Triterpenoid saponin biosynthesis genes and their expression patterns during the development of sea cucumber Apostichopus japonicus

Sea cucumbers are echinoderm species with a leathery skin and an elongated body.Natural compounds,including saponins,have been previously isolated from sea cucumber.The majority of saponins derived from this organism typically belong to the triterpene glycosides,which exhibit various biological effects(e.g.,antifungal,cytotoxic,hemolytic,and cytostatic immunomodulatory activities).In the present study,30 saponin biosynthesis enzymes,namely 2 AACTs,2 FPSs,2 HMGSs,2 OSCs,1 SS,2 SEs,and 19 UGTs,were found in the genome of sea cucumber Apostichopus japonicus.Compared with upstream saponin biosynthesis enzymes(i.e.,AACTs,FPSs,HMGSs,OSCs,and SSs),the downstream saponin biosynthesis enzymes(SEs and UGTs)shared lower amino acid sequence similarity with the corresponding genes in other echinoderms.It was proposed that the enzyme structures comprised bothα-helices andβ-sheets.Notably,only a low percentage ofβ-sheets were present in FPSs,OSCs,and SS.The saponin biosynthesis enzymes showed dynamic expression patterns during five critical developmental stages of A.japonicus(fertilized oocytes,blastula,gastrula,doliolaria,and penractula).The present study involved elucidation of putative saponin biosynthesis pathways in sea cucumber and provides a valuable platform for further investigation of saponin biosynthesis in echinoderms.

Functional characterization of triterpene synthases in Cibotium barometz

Cibotium barometz(Linn.)J.Sm.,a tree fern in the Dicksoniaceae family,is an economically important industrial exported plant in China and widely used in Traditional Chinese Medicine.C.barometz produces a range of bioactive triterpenes and their metabolites.However,the biosynthetic pathway of triterpenes in C.barometz remains unknown.To clarify the origin of diverse triterpenes in C.barometz,we conducted de novo transcriptome sequencing and analysis of C.barometz rhizomes and leaves to identify the candidate genes involved in C.barometz triterpene biosynthesis.Three C.barometz triterpene synthases(CbTSs)candidate genes were obtained.All of them were highly expressed in C.barometz rhizomes,consisting of the accumulation pattern of triterpenes in C.barometz.To characterize the function of these CbTSs,we constructed a squalene-and oxidosqualene-overproducing yeast chassis by overexpressing all the enzymes in the MVA pathway under the control of GAL-regulated promoter and disrupted the GAL80 gene in Saccharomyces cerevisiae simultaneously.Heterologous expressing CbTS1,CbTS2,and CbTS3 in engineering yeast strain produced cycloartenol,dammaradiene,and diploptene,respectively.Phylogenetic analysis revealed that CbTS1 belongs to oxidosqualene cyclase,while CbTS2 and CbTS3 belong to squalene cyclase.These results decipher enzymatic mechanisms underlying the origin of diverse triterpene in C.barometz.