High-Efficiency Synthesis of β-Caryophyllene in <i>Serratia marcescens</i> via an Aeropyrum-Type MVA Pathway and Enzyme Engineering

β-caryophyllene is a high-value natural bicyclic sesquiterpenoid compound. In this study, a highly efficient biosynthetic system for β-caryophyllene was first constructed using the terpene-tolerant <i>Serratia marcescens</i> as the chassis. By introducing the energy-efficient Aeropyrum-type mevalonate (MVA) pathway and rationally designing the active pocket of the β-caryophyllene synthase <i>Z</i>pTPS1 (Y25F-N535H double mutant), the affinity for the substrate farnesyl pyrophosphate (FPP) was significantly enhanced. Further fusion expression of FPP synthase and the mutant <i>Z</i>pTPS1 mediated by a flexible linker resulted in a shake flask fermentation yield of 5.54 g/L after 48 h. In a 5 L bioreactor, by employing a staged oxygen-limited fed-batch fermentation strategy, the maximum average yield of β-caryophyllene reached 60.32 g/L after 96 h of cultivation, representing the highest β-caryophyllene titer reported to date.