Functional characterization and key residue analysis of oxidosqualene cyclases involved in amyrin and cycloartenol formation in Parashorea chinensis

Dipterocarpaceae dammar resins contain diverse triterpenoids, including dammarane constituents and scaffolds other than dammarane, such as derivatives with oleanane, ursane or lupane scaffolds, but the oxidosqualene cyclases (OSCs) underlying this scaffold diversity remain poorly characterized. OSCs catalyze the cyclization of 2,3-oxidosqualene to generate distinct triterpene scaffolds. Genome mining identified 13 putative PcOSC genes in Parashorea chinensis . Guided by triterpenoid scaffold information from accessible leaf and stem tissues and by phylogenetic placement, we prioritized a PcOSC clade related to amyrin formation and a branch related to cycloartenol and sterol biosynthesis for functional analysis. Yeast expression assigned PcOSC10 as a cycloartenol synthase, PcOSC4 as a β -amyrin synthase, and PcOSC3 as a multifunctional α / β -amyrin synthase. Transient expression in Nicotiana benthamiana provided complementary evidence from a plant host for PcOSC3 and PcOSC4, whereas the cycloartenol signal associated with PcOSC10 expression was interpreted relative to the matched control because cycloartenol is an endogenous sterol intermediate in plant tissues. Key residue analysis identified residues associated with scaffold outcome and product output. PcOSC3 M729N shifted products from amyrins to lupeol, PcOSC4 K729N enabled germanicol formation, and PcOSC4 L483I increased β- amyrin output by approximately 4.3-fold under the tested yeast expression conditions. Together, these data define representative PcOSCs involved in amyrin formation and in the cycloartenol/sterol-related branch and identify residues associated with OSC scaffold choice and product output. Although the branch responsible for dammarane formation remains to be resolved by separate functional characterization, the present study provides an initial framework guided by metabolite information and phylogenetic analysis for dissecting triterpenoid scaffold diversification controlled by OSCs in Dipterocarpaceae.