Identification of Enzymes Involved in Sesterterpene Biosynthesis in Marine Fungi

Diverse and bioactive terpene compounds are produced by marine fungi. The biosynthesis of sesterterpene molecules containing a C25 skeleton is catalyzed by chimeric enzymes that carry out both chain elongation and terpene cyclization reactions. These bifunctional characteristics facilitate using different chain length polyisoprenoid diphosphates as precursors to yield the geranylfarnesyl diphosphate intermediate, which is then converted to a sesterterpene in one step. In this chapter, we describe the identification of sesterterpene synthase enzymes, together with other related enzymes such as diterpene and farnesyl diphosphate synthases, in a single fungal strain. The processes are based on genome sequencing, in silico analysis of terpene synthase, in vivo gene (cluster) deletion and complementation, and in vitro protein function verification, together with the methods of detecting terpenes using high-performance liquid chromatography (HPLC) and gas chromatography-mass spectrometry (GC-MS).