Functional characterization of a cycloartenol synthase and four glycosyltransferases in the biosynthesis of cycloastragenol-type astragalosides from Astragalus membranaceus

Astragalosides are the main active constituents of traditional Chinese medicine Huang-Qi,of which cycloastragenol-type glycosides are the most typical and major bioactive compounds.This kind of compounds exhibit various biological functions including cardiovascular protective,neuroprotective,etc.Owing to the limitations of natural sources and the difficulties encountered in chemical synthesis,re-engineering of biosynthetic machinery will offer an alternative and promising approach to producing astragalosides.However,the biosynthetic pathway for astragalosides remains elusive due to their complex structures and numerous reaction types and steps.Herein,guided by transcriptome and phylogenetic analyses,a cycloartenol synthase and four glycosyltransferases catalyzing the committed steps in the biosynthesis of such bioactive astragalosides were functionally characterized from Astragalus membranaceus.AmCAS1,the first reported cycloartenol synthase from Astragalus genus,is capable of catalyzing the formation of cycloartenol;AmUGT15,AmUGT14,AmUGT13,and AmUGT7 are four glycosyltransferases biochemically characterized to catalyze 3-O-xylosylation,3-O-glucosylation,25-O-glucosylation/O-xylosylation and 2’-O-glucosylation of cycloastragenol glycosides,respectively.These findings not only clarified the crucial enzymes for the biosynthesis and the molecular basis for the structural diversity of astragalosides in Astragalus plants,also paved the way for further completely deciphering the biosynthetic pathway and constructing an artificial pathway for their efficient production.

Cloning and characterization of squalene synthase and cycloartenol synthase from Siraitia grosvenorii

Mogrosides and steroid saponins are tetracyclic triterpenoids found in Siraitia grosvenorii.Squalene synthase(SQS) and cycloartenol synthase(CAS) are key enzymes in triterpenoid and steroid biosynthesis.In this study,full-length cDNAs of SgSQS and SgCAS were cloned by a rapid amplification of cDNA-ends with polymerase chain reaction(RACE-PCR) approach.The SgSQS cDNA has a 1254 bp open reading frame(ORF) encoding 417 amino acids,and the SgCAS cDNA contains a 2298 bp ORF encoding 765 amino acids.Bioinformatic analysis showed that the deduced SgSQS protein has two transmembrane regions in the C-terminal.Both SgSQS and SgCAS have significantly higher levels in fruits than in other tissues,suggesting that steroids and mogrosides are competitors for the same precursors in fruits.Combined in silico prediction and subcellular localization,experiments in tobacco indicated that SgSQS was probably in the cytoplasm or on the cytoskeleton,and SgCAS was likely located in the nucleus or cytosol.These results will provide a foundation for further study of SgSQS and SgCAS gene functions in S.grosvenorii,and may facilitate improvements in mogroside content in fruit by regulating gene expression.