The Investigations on Diterpene Biosynthesis through Substrate and Protein Engineering

Abstract

This cumulative doctoral dissertation "The Investigations on Diterpene Biosynthesis through Substrate and Protein Engineering" concentrates on diterpene biosynthesis from various aspects, including non-natural biotransformation, genome mining, mechanistic study and enzyme engineering. <br/> First, seven geranylgeranyl diphosphate (GGPP) analogs named iso-GGPPs, were synthesized chemically or enzymatically, of which double bonds were replaced by various modified methylene groups, leading to novel reactivities. The subsequent biotransformations with almost 20 characterized diterpene synthases resulted in the isolation of more than 50 nonnatural diterpenes featuring novel terpene skeletons. Among them, some compounds can be regarded as the derailment products in the cyclization cascade, while others are generated from distinct cyclization modes in comparison to its natural counterparts. Apart from that, four isotope labelled substrate analogs were stereoselectively synthesized and used in the enzymatic conversions to determine the absolute configurations of isolated non-natural diterpenes. Second, two new fungal terpene synthases were also functionally characterized after bioinformatic analysis, gene cloning and protein expression, whose products were wellinvestigated for understanding their formations through isotopic labelling experiments. Third, two previously reported diterpenes, benditerpe-2,6,15-triene and venezuelaene A, with tentative biosynthetic proposals presented in the original works, were investigated for the hydride shifts in their biosynthesis through isotopic labelling studies, non-natural biotransformations, product derivations and DFT calculations. In addition, site-directed mutagenesis studies in conjunction with molecular docking on spata-13,17-diene synthase and selina-4(15),7(11)-diene synthase disclosed the function of a specific hydrophobic tunnel in these two enzymes, which enable evolving the second enzyme from a sesquiterpene synthase to a diterpene synthase. Last but not least, the well-known diterpene taxa-4,11-diene was addressed in this thesis. Unique long-proton migrations were observed in the biosynthesis of taxaxenene and cyclophomactene, two compounds generated from a non-natural enzymatic conversion with wild type taxadiene synthase and an enzyme variant, respectively. <br/> All in all, this doctoral dissertation provides novel insights into the terpene field. The combination of various strategies enabled the discovery of novel terpenes and mechanistic investigations of utilized enzymes, which will be described in detail in this thesis.