Cara B. Marks

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

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.

A Biocatalytic Approach to Vitamin C Production

Although the primary focus of the biotechnology industry has been on the overproduction of new proteins, primarily for pharmaceutical purposes, there has been growing interest in other fields such as agriculture, diagnostics, and the biocatalytic production of organic chemicals. We have recently been working to develop a novel biosynthetic process for the production of vitamin C (L-ascorbic acid, ASA). This approach has involved the study of the enzymes, coenzymes, and metabolic pathways of different bacteria with the goal of creating new metabolic routes to make a new product (Fig. 6.1). This metabolic pathway engineering approach, which required the identification and characterization of several new enzymes and the cloning and expression of the gene coding for one of these enzymes, has led to a successful one-step bioconversion of D-glucose (G) into 2-keto-L-gulonic acid (2-KLG), a key intermediate in the synthesis of ascorbic acid.