Camellia sasanqua is an important economic plant that is rich in lipophilic triterpenols with pharmacological activities including antiallergic, anti-inflammatory, and anticancer activities. However, the key enzymes related to triterpene biosynthesis have seldom been studied in C. sasanqua. Oxidosqualene cyclases (OSCs) are the rate-limiting enzymes related to triterpene biosynthesis. In this study, seven putative OSC genes (CsOSC1-7) were mined from the C. sasanqua transcriptome. Six CsOSCs were characterized for the biosynthesis of diverse triterpene skeletons, including α-amyrin, β-amyrin, δ-amyrin, dammarenediol-II, ψ-taraxasterol, taraxasterol, and cycloartenol by the heterologous expression system. CsOSC3 was a multiple functional α-amyrin synthase. Three key residues, Trp260, Tyr262, and Phe415, are critical to the catalytic performance of CsOSC3 judging from the results of molecular docking and site-directed mutagenesis. These findings provide important insights into the biosynthesis pathway of triterpenes in C. sasanqua.

Many functionally promiscuous plant 2,3-oxidosqualene cyclases (OSCs) have been found, but complete functional reshaping is rarely reported. In this study, we have identified two new plant OSCs: a unique protostadienol synthase ( Ao PDS) and a common cycloartenol synthase ( Ao CAS) from Alisma orientale (Sam.) Juzep. Multiscale simulations and mutagenesis experiments revealed that threonine-727 is an essential residue responsible for protosta-13 (17),24-dienol biosynthesis in Ao PDS and that the F726T mutant completely reshapes the native function of Ao CAS into a PDS function to yield almost exclusively protosta-13 (17),24-dienol. Unexpectedly, various native functions were uniformly reshaped into a PDS function by introducing the phenylalanine → threonine substitution at this conserved position in other plant and non-plant chair-boat-chair–type OSCs. Further computational modeling elaborated the trade-off mechanisms of the phenylalanine → threonine substitution that leads to the PDS activity. This study demonstrates a general strategy for functional reshaping by using a plastic residue based on the decipherment of the catalytic mechanism.

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.

Bufo bufo gargarizans Cantor are precious medicinal animals in traditional Chinese medicine (TCM). Bufadienolides as the major pharmacological components are generated from the venomous glands of B. bufo gargarizans. Bufadienolides are one type of cardiac aglycone with a six-member lactone ring and have properties of antitumor, cardiotonic, tonsillitis, and anti-inflammatory. The biosynthesis of bufadienolides is complex and unclear. This study explored the transcriptome of three different tissues (skin glands, venom glands, and muscles) of B. bufo gargarizans by high-throughput sequencing. According to the gene tissue–specific expression profile, 389 candidate genes were predicted possibly participating in the bufadienolides biosynthesis pathway. Then, BbgCYP11A1 was identified as a cholesterol side chain cleaving the enzyme in engineering yeast producing cholesterol. Furthermore, the catalytic activity of BbgCYP11A1 was studied with various redox partners. Interestingly, a plant NADPH-cytochrome P450 reductase (CPR) from Anemarrhena asphodeloides showed notably higher production than BbgAdx-2A-BbgAdR from B. bufo gargarizans. These results will provide certainly molecular research to reveal the bufadienolides biosynthesis pathway in B. bufo gargarizans.