Discovery and Validation of a Novel Step Catalyzed by OsF3H in the Flavonoid Biosynthesis Pathway

SIMPLE SUMMARY: Flavonoids are important plant secondary metabolites mostly produced in the shikimate pathway. Kaempferol and quercetin are important anti-oxidant flavonoids, which enhance plant tolerance to environmental stresses. The biosynthesis of both the flavonoids largely depends on the expression of genes of the shikimate pathway. Therefore, we selected the OsF3H gene from rice and assessed its functional expression using the yeast expression system. We found that OsF3H regulates a very important step of the flavonoid biosynthesis pathway and enhances the accumulation of kaempferol and quercetin. The present research confirmed that overexpression of the OsF3H gene in rice could significantly increase the biosynthesis of flavonoids, which are essential for the plant defense system. ABSTRACT: 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 Saccharomyces cerevisiae using naringenin as a substrate. OsF3H 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 (F3H). 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 OsF3H activity in empty strains and confirmed high OsF3H expression in engineered strains. NMR spectroscopy confirmed only quercetin, while LCMS-MS results revealed that F3H is responsible for the conversion of naringenin to both kaempferol and quercetin.