Engineering lactate dehydrogenase from Lactiplantibacillus plantarum for artificial cofactors utilization with photocatalytic regeneration.

Lactate dehydrogenase (LDH) plays a key role in microbial fermentation production of d-lactic acid. To achieve efficient in vitro enzymatic pathway, robust synthetic nicotinamide cofactor biomimetics (NCBs) was explored to replace natural cofactor. Here, the cofactor preference of LpLDH, a lactate dehydrogenase from Lactiplantibacillus plantarum was successfully engineered to utilize synthetic NCBs. Based on structural analysis and high-throughput screening, eight variants displaying activity toward NCBs were identified, among which variant P85I could utilize both P2NAH and BNAH. Molecular docking between P2NAH and P85I reveals an additional alkyl-π interaction, and the intermolecular distance was validated by molecular dynamics simulation. Calculation of interaction energies further explains the activity of P85I toward P2NAH and BNAH. Finally, LpLDH was coupled with a photocatalytic cofactor regeneration system for d-lactic acid synthesis, and the conversion exceeded 50 % when utilizing NCBs as cofactors. Under elevated temperatures, NCBs showed better performance than that of NAD. After incubating at 60 °C for 20 h, the group using P2NA achieved 50 % conversion while merely 18.6 % conversion was observed with NAD group in photo-enzyme reaction with cofactor regeneration. This study demonstrates the potential of synthetic NCBs as substitutes for natural cofactors in photo-enzyme coupled process for synthesis of organic acids.