Terpenes are secondary natural products consisting of carbon five units produced by terpene synthase. They have been found to have functional importance ranging from medical uses (taxadiene), biofuel potential and taste and smell for cinnamon and mint. One terpene precursor substrate can produce up to 400 different products through various terpene synthase with some single enzymes producing over 50 different compounds. This broad chemical diversity is a critical focus for the engineering effort to produce novel terpenes.
Terpene synthase reactions are all initiated by the removal of a pyrophosphate to create a cation that is shaped in a hydrophobic section of the active site controlling the product structure. Little is known about how this hydrophobic region controls production, so this site is the focus of engineering work.
The aim of this project was to engineer Streptomyces pristinaespiralis selina-4(15),7(11)-diene synthase to produce novel compounds. Through this engineering, we could gain mechanistic insight into Streptomyces pristinaespiralis selina-4(15),7(11)-diene synthase and terpene synthases. Streptomyces pristinaespiralis selina-4(15),7(11)-diene synthase is a specific terpene synthase with selina-4(15),7(11)-diene comprising over 90% of the product profile and the second by-product germacrene B.
Using ligand binding software, a series of site directed mutants was produced with the aim of altering product binding and product distribution. An “in culture” GC/MS screen was developed to screen these targeted mutagenesis variants. Initially alanine scanning was performed at specific points in the active sites to identify potential hotspots for further targeted mutagenesis. While no novel products were found in the current screening, variant V187F showed a switch in specificity between the products and a Y152F showed a 50:50 ratio of wild-type products. The kinetic parameters of V187F and Y152F were determined and showed an increased Km suggesting the variant reduces the substrate affinity favouring specificity of germacrene B. It was concluded that, V187 and Y152 have a key roles in the conversion of germacrene B to selina-4(15),7(11)-diene and the formation of the two rings. This mechanistic insight may be used to engineer further terpene synthase to produce novel ring terpenes.
