Gene encoding phosphoglucoisomerase

Details Gene encoding phosphoglucoisomerase Gene encoding phosphoglucoisomerase

2000-07-21

2002-10-15

Patterson, Jr., Charles L. (Department: 1652)

Chemistry: molecular biology and microbiology

Enzyme , proenzyme; compositions thereof; process for...

Isomerase

C435S320100, C435S252300, C536S023200

Reexamination Certificate

active

06465238

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 process 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. Micorobiol.
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. Gen. 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 bacterial 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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Flores, N. 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).
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Marx, A. et al., “Determenation of the Fluxes in the Central Metabolism ofCorynebacterium glutamicumby Nuclear Magnetic Resonance Spectroscopy Combined with Metabolite Balancing,”Biotechnol. Bioengineering49:111-129 (1996).
Marx, A. et al., “Response of the Central Metabolism inCorynebacterium glutamicumto the use of an NADH-Dependent Glutamate Dehydrogenase,”Metabolic Engineering1:35-48 (Jan. 1999).
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Swiss-Prot Accession No. P77895, Swiss Prot ID No. G6PI_MYCTU (Jul. 15, 1998).
Dominguez, H. et a

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