Elucidation of Terpenoid Biosynthesis in Sclerotinia

Abstract

Terpenes are a large class of specialized metabolites found in plant and fungus species that have been widely studied for their medicinal and cytotoxic properties. The plant pathogenic fungus Sclerotinia homoeocarpa was previously shown to produce eleven nortetralabdane compounds that exhibit strong antiplasmodial and growth inhibitive properties. Our objective is to elucidate the biosynthesis of these unique terpene compounds in S. homoeocarpa. Genome mining identified two terpene cyclases as well as multiple clustered cytochromes P450 (CYPs). Combinatorial expression in Escherichia coli and GC‐MS analysis of extracts revealed that one cyclase, ShTPS1, is a bifunctional diterpene synthase that produces the 20‐carbon compound pimara‐8(14),15‐diene. This diterpene likely acts as a scaffold to be modified by downstream CYPs into nortetralabdanes. The second cyclase, ShTPS2, produces several 25‐carbon sesterterpenes, one of the least well‐characterized subsets of terpenes and a frontier for pharmaceutical research. The discovery of ShTPS2 is uniquely interesting due to its promiscuity and the rarity of characterized bifunctional sesterterpene synthases ‐ terpene synthases with a chimeric prenyltransferase domain. Homology modeling of ShTPS2 identified targets for site‐directed mutagenesis in the prenyltransferase and terpene cyclase domains. Mutations in these areas reveal both the functional plasticity of the enzyme and that the promiscuity may result from cationic intermediates of a single terpene cyclase pathway. This research contributes to understanding the diversity of terpene biosynthesis in fungi and allows for the biosynthesis of compounds of interest that are naturally produced at low levels, enabling future work to investigate their function.Support or Funding InformationThis work was supported by the Massachusetts Agricultural Experiment Station as well as the University of Massachusetts Amherst Commonwealth Honors College and Department of Biochemistry and Molecular Biology.