Engineering Plant Terpene Synthase Pt PS30 for Enhanced Biopesticide Precursor Synthesis: Synergistic Optimization via Homologous Sequence Alignment and Structure-Guided Engineering

Pinene synthase is a crucial biocatalyst for microbial production of (+)-α-pinene, a natural insect repellent and green pesticide enhancer. However, its low catalytic efficiency hinders industrial application. We employed a combination of homologous sequence alignment and structure-guided engineering to identify key mutations (H345F, S372C, and C527S) that significantly enhanced enzymatic activity. The double mutant H345F/S372C increased (+)-α-pinene titer to 273.66 mg/L, 3.38-fold higher than wild-type, with a 3.10-fold improvement in catalytic efficiency. Structural and computational analyses revealed that mutations optimized the substrate channel and reinforced hydrophobic pocket rigidity, improving substrate binding and enzyme stability. This engineered enzyme offers an efficient biosynthetic route for sustainable agrochemical production.