Discovery and Validation of a Novel Step Catalyzed by in the Flavonoid Biosynthesis Pathway.

Kaempferol and quercetin are the essential plant secondary metabolites that confer huge biological functions in the plant defense system. In this study, biosynthetic pathways for kaempferol and quercetin were constructed in using naringenin as a substrate. was cloned into pRS42K yeast episomal plasmid (YEp) vector and the activity of the target gene was analyzed in engineered and empty strains. We confirmed a novel step of kaempferol and quercetin biosynthesis directly from naringenin, catalyzed by the rice flavanone 3-hydroxylase (). The results were confirmed through thin layer chromatography (TLC) followed by western blotting, nuclear magnetic resonance (NMR), and liquid chromatography-mass spectrometry LCMS-MS. TLC showed positive results when comparing both compounds extracted from the engineered strain with the standard reference. Western blotting confirmed the lack of activity in empty strains and confirmed high expression in engineered strains. NMR spectroscopy confirmed only quercetin, while LCMS-MS results revealed that is responsible for the conversion of naringenin to both kaempferol and quercetin.