Identification and Engineering of UDP-rhamnosyltransferase from Trillium tschonoskii for Heterologous Biosynthesis of Polyphyllin II in Engineered Yeast

Polyphyllins, a class of isospirostan-type steroidal saponins, exhibit potent cytotoxicity against a variety range of cancer cells. Although extensive research efforts have been made, the complete biosynthetic pathway of these compounds remains unclear. In order to elucidate these pathways, different tissues from Trillium tschonoskii were collected for sequencing that yielded 173,382 high-quality unigene sequences, among which 353 were annotated as glycosyltransferases. Then, a novel rhamnosyltransferase gene, UGT738A3, was characterized, which catalyzes the conversion of triglycoside polyphyllin 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 through site-directed mutation that the mutant A158T/P101L exhibited improved catalytic activity towards polyphyllin III and pennogenin 3-O-beta-chacotrioside by 2.5-fold and 6.5-fold respectively. We thus reconstructed the biosynthesis pathway of polyphyllin II in yeast by introducing the UGT93M3(catalyzing the formation of polyphyllin VI) and UGT738A3 ^A158T/P101L3 . This study not only elucidates the pivotal role of UGT738A3 in catalyzing the formation of tetraglycoside, but also provides highly efficient enzymatic components essential for the heterologous biosynthesis of polyphyllin saponins.