Improvement of Biomass Digestibility through the manipulation of tricin biosynthesis pathway in rice

Abstract

Plant biomass is an abundant and sustainable raw material for biofuel ethanol production. However, the presence of lignin in cell wall impedes the release of sugar from cellulose for fermentation. Lignin is derived from oxidative couplings of monolignols. Interestingly, a wide range of monocots utilize tricin (a 3’, 5’-dimethoxyflavone) as a natural co-monomer with monolignols for lignification. Following the characterization of two P450 enzymes CYP93G1 and CYP75B4, we finalized the tricin biosynthesis pathway in rice as naringenin → apigenin → luteolin → chrysoeriol → selgin → tricin. CYP93G1 is a flavone synthase II which introduces the C2=C3 double bond to naringenin to form apigenin. Meanwhile, CYP75B4 serves a dual functional enzyme with apigenin 3’-hydroxylase and chrysoeriol 5’-hydroxylase activities. Importantly, both CYP93G1 and CYP75B4 are indispensable for generating tricin monomer for lignification. The respective rice mutants showed wild-type growth phenotypes with intact vascular tissues while NMR analysis demonstrated the depletion of tricin in cell wall lignin. Furthermore, both mutants showed reduced lignin content, altered S/G ratio, and enhanced enzymatic saccharification efficiency in biomass. Hence, genetic manipulation of tricin biosynthesis represents an attractive strategy to engineer grass lignin for improved biomass utilization without severely compromising plant fitness. Given that CYP93G1 and CYP75B4 are highly conserved in Poaceae, there is a strong potential to extend the application to bioenergy grass crops and other cereal crop residues.