Development of Photoaffinity Probe for the Discovery of Steviol Glycosides Biosynthesis Pathway in Stevia rebuadiana and Rapid Substrate Screening.

Functional discovery and characterization of the target enzymes responsible for the biosynthesis pathway coded for the genes is ongoing, and the unknown functional diversity of this class of enzymes has been revealed by genome sequencing. Commonly, it is feasible in annotating of biosynthetic genes of prokaryotes due to the existence of gene clusters of secondary metabolites. However, in eukaryotes, the biosynthetic genes are not compactly clustered in the way of prokaryotes. Hence, it remains challenging to identify the biosynthetic pathways of newly discovered natural products in plants. Steviol glycosides are one class of natural sweeteners found in high abundance in the herb Stevia rebaudiana. Here, we applied the chemoproteomic strategy for the proteomic profiling of the biosynthetic enzymes of steviol glycosides in Stevia rebaudiana. We not only identified a steviol-catalyzing UDP-glycosyltransferase (UGT) UGT73E1 involved in steviol glycoside biosynthesis but also built up a probe-based platform for the screening of potential substrates of functional uncharacterized UGT rapidly. This approach would be a complementary tool in mining novel synthetic parts for assembling of synthetic biological systems for the biosynthesis of other complex natural products.