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
2001-02-26
2002-02-26
Tate, Christopher R. (Department: 1651)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing alpha or beta amino acid or substituted amino acid...
C435S252310, C435S252500
Reexamination Certificate
active
06350596
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention relates to a method for producing L-phenylalanine, in particular, to a bacterium belonging to the genus Methylophilus and having an ability to produce L-phenylalanine and a method for producing L-phenylalanine using a bacterium belonging to the genus Methylophilus.
As a method for producing L-phenylalanine by using a microorganism by fermentation, those using recombinant bacteria belonging to the genus Escherichia (Japanese Patent Application Laid-Open Nos. 56-1890, 57-170184, 58-103398, 61-92565 and 1-104160, and International Publication No. WO 87/00202) are known. As a method for producing L-phenylalanine or L-tyrosine, one using a mutant belonging to the genus Corynebacterium (Japanese Patent Application Laid-Open No. 61-128897), and those using recombinant bacteria belonging to the genus Corynebacterium (Japanese Patent Application Laid-Open Nos. 60-34197, 60-24192, 61-260892, and 61-124375) are known.
However, production of L-phenylalanine by using a bacterium belonging to the genus Methylophilus is not known.
SUMMARY OF THE INVENTION
An object of the present invention is to provide a bacterium having an ability to produce L-phenylalanine and a method for producing L-phenylalanine by using the bacterium.
The present inventors have found that a bacterium belonging to the genus Methylophilus which is resistant to a phenylalanine analog, has a high ability to produce L-phenylalanine. Based on the finding, the present invention has been accomplished.
The present invention provides a bacterium belonging to the genus Methylophilus, which has an ability to produce L-phenylalanine and is resistant to a phenylalanine analog (hereinafter, also referred to as “bacterium of the present invention”).
The bacterium of the present invention is preferably resistant to the phenylalanine analog and L-phenylalanine.
The bacterium of the present invention is preferably resistant to DL-p-fluorophenylalanine.
The bacterium of the present invention is preferably obtained by selecting a strain which is resistant to the phenylalanine analog and L-phenylalanine from bacteria belonging to the genus Methylophilus, wherein said selection is performed at least once for each of the phenylalanine analog and L-phenylalanine in any order.
The bacterium of the present invention is more preferably resistant to DL-p-fluorophenylalanine and m-fluorophenylalanine, as well as to L-phenylalanine.
The bacterium of the present invention is more preferably obtained by selecting a strain which is resistant to DL-p-fluorophenylalanine, m-fluorophenylalanine and L-phenylalanine from bacteria belonging to the genus Methylophilus, wherein said selection is performed at least once for each of DL-p-fluorophenylalanine, m-fluorophenylalanine and L-phenylalanine in any order.
The bacterium of the present invention is preferably resistant to DL-p-fluorophenylalanine, m-fluorophenylalanine and cinnamic acid, as well as L-phenylalanine.
The bacterium of the present invention is more preferably obtained by selecting a strain which is resistant to DL-p-fluorophenylalanine, m-fluorophenylalanine, cinnamic acid and L-phenylalanine from bacteria belonging to the genus Methylophilus, wherein said selection is performed at least once for each of DL-p-fluorophenylalanine, m-fluorophenylalanine, cinnamic acid and L-phenylalanine in any order.
The bacterium of the present invention is preferably
Methylophilus methylotrophus.
The present invention also provides a method for producing L-phenylalanine, comprising the steps of:
culturing the bacterium of the present invention in a culture medium to produce and accumulate L-phenylalanine in a culture, and
recovering L-phenylalanine from the culture (hereinafter, also referred to as “method of the present invention).
DETAILED DESCRIPTION OF THE INVENTION
<1> The Bacterium of the Present Invention
The bacterium of the present invention is a bacterium belonging to the genus Methylophilus, which has an ability to produce L-phenylalanine and is resistant to a phenylalanine analog, and preferably to the phenylalanine analog and L-phenylalanine.
The bacterium belonging to the genus Methylophilus includes
Methylophilus methylotrophus.
The ability to produce L-phenylalanine means an ability to accumulate a significant amount of L-phenylalanine in a medium when the bacterium is cultured in the medium. Usually, it means an ability to accumulate not less than 0.5 g/l of L-phenylalanine under the conditions described in Examples mentioned below.
Examples of the phenylalanine analog are DL-p-fluorophenylalanine, m-fluorophenylalanine, &bgr;-amino-&bgr;-phenylpropionic acid, o-fluorophenylalanine, &bgr;-2-thienylalanine, &bgr;-3-thienylalanine, &bgr;-2-furylalanine, &bgr;-3-furylalanine, o-aminophenylalanine, p-aminophenylalanine, m-aminophenylalanine, &agr;-amino-&bgr;-phenylethanesulfonate, &bgr;-2-pyrrolalanine, 1-cyclopentene-1-alanine, 1-cyclohexene-1-alanine, &bgr;-4-pyridylalanine, &bgr;-4-pyrazolylalanine, p-nitro-phenylalanine, cyclohexylalanine, o-chlorophenylalanine, m-chlorophenylalanine, p-chlorophenylalanine, o-bromophenylalanine, m-bromophenylalanine, p-bromophenylalanine, &bgr;-4-thiazolealanine and the like.
To be resistant to an phenylalanine analog means that the bacterium can grow in the presence of the phenylalanine analog in the amount at which the wild type strain (for example, strain AS-1) can not grow. The amount varies depending on the kinds of the phenylalanine analog. In the case of DL-p-fluorophenylalanine, it is usually 2 g/l under the conditions described in Examples mentioned below, and in the case of m-fluorophenylalanine, it is usually 1 g/l under the conditions described in Examples mentioned below.
It is preferred that the bacterium of the present invention is resistant to at least two kinds of the phenylalanine analog. For example, it is preferred that it is resistant to DL-p-fluorophenylalanine and m-fluorophenylalanine.
To be resistant to L-phenylalanine means that the bacterium can grow in the presence of L-phenylalanine in the amount at which the wild type strain (for example, strain AS-1) can not grow. The amount is usually 8 g/l under the conditions described in Examples mentioned below.
The bacterium of the present invention is preferably resistant to a higher concentration (for example, 0.1 M) of L-phenylalanine.
The bacterium of the present invention can be obtained by conferring the required resistances to a bacterium belonging to the genus Methylophilus in order. The order of conferring the phenylalanine analog resistance and the L-phenylalanine resistance is not limited and the conferring may be made in any order.
The method of obtaining a bacterium belonging to the genus Methylophilus which is resistant to the phenylalanine analog and a bacterium belonging to the genus Methylophilus which is resistant to L-phenylalanine will be explained below.
The bacteria belonging to the genus Methylophilus which is resistant to a phenylalanine analog can be obtained by culturing bacteria belonging to the genus Methylophilus in a minimal medium containing the phenylalanine analog at the growth inhibitory concentration and selecting a growing strain.
The selection of the phenylalanine-analog-resistant strain may be performed with one kind of the phenylalanine analog, or with more kinds of the phenylalanine analogs. The selection of the strain may be performed once or more for one kind of the phenylalanine analog.
An amount of the phenylalanine analog which is to be added to the medium depends on a kind of the phenylalanine analog, but is preferably not less than 2 g/L in the case of DL-p-fluorophenylalanine. The bacteria belonging to the genus Methylophilus may be subjected to a mutation treatment prior to the selection. Mutation may be performed by ultraviolet irradiation or by treatment with a mutagen usually used for artificial mutagenesis such as N-methyl-N′-nitro-N-nitrosoguanidine (NTG) or nitrous acid and the like.
The bacteria belonging to the genus Methylophilus which is resistant to L-phenylalani
Abalakina Elena Georgievna
Iomantas Yurgis Antanas Vladovich
Ajinomoto Co. Inc.
Oblon & Spivak, McClelland, Maier & Neustadt P.C.
Tate Christopher R.
Winston Randall
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