Identification of P450 Candidates Associated with the Biosynthesis of Physalin-Class Compounds in Physalis angulata.

The Physalis genus has long been used as traditional medicine in the treatment of various diseases. Physalins, the characteristic class of compounds in this genus, are major bioactive constituents. To date, the biogenesis of physalins remains largely unknown, except for the recently established knowledge that 24-methyldesmosterol is a precursor of physalin. To identify the genes encoding P450s that are putatively involved in converting 24-methyldesmosterol to physalins, a total of 306 P450-encoding unigenes were retrieved from our recently constructed P. angulata transcriptome. Extensive phylogenetic analysis proposed 21 P450s that might participate in physalin biosynthesis. To validate the candidates, we developed a virus-induced gene silencing (VIGS) system for P. angulata, and four P450 candidates were selected for the VIGS experiments. The reduction in the transcripts of the four P450 candidates by VIGS all led to decreased levels of physalin-class compounds in the P. angulata leaves. Thus, this study provides a number of P450 candidates that are likely associated with the biosynthesis of physalin-class compounds, forming a strong basis to reveal the unknown physalin biosynthetic pathway in the future.

Molecular Cloning and Functional Characterization of a Sterol 3-O-Glucosyltransferase Involved in Biosynthesis of Steroidal Saponins in Trigonella foenum-graecum.

Fenugreek (Trigonella foenum-graecum), a pharmacologically important herb, is widely known for its antidiabetic, hypolipidemic, and anticancer effects. The medicinal properties of this herb are accredited to the presence of bioactive steroidal saponins with one or more sugar moieties linked to the C-3 OH position of disogenin or its C25-epimer yamogenin. Despite intensive studies regarding pharmacology and phytochemical profiles of this plant, enzymes and/or genes involved in synthesizing the glycosidic part of fenugreek steroidal saponins are still missing so far. This study reports the molecular cloning and functional characterization of a key sterol-specific glucosyltransferase, designated as TfS3GT2 here, from fenugreek plant. The recombinant TfS3GT2 was purified via expression in Escherichia coli, and biochemical characterization of the recombinant enzyme suggested its role in transferring a glucose group onto the C-3 hydroxyl group of diosgenin or yamogenin. The functional role of TfS3GT2 in the steroidal saponin biosynthesis was also demonstrated by suppressing the gene in the transgenic fenugreek hairy roots via the RNA interference (RNAi) approach. Down-regulation of TfS3GT2 in fenugreek generally led to reduced levels of diosgenin or yamogenin-derived steroidal saponins. Thus, Tf3SGT2 was identified as a steroid-specific UDP-glucose 3-O-glucosyltransferase that appears to be involved in steroidal saponin biosynthesis in T. foenum-graecum.