Abstract
D-Phenyllactic acid (D-PLA) is a potent antimicrobial typically synthesized through chemical methods. However, due to the complexity and large pollution of these reactions, a simpler and more eco-friendly approach was needed. In this study, a strain for D-PLA biosynthesis was constructed, but the efficiency was restricted by the activity of D-lactate dehydrogenase (DLDH). To address this issue, a DLDH mutant library was constructed and the Surface-Enhanced Raman Spectroscopy (SERS) was employed for the precise quantification of D-PLA at the single-cell level. The TB24 mutant exhibited a significant improvement in D-PLA productivity and a 23.03-fold increase in enzymatic activity, which was attributed to the enhanced hydrogen bonding and increased hydrophobicity within the substrate-binding pocket. By implementing multi-level optimization strategies, including the co-expression of glycerol dehydrogenase (GlyDH) with DLDH, chassis cell replacement, and RBS engineering, a significant increase in D-PLA yields was achieved, reaching 128.4g/L. This study underscores the effectiveness of SERS-based microdroplet high-throughput screening (HTS) in identifying superior mutant enzymes and offers a strategy for large-scale D-PLA biotransformation.
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