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

Philippe Le Bris,Clément Barbereau,Marion Dalmais,Yin Wang,Sébastien Antelme,Laurent Cézard,Mathias Schuetz,Lacey Samuels,Frédéric Legée,Angelina D’Orlando,Richard Sibout,Catherine Lapierre,Abdelhafid Bendahmane

doi:10.1186/s13068-019-1525-5

Abstract

BackgroundDedicated 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.ResultsLACCASE5 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.ConclusionThis 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.

Highlights

Dedicated lignocellulosic feedstock from grass crops for biofuel production is extensively increasing
The present study identifies and characterizes the function of LACCASE8 (LAC8, Bradi2g23370), which has an important function in the lignification of cell walls in Brachypodium distachyon stems
Considering that the lignin content of the lac5 mutant was found to be only moderately reduced relative to the wild type (WT) level [15], we hypothesized that another close paralog might be involved in lignification and that its activity might compensate for LAC5 deficiency

Summary

Introduction

Dedicated lignocellulosic feedstock from grass crops for biofuel production is extensively increasing. 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. It is well established that lignins negatively affect the cellulose-to-ethanol conversion process [2], but lignins represent potential sources of high-value products [1]. As grasses, such as Miscanthus spp. and switchgrass (Panicum virgatum) are considered promising bioenergy crops, there is increasing interest in their lignin composition and deposition. Lignins formed in grasses (Poaceae) have additional features, the main ones being their association to ferulic acid (FA) and p-coumaric acid (CA) [3,4,5,6,7,8]

Methods

Results

Conclusion