Inactivation of LACCASE8 and LACCASE5 genes in Brachypodium distachyon leads to severe decrease in lignin content and high increase in saccharification yield without impacting plant integrity

Abstract

Background: Dedicated lignocellulosic feedstock from grass crops for biofuel production is extensively increasing. However, the access to fermentable cell wall sugars by carbohydrate degrading enzymes is impeded by lignins. These complex polymers are made from reactive oxidized monolignols in the cell wall. Little is known about the laccase-mediated oxidation of monolignols in grasses, and inactivation of the monolignol polymerization mechanism might be a strategy to increase the yield of fermentable sugars. Results: LACCASE5 and LACCASE8 are inactivated in a Brachypodium double mutant. Relative to the wild type, the lignin content of extract-free mature culms is decreased by 20-30% and the saccharification yield is increased by 140%. Release of ferulic acid by mild alkaline hydrolysis is also 2.5-fold higher. Interfascicular fibers are mainly affected while integrity of vascular bundles is not impaired. Interestingly, there is no drastic impact of the double mutation on plant growth. Conclusion: This work shows that two Brachypodium laccases with clearly identified orthologs in crops are involved in lignification of this model plant. Lignification in interfascicular fibers and metaxylem cells is partly uncoupled in Brachypodium. Orthologs of these laccases are promising targets for improving grass feedstock for cellulosic biofuel production.