摘要Polyphyllins,a class of isospirostan-type steroidal saponins,exhibit cytotoxicity against a wide range of cancer cells.Despite extensive research,the complete biosynthetic pathway of these compounds remains elusive.To investigate these pathways,various tissues from Trillium tschonoskii were collected for sequencing,yielding 173,382 high-quality unigene sequences,including 353 annotated as glycosyltransferases.Subsequently,a novel rhamnosyltransferase gene,UGT738A3,was characterized,which catalyzes the conversion of triglycoside pol-yphyllin Ⅲ and pennogenin 3-O-beta-chacotrioside into tetraglycoside polyphyllin Ⅱ and polyphyllin Ⅶ.The key residues that affect the catalytic activity of UGT738A3 were identified using site-directed mutation.The catalytic activity of the A158T/P101L mutant toward polyphyllin Ⅲ and pennogenin 3-O-beta-chacotrioside improved by 2.5-and 6.5-fold,respectively.Therefore,we successfully reconstructed the biosynthesis pathway of polyphyllin Ⅱ in yeast by introducing UGT93M3,which catalyzes the formation of polyphyllin Ⅲ,and UGT738A3A158T/P101L,achieving a yield of 0.13 mg/L.This study not only investigated the pivotal role of UGT738A3 in the catalysis of tetraglycoside formation but also revealed highly efficient enzymatic components essential for the heterologous biosynthesis of polyphyllin saponins.