Characterization of a Group of 2,3-Oxidosqualene Cyclase Genes Involved in the Biosynthesis of Diverse Triterpenoids of Perilla frutescens

Abstract

Perilla frutescens (L.), a traditional edible and medicinal crop, contains diverse triterpenes with multiple pharmacological properties. However, the biosynthesis of triterpenes in perilla remains rarely revelation. In this study, nine putative 2,3-oxidosqualene cyclase (OSC) genes (PfOSC1-9) were screened from the P. frutescens genome and functionally characterized by heterologous expression. Camelliol C, a triterpenol with pharmacological effect, was first identified as abundant in perilla seeds, and the camelliol C synthase (PfOSC7) was first identified in P. frutescens utilizing a yeast system. In addition, PfOSC2, PfOSC4, and PfOSC9 were identified as cycloartenol, lupeol, and β-amyrin synthase, respectively. Molecular docking and site-directed mutagenesis revealed that changes in Leu253 of PfOSC4, Ala480 of PfOSC7, and Trp257 of PfOSC9 might lead to variations of catalytic specificity or efficiency. These results will provide key insights into the biosynthetic pathways of triterpenoids and have great significance for germplasm breeding in P. frutescens.

Genome-Wide Investigation of Oxidosqualene Cyclase Genes Deciphers the Genetic Basis of Triterpene Biosynthesis in Tea Plants

“…Finally, the CsOSC1 V484A mutant reduced enzyme activity by 65% and yielded camelliol C (1), a monocyclic triterpene, along with β-amyrin (5) at a ratio of 13:22 (Figures 6A,E, S7 and Table S8). Similar observations were made with the V784A mutant of PfOSC4 and V482A mutant of PfOSC9 from P. frutescens, 41 as well as the V483A/G mutants of EtAS, 44 which predominantly produced camelliol C. The conserved Val residue is known to be vital for B ring formation in triterpene scaffolds through steric hindrance. 45 Its substitution with the smaller Ala or Gly is thought to reduce the bulk size, improperly positioning 2,3-oxidosqualene within the reaction cavity.…”

“… Mutations of the corresponding residues, such as W260L in a multifunctional α-amyrin synthase from C. sasanqua and W257L in a β-amyrin synthase from Perilla frutescens, demonstrated similar effects. , …”

“…40 Mutations of the corresponding residues, such as W260L in a multifunctional αamyrin synthase from C. sasanqua and W257L in a β-amyrin synthase from Perilla frutescens, demonstrated similar effects. 23,41 The Y261H mutation in CsOSC1, also located within the MWCYCR motif, resulted in a rapid loss of the enzyme activity, where the β-amyrin production was completely abolished (Figures 6A,6E, S7 and Table S8). This is consistent with previous research that observed the loss of β-amyrin synthesis upon equivalent mutations of Tyr to aliphatic residues (Ala, Val, Leu, and Ile) or His in the β-amyrin synthase from Euphorbia tirucalli (EtAS).…”

Characterization of a Malabaricane-Type Triterpene Synthase from Astragalus membranaceus and Enzymatic Synthesis of Astramalabaricosides

“…29,30 Shen and co-workers demonstrated that L253 in lupeol synthase PfOSC4 and W257 in β-amyrin synthase PfOSC9 were the critical residues for their product specificity. 35 The F728H mutant of β-amyrin synthase EtAS has been reported to alter its product specificity for producing tricyclic, tetracyclic, and pentacyclic triterpenoids. 18 Recently, the triad motif VFM/N has been found to be the critical determinant for the formation of β-amyrin and cycloartenol.…”

“…There are a few reports on the altered product specificity of tetracyclic and pentacyclic triterpene synthases. , Shen and co-workers demonstrated that L253 in lupeol synthase PfOSC4 and W257 in β-amyrin synthase PfOSC9 were the critical residues for their product specificity . The F728H mutant of β-amyrin synthase EtAS has been reported to alter its product specificity for producing tricyclic, tetracyclic, and pentacyclic triterpenoids .…”