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
2000-06-22
2001-10-02
Leguyader, John L. (Department: 1635)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing alpha or beta amino acid or substituted amino acid...
C435S252330, C435S252800, C435S472000
Reexamination Certificate
active
06297031
ABSTRACT:
TECHNICAL FIELD
The present invention relates generally to microbiological industry and more specifically it concerns a novel bacterial strain of
Escherichia coli
BKIIM B-3996 as the producer of L-threonine.
L-threonine is known to be an essential amino acid applicable as the component of diverse nutritive mixtures of medical use. Besides, L-threonine can be used as an additive to animals' fodder, as well as a reagent for the pharmaceutical and chemical industries and as a growth factor for microorganisms producing some other amino acids, such as L-lysine and L-homoserine.
BACKGROUND ART
Known in the present state of the art are the L-threonine producing strains of microorganisms of a variety of species (e.g.,
Brevibacterium flavum, Serratia mercescens, Escherichia coli
, and others). It is the mutating strains of
E. coli
whose cells contain hybrid plasmids carrying the genes of the threonine operon (U.S. Pat. Nos. 4,278,785; 4,321,325) that prove to be the most efficacious L-threonine producers, of which the most productive is
Escherichia coli
strain VNIIgenetika M-1 (U.S. Pat. No. 4,321,325), which contains multicopy plasmid pYN7 obtained on the base of vector pBR322 and incorporating the threonine operon of
E. coli
strain K12 resistant to alpha-amino-beta-hydroxyvaleric acid, an analogue of threonine. The genes of the threonine operon of plasmid pYN7 code a bifunctional enzyme, viz., aspartate-kinase-homoserinedehydrogenase, which is insensitive to inhibition with L-threonine. Said strain M-1 is capable of accumulating L-threonine till a concentration of 30 g/l for a 40-hour fermentation period in laboratory fermenters when cultivated under conditions of feeding a sugar-ammonia additive to the nutrient medium in response to a signal sent by the pH sensor.
The aforesaid strain is featured by low productivity and inadequate stability of the plasmid, which compels one to make use of antibiotics to retain the plasmid in course of fermentation.
SUMMARY OF THE INVENTION
The strain proposed herein is a novel one and has not so far been described in literature.
It is therefore a primary and essential object of the present invention to provide a novel bacterial strain, which enables one to attain a high yield of L-threonine obtained within a shorter period of fermentation without adding any antibiotics during said period and featuring a high stability of the plasmid.
The aforesaid object is accomplished due to the fact that, according to the invention, proposed herein is a novel bacterial strain of
Escherichia coli
BKIIM B-3996 as the L-threonine producer, said strain containing recombinant plasmid pVIC40 and deposited on Nov. 19, 1987 in the collection of microorganism cultures at the USSR Antibiotics Research Institute under Reg. No. 1867.
The strain proposed herein is instrumental in producing 85 g/l L-threonine for a 36-hour fermentation period. The proposed strain contains recombinant plasmid pVIC40, which carries the same fragment of the
E. coli
chromosome as the pYN7 plasmid of the heretofore-known
E. coli
strain VNIIgenetika M-1 that codes the genes of L-threonine biosynthesis, and imparts to the cells resistance to an antibiotic streptomycin. Unlike the pYN7 the novel plasmid remains persistently in the cells when growing under nonselective conditions.
REFERENCES:
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Astaurova et al,Applied Biochemistry and Microbiology,vol. 21(5), pp. 485-490 (1986).
Chistoserdov et al,Plasmid,vol. 16, pp. 161-167 (1986).
Boylan et al,Biochemical and Biophysical Research Communications,vol. 85(1), pp. 190-197 (1978).
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Rex et al,Journal of Bacteriology,vol. 173, pp. 5944-5953 (1991).
Boylan et al,The Journal of Biological Chemistry,vol. 256(4), pp. 1809-1815 (1981).
V. Debabov in Proceedings of the IVthInternational Symposium on Genetics of Industrial Microorganisms, Y. Ikeda and T. Beppu, Eds., Tokyo, Japan, pp. 254-258 (1982).
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Arsatiants Raisa Alexandrovna
Bachina Tatyana Alexandrovna
Belareva Alla Valentinovna
Christoserdov Andrei Jurievich
Debabov Vladimir Georgievich
Ajinomoto Co. Inc.
Larson Thomas G.
LeGuyader John L.
Oblon & Spivak, McClelland, Maier & Neustadt P.C.
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