<i>De Novo</i> Biosynthesis of Polyphyllin V in <i>Nicotiana benthamiana</i> through Pathway Reconstruction and UDP-Sugar Engineering

Polyphyllin V (PPV) is a pharmaceutically valuable steroidal saponin with extremely low natural abundance, severely limiting its research and application. Here we established an efficient plant-based production platform for PPV in Nicotiana benthamiana through systematic optimization of glycosyltransferases and UDP-sugar metabolism. By comparing five candidate enzymes from Paris polyphylla and Dioscorea zingiberensis, we identified optimal combinations: PpUGT73CR1 with AtUGP1 (UDP-glucose pyrophosphorylase) for trillin synthesis (442.20 μg/g dry weight), and DzGT1 with AtRHM2 (rhamnose synthase) for PPV production (284.92 μg/g dw)─representing an ∼30-fold improvement over natural levels. Metabolite profiling revealed that UDP-sugar availability is a critical bottleneck, with AtUGP1 and AtRHM2 coexpression enhancing trillin and PPV yields by 1.27-fold and 1.45-fold, respectively. Scale-up production yielded 21.1 mg of purified PPV from 118 g of biomass in a 6 day production cycle. This work establishes a rapid, scalable platform for producing low-abundance steroidal saponins and demonstrates the importance of engineering both glycosyltransferases and sugar nucleotide metabolism for efficient heterologous biosynthesis.