A Redox-Neutral, Two-Enzyme Cascade for the Production of Malate and Gluconate from Pyruvate and Glucose

A triple mutant of NADP(H)-dependent malate dehydrogenase from thermotolerant Thermococcus kodakarensis has an altered cofactor preference for NAD+, as well as improved malate production compared to wildtype malate dehydrogenase. By combining mutant malate dehydrogenase with glucose dehydrogenase from Sulfolobus solfataricus and NAD+/NADH in a closed reaction environment, gluconate and malate could be produced from pyruvate and glucose. After 3 h, the yield of malate was 15.96 mM. These data demonstrate the feasibility of a closed system capable of cofactor regeneration in the production of platform chemicals.