Functional analysis of squalene epoxidases and oxidosqualene cyclases clarifies roles in Boswellia C3-epimeric...

Boswellia trees produce bioactive boswellic acids (BAs) in response to wounding, which are a rare class of C3-epimeric triterpenoids. Understanding BA biosynthesis, particularly knowing whether C3-epimerization occurs at the triterpenoid epoxidation/cyclization steps catalyzed by squalene epoxidase (SQE)/2,3-oxidosqualene cyclase (OSC) or at the later stage of scaffold modification, remains largely elusive. We identified four BsSQEs (BsSQE1-4) and six BsOSCs (BsOSC1-6) from the Boswellia serrata transcriptome, and functionally characterized them in vitro, in planta assays and using yeast SQE/OSC mutants to know the involvement of BsSQEs/BsOSCs in BA biosynthesis. Wound-inducible BsSQE1 and BsSQE3 formed (3S)-2,3-oxidosqualene, and a wound-inducible BsOSC3 produced α-amyrin and β-amyrin. BsSQEs did not make (3R)-2,3-oxidosqualene, a potential source of C3 epimers, and none of the BsOSCs formed C3 epimers when assayed using racemic (3R,S)-2,3-oxidosqualene. These results excluded the possibility of C3-epimerization at the squalene epoxidation/2,3-oxidosqualene cyclization steps. Remarkably, BsOSC3 overexpression in B. serrata leaves increased the contents of 3-epi-α-amyrin, 3-epi-β-amyrin and BAs, indicating that α/β-amyrin produced by BsOSC3 might get epimerized and subsequently fed into the BA biosynthetic pathway. Overall, the results suggested that 3-epi-α/β-amyrin is not the direct product of squalene epoxidation/2,3-oxidosqualene cyclization reactions; rather, 3-epi-α/β-amyrin might form via epimerization of α/β-amyrin, thus establishing a critical step in the BA biosynthetic pathway.