Independent recruitment of enzymes for phenylpropanoid sucrose ester biosynthesis in <i>Polygala tenuifolia</i>

Plants produce a vast diversity of specialized metabolites and can independently recruit enzymes from distinct evolutionary origins to catalyze identical reactions. Sibiricose A5 and A6, two phenylpropanoid sucrose esters (PSEs) with potent antidepressant and neuroprotective activities, are commonly isolated from the roots of the medicinal plant Polygala tenuifolia. Here, we confirmed the root-specific accumulation of these PSEs and assembled a high-quality, chromosome-scale genome for P. tenuifolia (678.38 Mb), achieving a BUSCO completeness of 99.30%. Through integrated phylogenetic and cross-tissue gene-metabolite correlation analyses, we selected root-specific BAHD acyltransferases candidates. Whole-cell feeding assays and in vitro enzyme assays subsequently identified Sucrose Hydroxycinnamoyl Transferases PtSHT1 and PtSHT2 as the enzymes responsible for the biosynthesis of both sibiricose A5 and A6. We further discovered a BAHD acyltransferase conserved within a syntenic genomic segment across angiosperms, representing the ancestral lineage of the sucrose ferulic acid transferases from Z. mays (ZmSFTs). However, these conserved enzymes lacked sibiricose A5/A6 synthetic activity, and the P. tenuifolia homolog was not expressed in roots. Phylogenetic analysis revealed that PtSHTs and ZmSFTs form two distinct monophyletic clades: PtSHTs belonging to acyltransferases clade 3, a eudicot-specific lineage, whereas ZmSFTs belong to clade 6. Furthermore, combinatorial expression of PtSHTs with auxiliary acyltransferases, selected based on syntenic context and correlation analysis, yielded several novel PSEs. These findings advance our understanding of PSE biosynthesis and the independent evolutionary recruitment of SHT enzymes with sibiricose A5 and A6 synthetic activity, while also facilitating the targeted production of specific PSEs for pharmaceutical applications.