Simulation-guided engineering enables a functional switch in selinadiene synthase towards hydroxylation

Engineering sesquiterpene synthases to form predefined alternative products is a major challenge due to their diversity in cyclisation mechanisms and our limited understanding of how amino acid changes affect the steering of these mechanisms. Here, we use a combination of atomistic simulation and site-directed mutagenesis to engineer a selina-4(15),7(11)-diene synthase (SdS) such that its final reactive carbocation is quenched by a trapped active site water, resulting in the formation of a complex hydroxylated sesquiterpene (selina-4-ol). Initially, the SdS G305E variant produced 20% selina-4-ol. As suggested by modelling of the enzyme-carbocation complex, selina-4-ol production could be further improved by varying the pH, resulting in selina-4-ol becoming the major product (48%) at pH 6.0. We incorporated the SdS G305E variant along with genes from the mevalonate pathway into bacterial BL21(DE3) cells and demonstrated production of selina-4-ol at a scale of 10 mg/L in batch fermentation. These results highlight opportunities for simulation-guided engineering of terpene synthases to produce predefined complex hydroxylated sesquiterpenes.