Functional analysis of squalene epoxidases and oxidosqualene cyclases clarifies roles in BoswelliaC3‐epimeric triterpenoid pathway

Abstract

Summary 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.