Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing alpha or beta amino acid or substituted amino acid...
Patent
1994-05-24
1997-04-08
Hendricks, Keith D.
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing alpha or beta amino acid or substituted amino acid...
4352523, 4353201, 435190, 435325, 435419, 435108, 935 10, 935 14, 536 232, C12N 904, C12N 1553
Patent
active
056187165
DESCRIPTION:
BRIEF SUMMARY
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to the general field of biosynthesis of serine and products related to serine, particularly tryptophan, and to methods and materials used in that biosynthesis.
2. The Prior Art
Serine is a primary intermediate in the biosynthesis of a wide variety of cellular metabolites including such economically important compounds as choline, glycine, cysteine and tryptophan. In addition, serine acts as a single carbon donor and is responsible for 60% to 75% of the total need of the cell for C.sub.1 units through the production of 5,10-methylenetetrahydrofolate from tetrahydrofolate. These C.sub.1 units are used in a wide variety of biosynthetic pathways including the synthesis of methionine, inosine monophosphate, other purines and some pyrimidines (e.g., thymidine and hydroxymethyl cytidine).
The serine biosynthetic pathway shown in FIG. 1 is generally available to a wide variety of tissues and microorganisms. The first committed step in that pathway is the conversion of 3-phospho-D-glyceric acid (PGA) to 3-phosphohydroxypyruvic acid (PHA) by means of the enzyme 3-phosphoglycerate dehydrogenase (PGD). The gene encoding PGD has been cloned and sequenced, and the amino acid sequence of the PGD subunit has been deduced. Tobey and Grant, J. Biol. Chem., 261:12179-12183 (1980).
In procaryotes (particularly bacteria) and microorganisms such as yeast, but not in higher eukaryotes, activity of wild-type PG is inhibited by cellular serine levels. This inhibition has been studied kinetically and reportedly proceeds in an allosteric manner. Tobey and Grant, J. Biol. Chem., 261:12179-12183 (1986); Dubrow and Pizer, J. Biol. Chem., 252:1527-1551 (1977); McKitrick and Pizer, J. Bacteriol., 141:235-245 (1980).
Tosa and Pizer, J. Bacteriol., 106:972-982 (1971), studied the effect of a normally toxic serine analog, L-serine hydroxamate, on an E. coli strain. Selection on a growth medium containing that analog yielded serine-resistant mutants. Some mutants were shown to have a modification in an enzyme unrelated to PGD, seryl-tRNA synthetase. Crude extract of one mutant showed PGD activity with reduced serine sensitivity (See, J. Bacteriol., 106:972-982 [1971]; FIG. 5; Table 6; and see p. 973 bottom left col., p. 977 bottom left col.).
SUMMARY OF THE INVENTION
One aspect of the invention generally features DNA encoding 3-phosphoglycerate dehydrogenase (PGD) with reduced sensitivity to inhibition by serine in comparison to wild-type PGD, i.e., the DNA encodes PGD which has at least some level of enzymatic activity useful for biosynthesis, and which retains that activity at higher serine levels than does the (unmodified) wild-type PGD.
In preferred embodiments, the wild-type PGD is microbial or yeast PGD. Also preferably, the engineered DNA encodes PGD which comprises an alteration in the C-terminal 25% of wild-type PGD, preferably in the C-terminal 50 amino acids. For example, the engineered DNA may encode PGD comprising a deletion in part of all of the C-terminus. Also preferably, the engineered DNA encodes PGD having an insertion in the C-terminus (e.g., between VAL 363 and ASN 364, or between ALA 392 and GLN 394) in addition to the deletion described above, or as a separate alteration.
The present invention is also directed to: a) PGD having the amino acid sequence of the above-described engineered DNA; b) expression vectors comprising the engineered DNA and regulatory DNA positioned and oriented for expression of the engineered DNA in a host expression system; c) cells comprising such expression vectors; and d) methods for producing serine or a serine-derived product by culturing such cells. As to c) above, the cell preferably is deleted for wild-type serA.
Yet another embodiment generally features a cell engineered (e.g., it includes a recombinant genetic construction) to produce a PGD-encoding mRNA transcript with an altered 3' end, which transcript is translated by the cell to yield PGD with reduced sensitivity to inhibition by serine in comparison
REFERENCES:
patent: 4743546 (1988-05-01), Backman et al.
patent: 4753883 (1988-06-01), Backman et al.
The Journal of Biological Chemistry, 1986 by the American Society of Biological Chemists, Inc., vol. 261, No. 26, pp. 12179-12183, Karen L. Tobey and Gregory A. Grant "The Nucleotide Sequence of the serA Gene of Escherichia coli and the Amino Acid Sequence of the Encoded Protein, D-3-Phosphoglycerate Dehydrogenase".
The Journal of Biological Chemistry, vol. 252, No. 8, pp. 1527-1551, 1977, Robert Dubrow and Lewis I. Pizer, "Transient Kinetic Studies on the Allosteric Transition of Phosphoglycerate Dehydrogenase".
Journal of Bacteriology, Jan. 1980, pp. 235-245, vol. 141, No. 1, John C. McKitrick and Lewis I. Pizer "Regulation of Phosphoglycerate Dehydrogenase Levels and Effect on Serine Synthesis in Escherichia coli K-12".
Journal of Bacteriology, Jun. 1971, pp. 972-982, vol. 106, No. 3, Tetsuya Tosa and Lewis I. Pizer, "Biochemical Bases for the Antimetabolite Action of L-Serine Hydroxamate".
T. M. Roberts et al., Gene 12 (1980), 123-127, Elsevier/North-Holland Biomedical Press "A plasmid cloning vehicle allowing a positive selection for inserted fragments".
Experiments in Molecular Genetics, Cold Spring Harbor Press, pp. 201-205, Miller (1972), "Generalized Transduction; Use of P1 in Strain Construction".
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Shevell et al. (1988), J. Bacteriol. 170, pp. 3294-3296, vol. 170, No. 7 "Construction of an Escherichia coli K-12 ada Deletion by Gene Replacement in a recD Strain Reveals a Second Methyltransferase That Repairs Alkylated DNA".
Experiments in Mol. Genetics, Cold Spring Harbor Lab., pp. 201-205 (1972), "Generalized Transduction; Use of P1 in Strain Construction".
Herrmann and Somerville (1983) (Amino Acids: Biosynthesis and Genetic Regulation).
Crueger and Crueger (1982) (Biotechnology: A Textbook of Industrial Microbiology).
Journal of Biological Chemistry, vol. 264, No. 5, 15 Feb. 1989, pp. 2645-2648, Baltimore, US, D.J. Schuller et al.: "Enhanced expression of the Escherichia coli serA gene in plasmid vector. Purification, crystallization, and preliminary X-ray data of D-3 phosphoglycerate dehydrogenase".
Bioscience Reports, vol. 1, No. 9, 1981, pp. 733-741, London, GB, G.A. Grant et al.: "D-3-phosphoglycerate dehydrogenase from Escherichia coli: Isolation by affinity chromatography and sequence comparison to other dehydrogenases".
Journal of Bacteriology, vol. 173, No. 5, Mar. 1991, p. 1571, Baltimore, US, K. O'Day et al.: "Physical location of bg1A and serA on the Escherichia coli K-12 chromosome".
Journal of Molecular Biology, vol. 186, No. 4, 20 Dec. 1985, pp. 707-713, London, GB, R. Cunin et al.: "Structure-function relationship in allosteric aspartate carbamoyltransferase from Escherichia coli. I. Primary structure of a pyrI gene encoding a modified regulatory subunit".
Hendricks Keith D.
Wacker-Chemie GmbH
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