GMP synthetase and gene coding for the same

Details GMP synthetase and gene coding for the same GMP synthetase and gene coding for the same

2000-04-14

2001-04-03

Achutamurthy, Ponnathapu (Department: 1652)

Chemistry: molecular biology and microbiology

Micro-organism, per se ; compositions thereof; proces of...

Bacteria or actinomycetales; media therefor

C435S183000, C435S320100, C435S252320, C536S023200

Reexamination Certificate

active

06210951

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a novel GMP synthetase and a DNA that codes for it. A microorganism that has GMP synthetase and its enzymatic activity can be used for the production of nucleic acid compounds such as guanosine 5′-monophosphate, which is useful as a raw material of seasonings and so forth.
2. Description of the Related Art
In the conventional production of nucleic acid compounds such as guanosine 51-monophosphate (also called 5′-guanylic acid, hereinafter abbreviated as “GMP”) and guanosine by fermentation, mutant strains that are imparted with adenine auxotrophy and nucleic acid analogue resistance have been utilized in a medium containing a limited amount of adenine compounds (Japanese Patent Publication (Kokoku) Nos. 55-2956/1980 and 55-45199/1980).
Usual mutagenesis treatments often introduce mutations in genes of resulting mutant strains other than a target gene. In addition, because complicated controlling mechanisms are present in the biosynthetic pathways of nucleic acid compounds, it is difficult to obtain a microorganism that produces a marked amount of a certain nucleic acid compound. Therefore, mutant strains obtained by conventional methods for breeding bacterial strains have not necessarily been satisfactory strains.
GMP is synthesized by an amination reaction catalyzed by GMP synthetase and utilizing L-glutamine as an amino group donor from 5′-inosinic acid (IMP) via xanthylic acid (XMP). Therefore, there has been proposed a method for producing GMP which utilizes
Escherichia coli
transformed with a GMP synthetase gene (guaA) of
Escherichia coli
(Japanese Patent Publication (Kokoku) No. 7-16431/1995). The guaA gene has been isolated from
Bacillus subtilis
(
J. Bacteriol.,
174, 1883-1890 (1992), EMBL/GenBank/DDBJ Accession M83691),
Corynebacterium ammoniagenes
(Korean Patent Publication No. 96-7743, EMBL/GenBank/DDBJ Accession Y10499),
Borrelia burgdorferi
(
J. Bacteriol.,
176, 6427-6432 (1994), EMBL/GenBank/DDBJ Accession L25883),
Dictyostelium discoideum
(
J. Biol. Chem.,
266, 16448-16452 (1991), EMBL/GenBank/DDBJ Accession M64282) and so forth.
However, no guaA gene of
Brevibacterium lactofermentum
(
Corynebacterium glutamicum
) has been known.
SUMMARY OF THE INVENTION
An object of the present invention is to provide a guaA gene of
Brevibacterium lactofermentum
(
Corynebacterium glutamicum
) and a transformant introduced with the gene.
The inventors of the present invention actively studied in order to achieve the aforementioned object. As a result, they found a plurality of regions conserved in amino acid sequences deduced from the known guaA genes, and based on the amino acid sequences of these regions, selected a region and a sequence suitable for amplifying a guaA gene of
Brevibacterium lactofermentum
(
Corynebacterium glutamicum
) by PCR. Then, they successfully isolated a part of the guaA gene by utilizing the selected sequence, and further obtained the guaA gene in its full length. Thus, they accomplished the present invention.
That is, the present invention provides the followings.
(1) A protein defined in the following (A) or (B):
(A) a protein which has the amino acid sequence of SEQ ID NO: 2, or
(B) a protein which has the amino acid sequence of SEQ ID NO: 2 including substitution, deletion, insertion, addition or inversion of one or several amino acids, and has GMP synthetase activity.
(2) A DNA which codes for a protein defined in the following (A) or (B):
(A) a protein which has the amino acid sequence of SEQ ID NO: 2, or
(B) a protein which has the amino acid sequence of SEQ ID NO: 2 including substitution, deletion, insertion, addition or inversion of one or several amino acids, and has GMP synthetase activity.
(3) The DNA according to (2), which is a DNA defined in the following (a) or (b):
(a) a DNA which has the nucleotide sequence comprising at least the nucleotide sequence of the nucleotide numbers 887 to 2455 in SEQ ID NO: 1; or
(b) a DNA which is hybridizable with a nucleotide sequence comprising at least the nucleotide sequence of the nucleotide numbers 887 to 2455 in SEQ ID NO: 1 under a stringent condition, and codes for a protein having GMP synthetase activity.
(4) The DNA according to (3), wherein the stringent condition means a condition that washing is performed at 1×SSC, 0.1% SDS and 60° C.
(5) A transformant which is introduced with the DNA according to any one of (2) to (4) in a form that allows expression of a protein encoded by the DNA.
PREFERRED EMBODIMENTS OF THE INVENTION
The present invention will be explained in detail hereafter.
The DNA of the present invention is a DNA that codes for GMP synthetase derived from
Brevibacterium lactofermentum
(
Corynebacterium glutamicum
).
Brevibacterium lactofermentum
has now been unified into
Corynebacterium glutamicum,
but it may be referred to as
Brevibacterium lactofermentum
in the present specification for convenience.
A part of the DNA of the present invention can be obtained from chromosomal DNA of
Brevibacterium lactofermentum
by PCR. Primers used for the PCR can be designed as follows. That is, an amino acid sequence of a region conserved in GMP synthetases can be retrieved by alignment of amino acid sequences of the GMP synthetases deduced from nucleotide sequences of the guaA genes which have been cloned from several microorganisms, for example,
Escherichia coli, Bacillus subtilis, Corynebacterium ammoniagenes, Borrelia burgdorferi, Dictyostelium discoideum
and so forth. Then, the primers can be designed based on the retrieved conserved region. In this procedure, the PCR primers are designed by selecting such a region that the number of corresponding codons should become as few as possible, and referring to occurring frequency of the codons in
Brevibacterium lactofermentum.
Chromosomal DNA of
B. lactofermentum
can be prepared by, for example, the method of Saito and Miura (
Biochem. Biophys. Acta.,
72, 619, (1963)), and the method of K. S. Kirby (
Biochem. J.,
64, 405, (1956)) or the like.
As preferred primers and PCR condition for amplification of the guaA gene, the combinations of primers and the reaction conditions used for Reactions 2, 3 and 4 shown in Table 1 can be mentioned. By using these reaction conditions, reaction products having sizes of about 0.5 kbp, about 1.2 kbp and about 0.7 kbp, respectively, can be obtained. Among these reaction products, the fragment of about 1.2 kbp overlaps the fragment of about 0.5 kbp.
Based on a partial sequence of the guaA gene obtained as described above, upstream and downstream flanking regions of the partial sequence can be obtained by the cassette-ligation mediated PCR method (
Molecular and Cellular Probes,
6, 467-475) or the like, and thus a full length guaA gene can be obtained. That is, a region flanking a known region on chromosomal DNA can be obtained by ligating a cassette to a chromosomal DNA fragment digested with a suitable restriction enzyme, and amplifying it by PCR utilizing a primer corresponding to the known region and a primer corresponding to the cassette. In this procedure, by using a dephosphorylated 5′ end of-the cassette, a nick is produced at the ligation site of the chromosomal DNA fragment and the 5′ end of the cassette. Therefore, the DNA synthesis started from the cassette primer is stopped at this ligation site, and thus only DNA synthesized from the synthetic primer serves as a template for the synthesis from the cassette primer to form a complementary strand. The nucleotide sequence of the obtained amplification product is determined, then specific primers for the newly determined nucleotide sequence are synthesized, and PCR is performed by using the chromosomal DNA fragment similarly ligated to the cassette as a template. By repeating this procedure, the upstream and downstream flanking regions can be obtained. A kit utilizing this method is commercially available (TAKARA LA PCR in vitro Cloning Kit, Takara Shuzo), and it can be utilized for obtaining th

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