β-法尼烯合酶杂合体的半理性工程改造用于在粘质沙雷氏菌中增强生产

Abstract

This study developed a novel hybrid β-farnesene synthase via semi-rational DNA shuffling and site-directed mutagenesis, utilizing the terpenoid-tolerant chassis Serratia marcescens HBQA7 as the host for β-farnesene biosynthesis. Initially, two wild-type enzymes from Artemisia annua and Matricaria chamomilla var. recutita were selected for domain swapping owing to their superior β-farnesene titers and 92.33% sequence identity. Specifically, hotspots within the optimal chimera CH11 were identified through sequence conservation analysis, molecular docking, and virtual saturation mutagenesis. Subsequently, six candidate residues were identified via alanine scanning, followed by site-saturation mutagenesis to evaluate superior single mutations. These beneficial mutations were further combined to construct double and triple variants. The optimal variant CH11 (K197S/F315L/E490D) yielded 8.5 g/L of β-farnesene in shake-flask fermentation, representing 1.50-fold and 1.66-fold increases relative to the parental enzymes. Molecular dynamics simulations revealed that the enhanced catalytic activity was attributable to improved substrate binding, facilitated by hydrogen bonding and the reinforcement of the hydrophobic network within the active site. In 5-L fed-batch fermentation, the β-farnesene titer produced by the triple mutant reached 63.9 g/L at 96 h. This work provides novel insights into the structure-function relationship of β-farnesene synthase and establishes a robust foundation for future enzyme engineering and industrial-scale β-farnesene biosynthesis. Graphical abstract