Abstract: A synthetic bioredox system for the oxidative decarboxylation of L-malate to pyruvate was presented, with which the enzyme purification steps can be omitted. The bioredox system consisted of ME-t (mutant malic enzyme ME L310R/Q401C) and non-natural coenzyme nicotinamide flucytosine dinucleotide (NFCD⁺) catalyzes the oxidation of L-malate to pyruvate and concurrently generates the reduced coenzyme, NFCDH. Escherichia coli BL21(DE3) pET24b-ME-t cell lysates catalyze the oxidation of L-malate and produce pyruvate only, indicating that the cell lysates have the same substrate selectivity as the purified enzyme. NADH oxidase (NOX) from Enterococcus faecalis can oxidize NFCDH to regenerate NFCD⁺. Compared with the reaction system containing NAD⁺, ME lysates, and NOX lysates, the system containing NFCD⁺, ME-t lysates, and NOX lysates converts L-malate to pyruvate in 9% higher yield and with significantly less lactate formation. These results suggest that the synthetic bioredox system composed of ME-t and NFCD⁺ is easy to use and has higher product selectivity than the corresponding natural system dependent on NAD⁺. The synthetic bioredox system has the potential to substitute the purified enzyme. The results provide new opportunities for design and application of redox biocatalysts.

Key words: biocatalysis, crude cell lysate, malic enzyme, pyruvate, synthetic bioredox system