Uncovering hidden sesquiterpene biosynthetic pathway through expression boost area-mediated productivity enhancement in basidiomycete.

Basidiomycetes are known to biosynthesize many biologically interesting compounds, including terpenoids. However, they are notoriously difficult to manipulate. Previously, we identified the gene cluster encoding enzymes responsible for the biosynthesis of lagopodins, cuparene-type sesquiterpenoid quinone natural products in Coprinopsis cinerea. In this study, we attempted to increase the productivity of lagopodin B (1) and related pathway products by overexpressing the terpene cyclase gene cop6 in C. cinerea to determine the details of the complex lagopodin and hitoyol biosynthetic pathway. Random integration of the cop6 into the genome of the ku70-deficient C. cinerea strain resulted in an ~2.4-fold increase in the production of 1. However, integration of cop6 into a highly transcribed position within the chromosome we designated as an expression boost area (EBA) resulted in an ~14-fold greater production of 1. Furthermore, the EBA-integration strain allowed us to isolate a previously undetected product 2, which we determined to be the known compound, hydroxylagopodin B. This finding expanded our understanding of the lagopodin-hitoyol biosynthetic pathway and allowed us to hypothesize a possible mechanism for the biosynthesis of a related homodimeric compound, lagopodin C. Our results demonstrate the potential of targeting EBA to integrate key biosynthetic genes into the genome for enhancing the production of difficult-to-obtain compounds for studying the biosynthesis of complex secondary metabolites in basidiomycetes and other complex eukaryotic organisms.