Characterization of a γ-Humulene Synthase from Fungus Ascobolus immersus

γ-Humulene is an 11-membered macrocyclic sesquiterpene widely distributed in plants and valued for its diverse biological activities. However, no dedicated γ-humulene synthase has been identified to date. In this study, a sesquiterpene synthase (AiSTS) from the fungus Ascobolus immersus was identified and functionally characterized. In vivo heterologous expression and in vitro enzymatic assays demonstrated that AiSTS specifically catalyzes the conversion of ( E, E )- farnesyl pyrophosphate (( E, E )-FPP) into γ-humulene. Furthermore, AiSTS also exhibits notable substrate promiscuity, accepting ( E ) - geranyl pyrophosphate (( E ) - GPP) and ( E, E, E )-geranylgeranyl pyrophosphate (( E, E, E )-GGPP) to generate diverse mono- and diterpene products. Site-directed mutagenesis revealed seven amino acid residues that are essential for enzyme folding, catalytic efficiency, and product selectivity. The discovery of AiSTS expands the repertoire of fungal terpene synthases and lays a solid foundation for enzyme engineering and sustainable microbial production of γ-humulene.