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
2000-07-21
2004-12-14
Pruity, 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...
C435S115000, C435S116000, C435S252320
Reexamination Certificate
active
06830903
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates, in general, to a method of producing L-amino acids and to a gene encoding phosphoglucoisomerase.
2. Background Information
Bacterial cells are used industrially to produce amino acids by fermentation processes (Ishino, S. et al.,
J. Gen Appl. Microbiol
. 37:157-165 (1991), Kinoshita, S., Nakayama, K. and Nagasaki, S.,
J. Gen. Appl. Microbiol
. 4:128-129 (1958)). Although numerous research reports and reviews have appeared concerning fermentation processes and the mechanisms of accumulation of amino acids, more progress needs to be made to increase the yields of amino acids from microorganisms (Ishino, S. et al.,
J. Gen. Appl. Microbiol
. 37:157-165 (1991), Aida, K. et al., eds., “Biotechnology of Amino Acid Production,” Kodansha (Tokyo)/Elsevier (New York) (1986) and Marx, A. et al.,
Metabolic Engineering
1:35-48 (1999)).
There has been some success in using metabolic engineering to direct the flux of glucose derived carbons toward aromatic amino acid formation (Flores, N. et al.,
Nature Biotechnol
. 14:620-623 (1996)). However, the successful application in producer strains has not yet been documented (Berry, A.,
TIBTECH
14:250-256 (1996)).
Metabolic engineering relates to manipulation of the flow of carbons of starting materials, such as carbohydrates and organic acids, through the variety of metabolic pathways during fermentation. Studies have been done, for example, on the central metabolism of
Corynebacterium glutamicum
using
13
C NMR studies (Ishino, S. et al.,
J. Get Appl. Microbiol
. 37:157-165 (1991), Marx, A. et al.,
Biotechnology and Bioengineering
49:111-129 (1996)). Additionally, also using
13
C NMR, Walker et al. (Walker, T. et al.,
J. Biol. Chem
. 257:1189-1195 (1982)) analyzed glutamic acid fermentation by
Microbacterium ammoniaphilum
, and Inbar et al. (Inbar, L. et al.,
Eur. J. Biochem
. 149:601-607 (1985)) studied lysine fermentation by
Brevibacterium flavum.
The present invention solves a problem of improving yields of amino acids during fermentation using metabolic engineering.
SUMMARY OF THE INVENTION
The present invention provides a method of producing L-amino acids by culturing altered bacteria cells having increased amounts of NADPH as compared to unaltered bacterial cells, whereby L-amino acid yields from said altered bacterial cells are greater than yields from unaltered bacterial cells.
The present invention also provides a method of producing a bacterial cell with a mutated phosphoglucose isomerase (pgi) gene comprising (a) subcloning an internal region of the pgi gene into a suicide vector; and (b) inserting said suicide vector into a bacterial genome, via homologous recombination, whereby a bacterial cell with an altered pgi gene is produced. The invention further provides an altered bacterial cell produced according to this method.
The invention also provides a vector useful according to this method.
The present invention further provides isolated nucleic acid molecules comprising a polynucleotide encoding the
Corynebacterium glutamicum
phosphoglucose isomerase polypeptide having the amino acid sequence shown in
FIG. 1
(SEQ ID NO:2) or one of the amino acid sequence encoded by the DNA clone deposited in a bacterial host as NRRL Deposit Number B-30174 on Aug. 17, 1999.
The present invention also relates to recombinant vectors, which include the isolated nucleic acid molecules of the present invention, and to host cells containing the recombinant vectors, as well as to methods of making such vectors and host cells and for using them for production of Pgi polypeptides or peptides by recombinant techniques.
The invention further provides an isolated Pgi peptide having an amino acid sequence encoded by a polynucleotide described herein.
Further advantages of the present invention will be clear from the description that follows.
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Berry, A., “Improving production of aromatic compounds inEscherichia coliby metabolic engineering,”TIBTECH 14:250-256 (1996).
Flores, W. et al., “Pathway engineering for the production of aromatic compounds inEscherichia coli,” Nature Biotechnol. 14:620-623 (1996).
Inbar, L. et al., “Natural-abundance13C nuclear magnetic resonance studies of regulation and overproduction of L-lysine byBrevibacterium flavum,” Eur. J. Biochem. 149:601-607 (1985).
Ishino, S. et al., “13C Nuclear Magnetic Resonance Studies of Glucose Metabolism in L-Glutamic Acid and L-Lysine Fermentation byCorynebacterium Glutamicum, ” J. Gen. Appl. Microbiol. 37:157-165 (1991).
Kinoshita, S. et al., “L-Lysine Production Using Microbial Auxotroph.”J. Gen. Appl. Microbiol. 4:128-129 (1958).
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Marx, A. et al., “Response of the Central Metabolism inCorynebacterium glutamicumto the use of an NADH-Dependent Glutamate Dehydrogenase,”Metabolic Engineering 1:35-48 (Jan. 1999).
Walker, T.E. et al., “13C Nuclear Magnetic Resonance Studies of the Biosynthesis byMicrobacterium ammoniaphilumof L-Glutamate Selectively Enriched with Carbon-13,”J. Biol. Chem. 257:1189-1195 (1982).
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Dominguez, H. et al., “Carbon-flux distribution in the central metabolic pathways ofCorynebacterium glutamicumduring growth on fructose,”Eur. J. Biochem. 254:96-102 (May 1998).
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O'Gera, J.P. and L.K. Dunican, “Direct Evidence for a Constitutive Internal Promoter in the Tryptophan Operon ofCorynebacterium glutamicum,” Biochem.&Biophys. Res. Comm. 203:820-827 (1994).
Vallino, J.J. and G. Stephanopoulos, “Metabolic Flux Distributions inCorynebacterium glutamicumDuring Growth and Lysine Overproduction,”Biotechnol.&Bioengineering 41:633-646 (1993).
Cocaign-Bousquet, M., and Lindley, N.D., “Pyruvate overflow and carbon flux within the central metabolic pathways ofCorynebacterium glutamicumduring growth on lactate,”Enzyme Microb. Technol. 17:260-267, Elsevier Science Inc. (1995).
Eikmanns, B.J., et al., “Cloning, Sequence Analysis, Expression, and Inactivation of theCorynebacterium glutamicum icdGene Encoding Isocitrate Dehydrogenase and Biochemical Characterization of the Enzyme,”J. Bacteriol. 177:774-782, America Society for Microbiology (1995).
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Boles, E. et al., “The role of the NAD-dependent glutamate dehydrogenase in restoring growth on glucose of aSaccharomyces cerevisiaephophoglucose isomerase mutant,”Eur. J. Biochem. 217:469-477, Federation of European Biochemical Societies (1993).
González Siso, M.I. et al.; “Reoxidation of the NADPH produced by the pentose phosphate pathway is necessary for the utilization of glucose byKluyveromyces lactis rag2mutants,”FEBS Lett. 387:7-10, Federation of European Biochemical Societies (1996).
Moritz, B. et al., “Kinetic propert
Hanke Paul D.
O'Donohue Michael R.
Archer-Daniels-Midland Company
Buchanan & Ingersoll PC
Cochenour Craig G.
Pruity Rebecca E.
Steadman David J
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