificial Sequence Synthetic DNA 10 gccggtacct
caatcctata attcttg 27
* * * * *