Fine-Tuning the Function of Farnesene Synthases for Selective Synthesis of Farnesene Stereoisomers

Farnesene synthase from Artemisia annua (AaFS) catalyzes the reaction from farnesyl pyrophosphate (FPP) to give the sesquiterpene beta-farnesene, a key building block for the biosynthesis of vitamin E. However, an insufficient yield of beta-farnesene precludes its industrialization. Understanding the mechanism would be essential for attaining beta-farnesene in high yield. Guided by structure-based enzyme engineering, we designed several potent variants, among which L326I increased the beta-farnesene yield from 450.65 to 3877.42 mg/L. Furthermore, we found that the function of beta-farnesene synthase AaFS can be modulated at two positions; W299 is responsible for tuning the enzyme's function to give its isomeric product alpha-farnesene and Y402 is the key residue for diverting from the linear farnesene products to the monocyclic alpha-bisabolol product. These findings provide valuable insights into the catalytic mechanism and functional modulation of farnesene synthases and set the basis for rational engineering of farnesene synthases for selective biosynthesis of diverse sesquiterpene natural products.