Efficient synthesis of L-malic acid by malic enzyme biocatalysis with CO₂ fixation

The malic enzyme (ME) catalyzes the synthesis of L-malic acid (L-MA) from pyruvic acid and CO₂ with NADH as the reverse reaction of L-MA decarboxylation. Carboxylation requires excess pyruvic acid, limiting its application. In this study, it was determined that CO₂ was the carboxyl donor by parsing the effects of HCO₃^- and CO₂, which provided a basis for improving the L-MA yield. Moreover, the concentration ratio of pyruvic acid to NADH was reduced from 70:1 to 5:1 using CO₂ to inhibit decarboxylation and to introduce the ME mutant A464S with a 2-fold lower K_m than that of the wild type. Finally, carboxylation was coupled with NADH regeneration, resulting in a maximum L-MA yield of 77 % based on the initial concentration of pyruvic acid. Strategic modifications, including optimal reactant ratios and efficient mutant ME, significantly enhanced L-MA synthesis from CO₂, providing a promising approach to the biotransformation process.