Biochemistry andChemoinformatics Guided Classificationof Hirsutane Sesquiterpenes Isolated from Mushroom

Hirsutanes are mushroom-derived sesquiterpenes with a characteristic 5-5-5 tricyclic ring skeleton. To date, more than 70 derivatives have been isolated from nature. In this study, we applied heterologous expression, in vitro enzymatic reactions, and biotransformation to characterize the function of nine enzymes involved in the synthesis of anhydroarthrosporone and six novel hirsutanes. The elucidated biosynthesis involves oxidative modifications of the A-ring followed by structural diversification of the B- and C-rings. Most importantly, biosynthetic pathways provide crucial insights into the classification and organization of isolated hirsutanes. We successfully classified 69 natural hirsutanes into three groups based on their A-ring modification patterns. Our classification covered 92% of the natural hirsutanes. A comprehensive understanding of their biosynthesis will provide opportunities to isolate structurally diverse hirsutanes using genetic engineering techniques.