Chemistry: molecular biology and microbiology – Micro-organism – per se ; compositions thereof; proces of... – Bacteria or actinomycetales; media therefor
Reexamination Certificate
1991-03-20
2001-07-17
Marx, Irene (Department: 1651)
Chemistry: molecular biology and microbiology
Micro-organism, per se ; compositions thereof; proces of...
Bacteria or actinomycetales; media therefor
C435S832000, C435S106000, C435S115000, C435S441000, C435S446000
Reexamination Certificate
active
06261825
ABSTRACT:
BACKGROUND OF THE INVENTION
This invention relates to production of amino acids using auxotrophic mutants of a methylotrophic Bacillus.
Microorganisms that utilize one-carbon compounds more reduced than carbon dioxide (methylotrophs) are diverse and ubiquitous. Anthony, The Biochemistry of methylotrophs, p 3 (Academic Press, London 1982); Hanson,
Adv. Appl. Microbiol
., 26:3 (1980). Those methylotrophic bacteria reported to utilize methane are all gram-negative and nearly all have an obligate requirement for one-carbon compounds as energy sources (Anthony, supra; Whittenburg et al.
J Gen. Microbiol
. 61: 219-226 (1970)). Bacteria that grow on methanol and methylamines but not methane include several facultative as well as obligate methylotrophs (Anthony, supra; Hanson, supra. All the obligate methylotrophs unable to utilize methane are gram-negative aerobic bacteria (Anthony, supra.; Whittenburg, supra). Of the facultative methylotrophs isolated that utilize methanol, methylamine or both, only a few were gram positive and were assigned to the genera Bacillus, Corynebacterium, Arthrobacter, or Nocardia (Akiba et al,
J. Ferment. Technol
., 48:323-328 (1970); Clement et al.
Abstracts of the Fifth International Symposium Microbiol. Growth on C
1
Compounds
, p. 69 (Free Univ. Press, Amsterdam 1986); Hazen et al,
Arch. Microbiol
., 135: 205-210 (1983); Mimura et al.,
J. Ferment. Technol
., 56: 243-252 (1978).
Production of single cell protein and selected amino acids by microbial fermentation is known, e.g., U.S. Pat. No. 4,652,527 to Stirling. One amino acid which has been produced on an industrial scale is lysine, see Tosaka et al.,
Trends in Biotechnology
, 1: 70-74 (1983), Tosaka and Takinami,
Progress in Industrial Microbiology
, Ch. 24, p. 152-172 (Aida et al., 1986). Bacillus species have been used in fermentation processes to produce amino acids, Tosaka et al., supra.; Tosaka and Takinami, supra. However, to date no production of amino acids using an isolated Bacillus species capable of rapid growth on methanol at temperatures above 50° C. has occurred.
The industrial advantages of a thermophilic methanol utilizing fermentation process at elevated temperatures have been described, Snedecor and Cooney,
Appl. Microbiol
., 27: 112-1117 (1974). For example, use of elevated temperatures can significantly reduce cooling costs. A methanol utilizing, thermophilic mixed culture that included an endosporeforming species was selected by Snedecor and Cooney; however, Snedecor and Cooney, were unable to isolate a pure culture capable of growth on methanol. It is extremely difficult or impossible to isolate appropriate mutants from mixed or impure cultures.
Accordingly, there is a need for a method of producing amino acids using a type I methylotrophic bacterium of the genus Bacillus which exhibits sustained growth at 50° C. in medium having a nitrogen source, vitamin B
12
and methanol as a source of carbon and energy.
SUMMARY OF THE INVENTION
We have discovered a biologically pure strain of a type I methylotrophic bacterium of the genus Bacillus which exhibits sustained growth at 50° C. in nutrient media comprising methanol as a source of carbon and energy, vitamin B
12
and biotin. The bacterium grows at temperatures from about 45° C. to about 55° C. and contains a soluble NAD
+
dependent alcohol dehydrogenase.
We have further discovered that amino acid auxotrophs of the biologically pure strain mentioned above are useful for producing substantial amounts of amino acids. In a preferred embodiment, an amino acid auxotroph of the biologically pure strain type I methylotrophic bacteria of the genus Bacillus produces at least one amino acid when cultured at 50° C. in an aqueous nutrient media having a carbon and energy source, preferably methanol, a nitrogen source, vitamin B
12
, and biotin.
In a further preferred embodiment the bacterium of the present invention is capable of simultaneous production of multiple amino acids useful as animal feeds and animal feed supplements or as nutritional supplements for animal feeds. The amino acid(s) produced according to the present invention can be subsequently separated from the culture media. Preferably, the culture media containing the amino acids can be dried and used directly as a valuable animal feed or animal feed supplement.
A preferred auxotrophic bacterium of the present invention is a mutant of biologically pure strain MGA3 and morphological variants thereof. Most preferably, the amino acid auxotrophs of the present invention are also resistant to amino acid analogues.
A preferred nutrient media for culturing the bacterium of the present invention to produce amino acids includes a carbon and energy source, preferably methanol a nitrogen source, vitamin B
12
, and biotin together with effective amounts of a phosphate source, a sulfate source, a calcium source and trace elements. Amino acid production by auxotrophic bacterium of the present invention is enhanced by automatically feeding the culture media with effective amounts of methanol and trace elements together with required amino acids. lost preferably amino acid production is maximized when cells grow to high cell density by using a continuous culture process including effective amounts of methanol, trace elements and required amino acids. In preferred, semi-continuous (fed batch) or continuous culture methods, production of amino acids is non-growth associated at constant cell density.
We have observed that using the method of the present invention, auxotrophic bacteria of a biologically pure strain of type I methylotrophic Bacillus excrete substantial amounts of lysine. In a preferred embodiment we have observed an amino acid auxotroph excreting from about 3-10 grams/per liter L-lysine. A more preferred auxotrophic mutant for use in production of lysine is a homoserine auxotroph that is resistant to growth inhibition by S-2-aminoethyl-cysteine and analogs of threonine and methionine. A most preferred auxotroph, is a homoserine auxotroph that is resistant to inhibition by S-2-aminoethyl-cysteine and is also a mutant requiring phenylalanine and tyrosine which is resistant to tryptophan, tyrosine and phenylalanine analogs.
The present invention also is directed to a method of obtaining amino acid producing mutants of a biologically pure strain of a type I methylotrophic bacterium of the genus Bacillus involving the steps of isolating a biologically pure strain of a type I methylotrophic bacterium of the genus Bacillus that exhibits sustained growth at 50° C. in an aqueous nutrient media comprising a carbon and energy source, preferably methanol, vitamin B
12
and biotin and treating the isolated bacterium with an amount of mutagenic agent effective to produce a mutant exhibiting increased amino acid production. Amino acid producing mutants are selected based on the ability to grown on media containing one or more desired amino acids or biosynthetic intermediates. In a preferred embodiment, isolated type I methylotrophic Bacillus of the present invention are treated with either or both a chemical mutagen such as ethyl methane sulfonate or N-methyl-N-nitro-N′-nitrosoguanine or an amino acid analog such as S-2-aminoethyl-L-cysteine to increase amino acid production by the bacterium.
Other features and advantages of the invention will be apparent from the following detailed description and appended claims.
REFERENCES:
patent: 4411991 (1983-10-01), Hirakawa et al.
patent: 4652527 (1987-03-01), Stirling
Applied and Environmental Microbiology, vol. 56, No. 4, Apr. 1990, American Society for Microbiology, (Washington, DC, US), F.J. Schendel et al: “L-Lysine production at 50 degrees C. by mutants of a newly isolated and characterized methylotrophic bacillus sp.”, pp. 963-970.
T. Akiba et al.,J. Ferment. Technol., 48, 323-328 (1970).
C. Anthony,The Biochemistry of Methylotrophs, Academic Press, London (1982), p. 3.
R. S. Hanson,Adv. Appl. Microbiol., 26, 3-39 (1980).
A. Mimura et al.,J. Ferment. Technol., 56, 243-252 (1978).
R. Whittenbury et al.,J. Gen. Microbiol., 61,
Flickinger Michael C.
Guettler Michael V.
Hanson Richard S.
Schendel Frederick J.
Marx Irene
Merchant & Gould P.C.
Regents of the University of Minnesota
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