Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process...
Reexamination Certificate
1999-07-22
2001-07-10
Saidha, Tekchand (Department: 1652)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
C435S015000, C435S066000, C435S193000, C435S252300, C435S320100, C435S840000, C435S843000, C435S848000, C536S023200
Reexamination Certificate
active
06258554
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to a method for the production of useful substances which are synthesized by a fermentation method on the biosynthetic pathway from phosphoribosyl pyrophosphate (referred to as “PRPP” hereinafter) using a metabolically modified strain which is entirely transketolase activity-deficient or which has reduced transketolase activity compared to the parent strain.
BACKGROUND OF THE INVENTION
Examples of useful substances synthesized from PRPP on the microbial metabolic pathway include nucleic acid-related substances such as purine nucleotides, pyrimidine nucleotides, purine nucleosides, pyrimidine nucleosides, purine bases, pyrimidine bases and flavin nucleotides, as well as L-histidine and riboflavin.
These nucleic acid related substances, L-histidine, riboflavin and the like have been produced industrially by a fermentation method or by a combination of a fermentation method with a synthetic method. See Applied Microbiology, edited by Shoichi Takao et al., Buneido Shuppan (1996); Amino Acid Fermentation, edited by Hiroshi Aida et al., Gakkai Shuppan Center (1986); JP-A-6-225776 (“JP-A” as used herein means an “Japanese Published Unexamined Patent Application”).
In microorganisms, PRPP is biologically synthesized by a PRPP synthetase from ribose 5-phosphate which is an intermediate in the pentose phosphate pathway.
As the biosynthetic pathways of ribose 5-phosphate, an oxidative pentose phosphate pathway via glucose 6-phosphate dehydrogenase is known, and it is expected that a non-oxidative pentose phosphate pathway is also involved in its biosynthesis because all of the enzymes on the non-oxidative pentose phosphate pathway via transketolase reaction are reversible (see FIG.
1
).
It has been determined by a study of the metabolism of labeled glucose that both of these pathways are concerned in the biosynthesis of ribose 5-phosphate in
Escherichia coli,
see
Biochemistry,
12, 10, 1969 (1973). Also, it has been shown by genetic studies that ribose 5-phosphate is provided by the non-oxidative pentose phosphate pathway via transketolase reaction, because glucose 6-phosphate dehydrogenase-deficient strains of
Escherichia coli
and
Corynebacterium glutamicum
do not show ribose auxotrophy, see D. G. Fraenkel and R. T. Vinopal,
Ann. Rev. Microbiol.,
27, 69-100 (1973); E. D. Ihnen and A. L. Demain,
J. Bacteriol.,
98, 1151-1158 (1969).
This information suggests that increase in the activity of transketolase enables increased supply of ribose 5-phosphate and PRPP from the non-oxidative pathway and therefore the amount of substances biologically synthesized from PRPP is increased.
In addition, it has been reported that a transketolase activity-deficient strain in inosine-producing strains belonging to the genus Bacillus such as
Bacillus subtilis
accumulates a considerable amount of ribose in the culture medium so that the yield of purine nucleotide production is reduced, see K. Sasajima and M. Yoneda,
Biotechnol. Genet. Eng. Rev.,
2, 175-213 (1984).
This information also suggests that the activity of transketolase is necessary for the production of substances biologically synthesized from PRPP. However, to date, with respect to all microorganisms, it has not been known that the production yield of metabilites to be biologically synthesized via PRPP is improved by having transketolase activity lost or reduced.
Since demands have been increasing for metabolites biologically synthesized via PRPP, particularly nucleic acid-related substances, L-histidine and riboflavin and the like, the development of an industrially advantageous method for their production is strongly desired.
SUMMARY OF THE INVENTION
The object of the present invention is to provide an industrially more advantageous method for the production of metabolites biologically synthesized via PRPP.
In conventional studies on the breeding of microorganisms, great achievement has been made by the enhancement of metabolism of the terminal biosynthetic pathway concerned in the biosynthesis of each metabolite of interest.
The inventors of the present invention have considered that other modification than metabolic enhancement of terminal biosynthetic pathway, namely modification of the central metabolism capable of increasing supply and pooling of the starting substrate, would be necessary for further improvement of the productivity of metabolites, and conducted extensive studies on the influence of carbon flow upon various metabolites by manipulating the central metabolic pathway in microorganisms.
As a result, it was found that the production efficiency of metabolites biologically synthesized via PRPP in the biosynthetic pathway can be improved by making transketolase activity to be deficient or reduced in comparison with that of a parent strain thereof, thus resulting in the accomplishment of the present invention.
Accordingly, it is an object of the present invention to provide:
(1) a method for increasing productivity of metabolites biologically synthesized via phosphoribosyl pyrophosphate on the metabolic pathway in a microorganism, which comprises making transketolase activity in a microorganism belonging to the genus Corynebacterium, Brevibacterium or Escherichia to be deficient or reduced in comparison with that of a parent strain thereof;
(2) a method for producing metabolites, which comprises culturing in a medium a microorganism belonging to the genus Corynebacterium, Brevibacterium or Escherichia in which transketolase activity is deficient or reduced in comparison with a parent strain thereof until one or a plurality of metabolites selected from metabolites biologically synthesized via phosphoribosyl pyrophosphate on the metabolic pathway is formed and accumulated in the culture, and recovering said metabolites therefrom;
(3) the method according to (1) or (2) above, wherein the metabolite is selected from the group consisting of purine nucleotides, pyrimidine nucleotides, purine nucleosides, pyrimidine nucleosides, purine bases, pyrimidine bases, flavin nucleotides, L-histidine and riboflavin;
(4) the method according to (1) or (2) above, wherein said microorganism is selected from the group consisting of
Corynebacterium glutamicum
TKT6,
Corynebacterium glutamicum
TKT6/pPH8,
Corynebacterium glutamicum
TKT6/pFM41 and
Escherichia coil
AI80/pFM201;
(5) a microorganism belonging to the genus Corynebacterium or Brevibacterium, in which transketolase activity is deficient or reduced in comparison with a parent strain thereof; and
(6) the microorganism according to (5) above, wherein said microorganism is selected from the group consisting of
Corynebacterium glutamicum
TKT6,
Corynebacterium glutamicum
TKT6/pPH8 and
Corynebacterium glutamicum
TKT6/pFM41.
REFERENCES:
patent: 5168056 (1992-12-01), Frost
patent: 5589355 (1996-12-01), Koizumi et al.
patent: 19644567 (1998-04-01), None
patent: 600 463 (1994-06-01), None
patent: 98/18937 (1998-05-01), None
Biotechnology & Genetic Engineering Reviews, vol. 2, pp. 175-213 (1984).
J. Bacteriol, vol. 175, No. 17, p. 5375-5383 (1993).
Biochimica et Biophysica Acta, vol. 1216, pp. 307-310 (1993).
Biochemistry, vol. 12, No. 10, pp. 1969-1971 (1973).
J. Bacteriol, vol. 98, No. 3, pp. 1151-1158 (1969).
Chemical Abstracts, vol. 67, No. 9, Aug. 28, 1967, No. 41631, XP-002119105.
Ikeda Masato
Kamada Nozomu
Nakano Tetsuo
Okamoto Kazuyuki
Fitzpatrick ,Cella, Harper & Scinto
Kyowa Hakko Kogyo Co. Ltd.
Saidha Tekchand
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