The engineering of Corynebacterium 2,5-diketo-D-gluconic acid reductase and modeling its use in in vitro vitamin C biosynthesis

L-Ascorbate, or vitamin C, is the largest specialty chemical produced, with worldwide consumption exceeding 53,000 tons per year. It is a valuable food preservative and a lack of vitamin C in the human diet can cause scurvy. Therefore, considerable effort has been dedicated to improving methods for vitamin C production.; Through the use of metabolic engineering, two novel pathways for vitamin C biosynthesis have recently been described. Both pathways require the use of the 2,5-diketo-D-gluconic acid (2,5-DKG) reductase enzyme, which was cloned from Corynebacterium. This enzyme is a member of the aldo-keto reductase superfamily of proteins, and it is strongly NADPH-dependent. Unfortunately, this cofactor preference may not be optimal in either metabolic pathway. Therefore, a 2,5-DKG reductase enzyme with improved activity with NADH as a cofactor may be a valuable industrial catalyst.; In this work we have taken several approaches to broaden the cofactor specificity of the 2,5-DKG reductase to better use NADH. NADH only differs from NADPH by the lack of a 2′-phosphate group on its adenosine moiety. Site-directed mutations were made at every amino acid side chain in the enzyme that interacts with the 2′-phosphate group. Beneficial mutations were combined with each other and with another pair of successful mutations. The best new mutant enzymes were kinetically characterized and the most active mutant was found to be more active with NADH than the wild type is with NADPH, at high cofactor concentrations.; In order to gain further improvements, homology mutagenesis and combinatorial mutagenesis were also attempted. Both approaches led to improvements in NADH-mediated catalysis, but no mutants were produced that were better than what was obtained through rational protein design. In order to attempt combinatorial mutagenesis, a screening protocol was developed that could easily be extended to improve other aspects of the 2,5-DKG reductase.; Finally, the impact of the new NADH-active 2,5-DKG reductase mutants on vitamin C production was predicted using a mathematical model of the in vitro 2-KLG process. This model was used to demonstrate that the best new mutant 2,5-DKG reductase enzyme could bring a significant cost savings to the in vitro biosynthesis of vitamin C.