Engineered L-phenylserine aldolase enhances L-norvaline synthesis within an enzyme cascade

L-Norvaline is a crucial intermediate in the synthesis of antihypertensive agents, and its production via biotechnological methods has garnered significant interest and commercial value in recent years. Here, for the enzymatic cascade synthesis of L-norvaline from propionaldehyde and glycine, an L-phenylserine aldolase gene (designated as ppLPA) from Pseudomonas putida was selected. Following the identification of potential mutation sites via error-prone PCR, coupled with site-directed mutagenesis, a single-site mutant, I18T, was identified, exhibiting a 1.4-fold increase in enzyme activity. Then, the ppLPA mutant I18T was combined with L-threonine deaminase, L-leucine dehydrogenase, and alcohol dehydrogenase to construct a one-pot, multi-enzyme cascade catalytic system for L-norvaline synthesis. The reaction conditions were systematically optimized. To mitigate the inhibitory effects of propionaldehyde, we employed a pH-stat substrate feeding strategy. Under optimal reaction conditions, L-norvaline production achieved a maximum yield of 116.5 g/L after 14 h of reaction, with a conversion rate exceeding 99 % in a 1 L reaction volume. This study highlights significant advancements in improving L-norvaline production, providing potential for more efficient biomanufacturing processes and broader industrial applications.