Hu Lin, Luo Ruoshi, Wang Dan, Lin Fanzhen, Xiao Kaixing, Kang Yaqi
Department of Chemical Engineering, School of Chemistry and Chemical Engineering, Chongqing University, Chongqing, China.
Front Bioeng Biotechnol. 2024 Sep 20;12:1470830. doi: 10.3389/fbioe.2024.1470830. eCollection 2024.
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 128.4 g/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.
D-苯乳酸（D-PLA）是一种通常通过化学方法合成的强效抗菌剂。然而，由于这些反应的复杂性和大量污染，需要一种更简单、更环保的方法。在本研究中，构建了一种用于D-PLA生物合成的菌株，但效率受到D-乳酸脱氢酶（DLDH）活性的限制。为了解决这个问题，构建了一个DLDH突变体文库，并采用表面增强拉曼光谱（SERS）在单细胞水平上对D-PLA进行精确量化。TB24突变体在D-PLA生产力方面有显著提高，酶活性提高了23.03倍，这归因于底物结合口袋内氢键增强和疏水性增加。通过实施多级优化策略，包括甘油脱氢酶（GlyDH）与DLDH共表达、底盘细胞替换和核糖体结合位点（RBS）工程，D-PLA产量显著提高，达到128.4 g/L。本研究强调了基于SERS的微滴高通量筛选（HTS）在鉴定优良突变酶方面的有效性，并为大规模D-PLA生物转化提供了一种策略。
