Elucidation and engineering of arabinofuranosyltransferase to enable total de novo biosynthesis of paris saponins in yeast

Abstract Paris saponins (PSs) are structurally complex steroidal saponins that, due to their diverse glycosylation patterns, exhibit a range of significant pharmacological activities, including anti-tumor and antibacterial effects. However, incomplete characterization of the key enzymes responsible for glycosylation modifications has hindered their efficient heterologous biosynthesis. In this study, we reprogrammed the sugar donor specificity of a steroidal rhamnosyltransferase (UGT93M3) to enable the transfer of arabinofuranose (Ara f ). Through structural analysis, we identified key amino acid residues (368H/Q) that play an important role in determining Ara f donor specificity. Guided by this insight, we successfully reconstructed the paris saponin I (PSI) biosynthetic pathway in Saccharomyces cerevisiae using engineered enzymes. To address challenges related to donor availability, we introduced UDP-sugar biosynthetic modules (UDP-Rha and UDP-Ara f ) into yeast. With this integrated platform, we were able to de novo produce a range of paris saponins, including diosgenin-3- O -glucosyl-(1→6)-glucoside (DGG), diosgenin-3- O -rhamnosyl(1→2) [glucosyl(1→6)]glucoside (DRGG) and paris saponin II. This work establishes a novel microbial platform for the sustainable production of paris saponins, particularly PSI, advancing the biosynthesis of steroidal glycosides and providing a potential strategy for the industrial-scale production of bioactive saponins. Teaser In this study, we report the first complete de novo biosynthesis of four bioactive PSs, PSI, PSII, DGG and DRGG, in Saccharomyces cerevisiae from simple carbon source. Key advances include: (i) A single amino-acid switch (N368H) in the rhamnosyltransferase UGT93M3 endowed high-efficiency transfer of the rare five-membered arabinofuranose, solving a bottleneck in PS I biosynthesis; (ii) Elucidation of the molecular basis for sugar-donor specificity through AlphaFold3 docking and 300-ns molecular-dynamics simulations, revealing a histidine “latch” that stabilizes UDP-Ara f in the catalytic pose; (iii) Construction of a 16-gene yeast chassis that integrates plant P450s, optimized glycosyltransferases, and de novo modules for UDP-rhamnose and UDP-arabinofuranose supply, achieving de novo microbial production of PS II, DGG, DRGG and PS I from glucose alone.