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Science 11 October 1985:
Vol. 230. no. 4722, pp. 144 - 149
DOI: 10.1126/science.230.4722.144
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Articles

Production of 2-Keto-L-Gulonate, an Intermediate in L-Ascorbate Synthesis, by a Genetically Modffied Erwinia herbicola

Stephen Anderson 1, Cara Berman Marks 1, Robert Lazarus 1, Jeffrey Miller 2, Kevin Stafford 3, Jana Seymour 1, David Light 1, William Rastetter 1, and David Estell 2

1 Department of Biocatalysis at Genentech, Inc., 460 Point San Bruno Boulevard, South San Francisco, California 94080
2 Genencor, Inc., 180 Kimball Way, South San Francisco, California 94080.
3 Department of Fermentation Development at Genentech, Inc., 460 Point San Bruno Boulevard, South San Francisco, California 94080

A new metabolic pathway has been created in the microorganism Erwinia herbicola that gives it the ability to produce 2-keto-L-gulonic acid, an important intermediate in the synthesis of L-ascorbic acid. Initially, a Corynebacterium enzyme that could stereoselectively reduce 2,5-diketo-D-gluconic acid to 2-keto-L-gulonic acid was identified and purified. DNA probes based on amino acid sequence information from 2,5-diketo-D-gluconic acid reductase were then used to isolate the gene for this enzyme from a Corynebacterium genomic library. The 2,5-diketo-D-gluconic acid reductase coding region was fused to the Escherichia coli trp promoter and a synthetic ribosome binding site and was then introduced into E. herbicola on a multicopy plasmid. Erwinia herbicola naturally produces 2,5-diketo-D-gluconic acid via glucose oxidation, and when recombinant cells expressing the plasmid-encoded reductase were grown in the presence of glucose, 2-keto-L-gulonic acid was made and released into the culture medium. The data demonstrate the feasibility of creating novel in vivo routes for the synthesis of important specialty chemicals by combining useful metabolic traits from diverse sources in a single organism.

Submitted on June 19, 1985
Accepted on September 13, 1985


THIS ARTICLE HAS BEEN CITED BY OTHER ARTICLES:
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Alteration of the specificity of the cofactor-binding pocket of Corynebacterium 2,5-diketo-D-gluconic acid reductase A.
S. Banta, B. A. Swanson, S. Wu, A. Jarnagin, and S. Anderson (2002)
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DNA from Uncultured Organisms as a Source of 2,5-Diketo-D-Gluconic Acid Reductases.
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S. Khurana, D. B. Powers, S. Anderson, and M. Blaber (1998)
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An unlikely sugar substrate site in the 1.65 A structure of the human aldose reductase holoenzyme implicated in diabetic complications.
D. Wilson, K. Bohren, K. Gabbay, and F. Quiocho (1992)
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Toward a science of metabolic engineering.
J. Bailey (1991)
Science 252, 1668-1675
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