Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing alpha or beta amino acid or substituted amino acid...
Reexamination Certificate
1998-12-30
2001-07-10
Prouty, Rebecca E. (Department: 1652)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing alpha or beta amino acid or substituted amino acid...
C435S252300, C435S252320, C435S190000, C435S320100, C435S106000
Reexamination Certificate
active
06258573
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a method of producing L-serine for use in the production of amino acid mixtures utilized in the field of pharmaceuticals, chemicals, and cosmetics, to coryneform bacteria constituting the method, to D-3-phosphoglycerate dehydrogenase (hereafter, sometimes referred to as “3-PGDH”), and to DNA coding for the 3-PGDH.
BACKGROUND OF THE INVENTION
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 medium containing 30 g/L of glycine to produce at most 14 g/L of L-serine. The conversion yield of glycine into L-serine by this method 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 a bacterium belonging to the genus 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, &agr;-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, p201-208, 1974). Accordingly, a further strain improvements of 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), and the possibility of growing L-threonine or L-isoleucine producing bacteria (U.S. Pat. Nos. 4,452,890 and 4,442,208). Also, regarding the growth of L-lysine producing bacteria, there has been known a technology involving the incorporation of a gene participating in the biosynthesis of L-lysine into a vector plasmid and the amplification of the plasmid in the cell (Japanese Patent Application Laid-open No. 56-160997).
In the case of
Escherichia coli,
the enzymes participating in the biosynthesis of L-serine include an enzyme that is susceptible to feedback inhibition relative to L-serine production in the wild type and an example has been known in which the introduction of a mutant gene that has been mutated so that the feedback inhibition could be desensitized resulted in an enhancement in the L-serine (Japanese Patent No. 2584409). As such genes, there has been known specifically 3-PGDH gene (hereafter, the gene coding for 3-PGDH protein will also be referred to “serA”).
Further, in the case of coryneform bacteria, an example has been known in which the amplification of 3-PGDH gene influences the productivity of L-tryptophane (Japanese Patent Application Laid-open No. 3-7591).
SUMMARY OF THE INVENTION
An object of the present invention is to provide a microorganism that converts a sugar into L-serine and to provide a method of accumulating L-serine in a culture medium utilizing the ability of the microorganism to convert the sugar into L-serine, i.e., a method of producing L-serine that is advantageous in practicing on an industrial scale.
As a result of intensive investigation on the method of producing L-serine with view to achieving the above object, it has now been discovered by the present inventors that screening a coryneform bacterium having L-serine productivity, particularly preferably a mutant strain exhibiting resistance to azaserine or &bgr;-(2-thienyl)-DL-alanine derived from a strain of the coryneform bacterium but is deficient in L-serine decomposing activity as a parent strain and conducting L-serine fermentation using the screened strain will enhance the accumulation of L-serine drastically. The present invention has been completed based on this discovery.
That is, the present invention relates to a coryneform bacterium having resistance to azaserine or &bgr;-(2-thienyl)-DL-alanine and having L-serine productivity.
Further, the present invention relates to D-3-phosphoglycerate dehydrogenase derived from a coryneform bacterium, in which feedback inhibition by L-serine is desensitized; to the D-3-phosphoglycerate dehydrogenase as described above, obtainable from a coryneform bacterium having resistance to azaserine or &bgr;-(2-thienyl)-DL-alanine and having L-serine productivity; to D-3-phosphoglycerate dehydrogenase having an amino acid sequence amino acid sequence depicted in SEQ ID NO: 12 in Sequence Listing or the sequence including substitution, addition or deletion of one or more amino acids, wherein an amino acid residue corresponding to the 325th glutamic acid residue of the amino acid sequence in the SEQ ID NO: 12 is replaced with an amino acid other than glutamic acid; and to the D-3-phosphoglycerate dehydrogenase as described above that has an depicted in SEQ ID NO: 14 in Sequence Listing.
Still further, the present invention relates to a DNA coding for the D-3-phosphoglycerate dehydrogenase described above and to the DNA described above having a base sequence as depicted in SEQ ID NO: 13 in Sequence Listing.
Yet further, the present invention relates to a coryneform bacterium that harbors a recombinant DNA containing the DNA described above.
Further, the present invention relates to a method of producing L-serine, comprising the steps of cultivating the bacterium as described above in a medium to allow accumulation of L-serine in the medium, and collecting the L-serine from the medium.
Specific examples of the coryneform bacterium having resistance to azaserine or &bgr;-(2-thienyl)-DL-alanine and having L-serine productivity include
Brevibacterium flavum
AJ13324 and AJ13327 or
Brevibacterium flavum
AJ13325.
The present invention provides coryneform bacteria that produce L-serine from a sugar. The coryneform bacteria can be utilized in a method of producing L-serine that is industrially advantageous.
REFERENCES:
patent: 5407824 (1995-04-01), Katsumata et al.
patent: 5447857 (1995-09-01), Katsumata et al.
patent: 5618716 (1997-04-01), Burlingame
patent: 5856148 (1999-01-01), Burlingame
patent: 3-7591 (1991-01-01), None
patent: 2584409 (1997-02-01), None
patent: 10248588 (1998-09-01), None
patent: WO 93/12235 (1993-06-01), None
Hibino Wataru
Ito Mika
Nakamatsu Tsuyoshi
Osumi Tsuyoshi
Suga Mikiko
Ajinomoto Co. Inc.
Oblon & Spivak, McClelland, Maier & Neustadt P.C.
Prouty Rebecca E.
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