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

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.

Functional Characterization of F3H Gene and Optimization of Dihydrokaempferol Biosynthesis in Saccharomyces cerevisiae

“…It was found that the silencing of the apple MdF3H gene using antisense technology resulted in an increase in flavanones but a decrease in their downstream products compared to the wild type, suggesting that mutations in the F3H gene inhibit the conversion of flavanones to downstream metabolites. Jan et al showed that overexpression of OsF3H gene in rice (Oryza sativa L.) significantly increased flavonoid biosynthesis [35]. Thus, repression or overexpression of the F3H gene leads directly to the down-or up-regulation of flavonoid metabolism synthesis.…”

The parallel biosynthesis routes of hyperoside from naringenin in Hypericum monogynum

“…6 ) implied that HmFLS1 may evolve much later with more specific activity in biosynthesizing flavonols than HmFLS2–3, which produced plentiful flavones as well. Previous studies found that OsF3H could catalyze dihydroflavonols into flavonols [ 29 ]; GbF3H, PrF3H, and PsF3H could catalyze naringenin into DHK and slight amounts of apigenin [ 28 ]; AtFLS1, CsFLS2, and AtANS could catalyze naringenin into DHK, kaempferol and apigenin [ 22 , 30 , 31 ]. These phenomena implied a deeper connection among F3Hs, FNSIs, FLSs, and ANSs – that the four types of 2-ODD had overlapped functions with each other in different degrees by evolving from a mutual gene or possibly two isozyme genes, which were partly silent or activated through mutation during plant evolution.…”

Optimizing the Biosynthesis of Dihydroquercetin from Naringenin in Saccharomyces cerevisiae

“…10 Model microorganisms such as Escherichia coli and Saccharomyces cerevisiae have been successfully engineered to biosynthesize a range of flavonoids, including members of the anthocyanin group, 11,12 breviscapine, 13 catechin, 14 and kaempferol. 15 Research has shown that DHQ can be synthesized by hydroxylation of the key metabolite naringenin (NAR) at specific positions. Through the action of flavanone-3-hydroxylase (F3H), (2S)-NAR is converted to (2R,3R)-dihydrokaempferol (DHK), which is then hydroxylated at the 3-position of its B ring to produce (2R,3R)-DHQ.…”

“…Moreover, downstream processing in biosynthetic methods tends to be simpler than plant extraction techniques . Model microorganisms such as Escherichia coli and Saccharomyces cerevisiae have been successfully engineered to biosynthesize a range of flavonoids, including members of the anthocyanin group, , breviscapine, catechin, and kaempferol . Research has shown that DHQ can be synthesized by hydroxylation of the key metabolite naringenin (NAR) at specific positions.…”