Combinatorial engineering of enzyme and pathway for efficient β-farnesene bioproduction in Yarrowia lipolytica

β-farnesene, a natural sesquiterpene compound, has gained significant attention due to its versatile applications in agriculture, industry, biofuels, and related fields. Microbial biosynthesis offers an environmentally sustainable approach for its commercial-scale production. In order to enhance its production efficiency, further exploration of key rate-limiting steps is required. Here, through directed evolution of the essential β-farnesene synthase, we obtained an optimal variant (AaFS^{T196A/M356T/E380G}), demonstrating 2.29-fold enhancement in β-farnesene titer relative to wild-type. Structural elucidation revealed that the distal mutations mediate allosteric modulation of the catalytic core significantly improving the conversion efficiency of farnesyl diphosphate (FPP) to β-farnesene. Then comprehensive pathway engineering, including the mevalonate pathway amplification, acetyl-CoA precursor enhancement, competitive pathway elimination, and auxotrophic restoration, were carried out in Yarrowia lipolytica, resulting in a high-performance strain FS18 capable of producing 3.41 g/L β-farnesene in shake-flask cultures. Notably, scale up fermentation in 5 L bioreactors yielded a titer of 45.69 g/L, the highest concentration reported in Y. lipolytica to date. This study provided mechanistic insights into terpene synthase engineering and a practical framework for high-level terpenoid biosynthesis in Y. lipolytica.