Specific flavonoid B-ring hydroxylases (CYP75B members) are recruited for different biosynthesis pathways of flavone-derived metabolites in grass biomass

Abstract

Members in the cytochrome P750 75B subfamily are hydroxylases that modify the B-ring substitution patterns in flavonoids, impacting their structure and functions. In rice biomass, OsF2H and OsFNSII function as key enzymes directing flavanones to separate biosynthetic routes for extractable flavone C-glycosides and tricin-derived metabolites (soluble and lignin-bound), respectively. Here, CYP75B3 and CYP75B4 showed co-expression pattern with OsF2H and OsFNSII, respectively, in vegetative tissues. CRISPR/Cas-9 homozygous mutants and T-DNA insertion lines were generated (or obtained) to investigate their flavonoid profile, cell wall structure, and biomass digestibility. Accordingly, we established CYP75B3 as the sole F3´H in flavone C-glycosides biosynthesis and CYP75B4 as an indispensable 3´,5´-hydroxylase to produce soluble tricin derivative and tricin-bound lignin. Hence, CYP75B3 and CYP75B4 represent two different pathway-specific enzymes co-evolved with OsF2H and OsFNSII, respectively. Interestingly, the OsF2H-CYP75B3 and OsFNSII-CYP75B4 pairs are highly conserved in the grass family. Furthermore, CYP75B4 disruption resulted in reduced lignin content, altered S/G ratio, and enhanced enzymatic saccharification efficiency in biomass. Therefore, genetic manipulation of tricin biosynthesis presents a novel approach to engineer grass lignin for improved biomass utilization.