Systemically functional characterization of regiospecific flavonoid O-methyltransferases from Glycine max

Plants produce diverse flavonoids for defense and stress resistance, most of which have health benefits and are widely used as food additives and medicines. Methylation of the free hydroxyl groups of flavonoids, catalyzed by S-adenosyl-l-methionine-dependent O-methyltransferases (OMTs), significantly affects their physicochemical properties and bioactivities. Soybeans (Glycine max) contain a rich pool of O-methylated flavonoids. However, the OMTs responsible for flavonoid methylation in G. max remain largely unknown. We screened the G. max genome and obtained 22 putative OMT-encoding genes that share a broad spectrum of amino acid identities (25-96%); among them, 19 OMTs were successfully cloned and heterologously expressed in Escherichia coli. We used the flavonoids containing the free 3, 5, 7, 8, 3', 4' hydroxyl group, such as flavones (luteolin and 7, 8-dihydroxyflavone), flavonols (kaempferol and quercetin), flavanones (naringenin and eriodictyol), isoflavonoids (daidzein and glycetein), and caffeic acid as substrates, and 15 OMTs were proven to catalyze at least one substrate. The methylation activities of these GmOMTs covered the 3, 7, 8, 3', 4'- hydroxyl of flavonoids and 7, 4'- hydroxyl of isoflavonoids. The systematic characterization of G. max flavonoid OMTs provides insights into the biosynthesis of methylated flavonoids in soybeans and OMT bioparts for the production of methylated flavonoids via synthetic biology.