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
2000-06-30
2002-09-17
Yucel, Remy (Department: 1636)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing alpha or beta amino acid or substituted amino acid...
C435S252320, C435S254100, C435S254110, C435S419000, C800S295000
Reexamination Certificate
active
06451564
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates, in general, to the field of production of amino acids. More specifically, the present invention relates to the over-production of isoleucine by nonhuman organisms.
2. Related Art
Corynebacteria have a long history of use in the industrial production of amino acids, which are used as food additives (most notably lysine and other essential amino acids) and as flavor enhancers (monosodium glutamate, or MSG). The overall global market for amino acids as animal feed additives is estimated to be worth more than $2 billion and totals about 700,000 metric tons of material. Lysine and methionine account for an overwhelming majority of the market, which also includes lower-volume products like threonine (Lessard, P. A., et al., “Corynebacteria, Brevibacteria,” in
The Encyclopedia of Bioprocess Technology: Fermentation, Biocatalysis and Bioseparation
, John Wiley & Sons, New York, N.Y. (April 1999), Volume 2, pp. 729-740). The current production of isoleucine is less than 400 metric tons per year. This amino acid is currently used as a constituent of infusions and special dietary products. As with other amino acids the demand for isoleucine is increasing and its industrial production is expected to open up additional markets as an animal feed additive. Due to the tight control of isoleucine biosynthesis in bacteria, this amino acid is still in part produced commercially by direct extraction from protein hydrolysates.
The Gram positive
Corynebacterium glutamicum
is currently used in industry to produce over 100 grams of lysine per liter of culture. The flux of carbon through metabolic pathways can be diverted from the production of lysine to make other related amino acids by the processes and methods of metabolic engineering. Traditional metabolic manipulation involved random mutagenesis and screening for the desired changes in physiology, but transformation and genetic manipulation tools have been developed in the last ten years to allow more direct engineering of specific pathway elements in Corynebacterium (Jetten, M. S. M., et al., “Molecular organization and regulation of the biosynthetic pathway for aspartate-derived amino acids in
Corynebacterium glutamicum
,” in
Industrial microorganisms: basic and applied molecular genetics
, Baltz, R. H., et al., eds., Am. Soc. Microbiol., Washington, D.C. (1993), pp. 97-104; Lessard, P. A., et al., “Corynebacteria, Brevibacteria,” in
The Encyclopedia of Bioprocess Technology: Fermentation, Biocatalysis and Bioseparation
, John Wiley & Sons, New York, N.Y. (April 1999), Volume 2, pp. 729-740).
L-Isoleucine belongs to the aspartate-derived family of amino acids, as do lysine, homoserine, threonine and methionine. The enzymes that synthesize this family of amino acids have been well characterized in Corynebacterium, as has their regulation (FIG.
1
). The first important regulatory point in the production of isoleucine by
C. glutamicum
is the end-product inhibition of the first committed enzyme, threonine dehydratase (E.C. 4.2.1.16), encoded by the gene ilvA. Overproduction of isoleucine has been accomplished by introducing excess threonine dehydratase (encoded by ilvA) into threonine producer strains (Colón, G. E., et al.,
Appl. Microbiol. Biotechnol
. 43:482-488 (1995)). Threonine dehydratase is normally feedback inhibited by isoleucine. Mutant derivatives of threonine dehydratase with reduced sensitivity to isoleucine provided an additional dividend in this isoleucine production system (Hashiguchi, K., et al.,
Biosci. Biotechnol. Biochem
. 61:105-108 (1997); Morbach, S. et al.,
Appl. Env. Microbiol
. 61:4315-4320 (1995)). Despite these gains, it appears that amino acid export has been a serious limitation to the effectiveness of amino acid production (Kelle, R., et al.,
J. Biotechnol
. 50:123-136 (1996)).
Work on artificial enzyme evolution has shown that it is difficult to subtly alter a task for which an enzyme was specifically evolved, while it is easier to coopt an enzyme for a completely new task (Benner, S. A., Chem. Rev. 89:789-806 (1989)).
One such alternative enzyme might be the catabolic threonine dehydratase, also called biodegradative threonine dehydratase, (E.C. 4.2.1.16), which is also known as threonine deaminase. This threonine dehydratase is produced in
E. coli
cells when the organism is grown anaerobically in a medium containing high concentrations of amino acids and no glucose (Umbarger, H. E. & Brown, B.,
J. Bacteriol
73:105-112 (1957)). In contrast to the threonine dehydratase encoded by ilvA, an anabolic threonine dehydratase, the enzyme encoded by tdcB in
E. coli
is insensitive to inhibition by L-isoleucine and is activated by adenosine 5′-monophosphate. The tdcB gene from
E.coli
has already been cloned and sequenced (Goss, T. J. & Datta, P.,
Mol. Gen. Genet
. 201:308-314 (1985)).
In the past, overproduction of isoleucine has been accomplished by introduced excess threonine dehydratase encoded by ilvA, an anabolic threonine dehydratase, into threonine producer strains (colón, G. E., et al.,
Appl. Microbiol. Biotechnol
. 43:482-488 (1995)). Although the conventional methods have considerably enhanced the production of isoleucine, the development of a more efficient, cost-effective technique is required in order to meet increasing demand for L-isoleucine.
BRIEF SUMMARY OF THE INVENTION
It is a general object of the invention to provide a method for over-producing L-isoleucine.
It is a specific object of the invention to provide a method for producing L-isoleucine comprising growing a transformed nonhuman organism under conditions that provide for synthesis of L-isoleucine, wherein said nonhuman organism comprises one or more copies of a transgene comprising at least one nucleotide sequence encoding catabolic threonine dehydratase, wherein said L-isoleucine is synthesized by said transformed nonhuman organism, said synthesis being greater than that of the corresponding non-transformed nonhuman organism, and recovering said L-isoleucine from said culture medium in which said transformed nonhuman organism was cultured.
It is a specific object of the invention to provide a method for producing L-isoleucine comprising growing an L-isoleucine producing nonhuman organism comprising one or more copies of a transgene comprising at least one nucleotide sequence encoding catabolic threonine dehydratase, wherein said nonhuman organism synthesizes L-isoleucine, said synthesis being greater than that of a corresponding nonhuman organism which does not comprise one or more copies of a transgene comprising at least one nucleotide sequence encoding catabolic threonine dehydratase, and recovering said L-isoleucine from said culture media in which said L-isoleucine producing nonhuman organism was cultured.
It is another specific object of the invention to provide a transformed non-human organism, wherein said transformed nonhuman organism is an L-isoleucine producing nonhuman organism, comprising one or more copies of a transgene comprising at least one nucleotide sequence encoding catabolic threonine dehydratase, wherein said transformed nonhuman organism synthesizes L-isoleucine, said synthesis being greater than that of the corresponding non-transformed nonhuman organism.
It is another object of the invention to provide an L-isoleucine producing nonhuman organism comprising one or more copies of a transgene comprising at least one nucleotide sequence encoding catabolic threonine dehydratase, wherein said nonhuman organism synthesizes L-isoleucine, said synthesis being greater than that of a corresponding nonhuman organism which does not comprise one or more copies of a transgene comprising at least one nucleotide sequence encoding catabolic threonine dehydratase.
It is another specific object of the invention to provide a transformed organism,
Corynebacterium glutamicum
ATCC21799 comprising pAPE7, ATCC deposit no. PTA-981. It is another specific object of the invention to provide a transformed organism,
Corynebacterium gluta
Guillouet Stephane
Lessard Philip A.
Rodal Avital A.
Sinskey Anthony J.
Loeb Bronwen M.
Massachusetts Institute of Technology
Sterne Kessler Goldstein and Fox P.L.L.C.
Yucel Remy
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