Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Reexamination Certificate
2001-01-18
2002-05-28
Achutamurthy, Ponnathapu (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
C435S252300, C435S320100, C435S440000, C536S023200
Reexamination Certificate
active
06395528
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to the DNA coding for phosphoserine phosphatase of coryneform bacteria. The DNA may be utilized for microbiologic industry, such as breeding L-serine producing coryneform bacteria.
2. Description of the Related Art
As a conventional method of producing L-serine by fermentation, there has been reported the method in which a bacterial strain capable of converting glycine and sugar into L-serine is used in a mediumcontaining 30 g/L of glycine to produce at most 14 g/L of L-serine. The conversion yield amounted to 46% (Kubota, K.,
Agricultural Biological Chemistry,
49, 7-12 (1985)). Using a bacterial strain capable of converting glycine and methanol into L-serine, 53 g/L of L-serine can be produced from 100 g/L of glycine (T. Yoshida et al.,
Journal of Fermentation and Bioengineering,
Vol. 79, No. 2, 181-183, 1995). In the method using Nocardia, it has been known that the L-serine productivity of the bacterium can be improved by breeding those strains resistant to serine hydroxamate, azaserine or the like (Japanese Patent Publication No. 57-1235). However, these methods involve use of glycine that is a precursor of L-serine and include complicated operation and is disadvantageous from the viewpoint of costs.
As strains that can ferment L-serine directly from a sugar and do not need addition of the precursor of L-serine to the medium, there has been known
Corynebacterium glutamicum
that is resistant to D-serine, á-methylserine, o-methylserine, isoserine, serine hydroxamate, and 3-chloroalanine but the accumulation of L-serine is as low as 0.8 g/L (
Nogei Kagakukaishi,
Vol. 48, No. 3, p.201-208, 1974). Accordingly, further strain improvements are needed for direct fermentation of L-serine on an industrial scale.
On the other hand, regarding coryneform bacteria, there have been disclosed a vector plasmid that is capable of autonomous replication in the cell and having a drug resistance marker gene (cf. U.S. Pat. No. 4,514,502) and a method of introducing a gene into the cell (Japanese Patent Application Laid-open No. 2-207791). These techniques have been utilized for breeding L-amino acid producing bacteria. As for L-serine, it has been found that L-serine productivity of coryneform bacteria having the L-serine producing ability is improved by introduction of a gene coding for D-3-phosphoglyceratedehydrogenase whose feedback inhibition by L-serine is desensitized (serA gene) (European Patent Application Laid-Open No. 943687), or amplification of a gene coding for phosphoserine phosphatase (serB) or phosphoserine transaminase (serC) (European Patent Application Laid-Open No. 931833). There has been known serB gene in
Escherichia coli
(GenBank accession X03046, M30784), Yeast (GenBank accession U36473),
Helicobacter pylori
(GenBank accession AF006039), however, serB gene of coryneform bacteria has not been known.
SUMMARY OF THE INVENTION
An object of the present invention, in view of the aforementioned points, is to provide the DNA coding for phosphoserine phosphatase of coryneform bacteria.
The present inventors obtained the DNA fragment from a chromosome DNA library of
Brevibacterium flavum,
which complimented serB deficiency of
Escherichia coli.
The open reading frame having homology with known serB gene of
Escherichia coli
was subcloned from the DNA fragment and introduced into serB deficient mutant strain of aforementioned
Escherichia coli.
However, serB deficiency was not complemented. It was found that serB deficiency was complemented with the aforementioned ORF which was forcedly expressed utilizing lac promoter. Thus, it was confirmed that the aforementioned ORF was serB homologue of
Brevibacterium flavum.
It was indicated that the aforementioned ORF did not have its own promoter because of forming operon.
The present invention was accomplished as described above, and provides the followings.
(1) A protein defined in the following (A) or (B):
(A) A protein which comprises an amino acid sequence of SEQ ID: 2 in Sequence Listing; or
(B) A protein which comprises an amino acid sequence including substitution, deletion, insertion, addition or inversion of one or several amino acids in the amino acid sequence of SEQ ID NO: 2 in Sequence Listing, and which has phosphoserine phosphatase activity.
(2) A DNA coding for a protein as defined in the following (A) or (B):
(A) A protein which comprises an amino acid sequence of SEQ ID: 2 in Sequence Listing; or
(B) A protein which comprises an amino acid sequence including substitution, deletion, insertion, addition or inversion of one or several amino acids in the amino acid sequence of SEQ ID NO: 2 in Sequence Listing, and which has phosphoserine phosphatase activity.
(3) A DNA coding for a protein having phosphoserine phosphatase activity which hybridizes under stringent conditions to a DNA sequence encoding a protien which comprises an amino acid sequence of SEQ ID NO: 2.
(4) The DNA according to (3), the stringent conditions comprise washing at 60° C. and at a salt concentration corresponding to 1×SSC and 0.1% SDS.
(5) The DNA according to (2), which is DNA as defined in the following (a) or (b):
(a) A DNA which comprises a nucleotide sequence corresponding to nucleotide numbers of 210-1547 of nucleotide sequence of SEQ NO: 1 in Sequence Listing; or
(b) A DNA which is hybridizable with a probe which comprises the nucleotide sequence corresponding to nucleotide numbers of 210-1547 of nucleotide sequence of SEQ NO: 13 in Sequence Listing or a partial nucleotide sequence under stringent conditions, and which codes for the protein having the phosphoserine phosphatase activity.
(6) The DNA according to (5), the stringent conditions comprise washing at 60° C. and at a salt concentration corresponding to 1×SSC and 0.1% SDS.
(7) A vector comprising the DNA according to any of (1) to (6).
(8) A bacterial cell in which phosphoserine phosphate activity encoded by the DNA according to any of (1) to (16) is increased.
(9) A method of producing L-serine comprising the steps of cultivating the bacterium according to (8) ina medium to produce and accumulate L-serine in the medium, and collecting L-serine from the medium.
Hereafter, the present invention will be explained in detail.
The DNA of the present invention can be obtained through PCR (polymerase chain reaction) utilizing chromosomal DNA of
Brevibacterium flavum,
for example, the
Brevibacterium flavum
strain ATCC14067, as a template, as well as a primer having the nucleotide sequence of SEQ ID NOs: 3 and 4 shown in sequence listing. Because each of these primers has a restriction enzyme recognition site of EcoRI or SalI in their 5′ sequences, the amplification product digested with these restriction enzymes can be inserted into a vector having EcoRI and SalI digested ends.
The nucleotide sequences of the aforementioned primers were designed based on the nucleotide sequence of the DNA fragment which complements serB deficient mutant strain
Escherichia coli
ME8320 (thi, serB, zhi-1::Tn10) (available from national genetics institute). By using these primers, a DNA fragment containing the coding region of the serB homologue and its flanking region (5′ non-translation region of about 200 bp and 3′ non-translation region of about 300 bp) can be obtained.
The nucleotide sequence of the coding region of the DNA of the present invention obtained as described above and an amino acid sequence which may be encoded by the sequence are shown in SEQ ID NO: 1. The amino acid sequence alone is shown in SEQ ID NO: 2.
The aforementioned serB homologue was found as the open reading frame (ORF) having homology with serB genes of
Escherichia coli
and Yeast (
Saccharomyces cerevisiae
), which existed in the DNA fragment complementing serB deficiency of strain ME8320. The DNA fragment having enough length to contain the ORF and the promoter was obtained from
Brevibacterium flavum
ATCC14067 by PCR utilizing aforementioned primers having the nucleotide sequence of SEQ ID NOs:
Asakura Yoko
Ito Hisao
Suga Mikiko
Sugimoto Masakazu
Achutamurthy Ponnathapu
Ajinomoto Co. Inc.
Pak Yong
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