Isolating and Assaying Unspecific Peroxygenase and Flavin Binding Enzymes for in vitro Terpenoid Biosynthesis

Abstract

Terpenoids are an exceptionally large family of natural products, and contain numerous bioactive members that are pharmaceutically important. While most research into terpenoids and their metabolism has thus far occurred in non-fungal organisms, chiefly plants, Basidiomycota (mushroom forming fungi) are well known as prolific producers of bioactive sesquiterpenoids, such as the potent anticancer compounds Illudin M and S. While natural products have traditional been a huge driver of pharmaceutical discovery, this natural abundance is often hampered by very low expression in the native host and slow growth or rarity of the host itself. This drives up financial and environmental costs, and in many cases makes the production of otherwise useful natural products impracticable. One potential solution to this quandary is heterologous production of secondary metabolites in non-native hosts such as Escherichia coli and Saccharomyces cerevisiae. Another avenue is in vitro biocatalysis, wherein the biosynthetic enzymes of the native host are heterologously expressed, isolated, and used to perform synthesis outside of the cell. This approach avoids the fragility of the in vivo system and would allow the creation of combinatorial enzymatic pathways to create novel bioactive structures. With regards to constructing a sesquiterpenoid biosynthetic pathway, many stable terpene synthases have been isolated and shown to be active in vitro. However, the most well studied terpene scaffold modifying enzymes, cytochrome P450s, are notoriously difficult to heterologously express in active form. In order to obtain scaffold modifying oxygenases capable of being part of an in vitro terpenoid biosynthetic pathway, fungal oxygenases aside from cytochrome P450s were investigated. Specifically, unspecific peroxygenase from Agrocybe aegerita and flavin binding oxidoreductases from Δ6-protoilludene biosynthetic gene clusters were expressed and assayed against the sesquiterpene scaffold.