Removal of glucuronic acid from xylan is a strategy to improve the conversion of plant biomass to sugars for bioenergy

Abstract

BackgroundPlant lignocellulosic biomass can be a source of fermentable sugars for the production of second generation biofuels and biochemicals. The recalcitrance of this plant material is one of the major obstacles in its conversion into sugars. Biomass is primarily composed of secondary cell walls, which is made of cellulose, hemicelluloses and lignin. Xylan, a hemicellulose, binds to the cellulose microfibril and is hypothesised to form an interface between lignin and cellulose. Both softwood and hardwood xylan carry glucuronic acid side branches. As xylan branching may be important for biomass recalcitrance and softwood is an abundant, non-food competing, source of biomass it is important to investigate how conifer xylan is synthesised.ResultsHere, we show using Arabidopsis gux mutant biomass that removal of glucuronosyl substitutions of xylan can allow 30% more glucose and over 700% more xylose to be released during saccharification. Ethanol yields obtained through enzymatic saccharification and fermentation of gux biomass were double those obtained for non-mutant material. Our analysis of additional xylan branching mutants demonstrates that absence of GlcA is unique in conferring the reduced recalcitrance phenotype. As in hardwoods, conifer xylan is branched with GlcA. We use transcriptomic analysis to identify conifer enzymes that might be responsible for addition of GlcA branches onto xylan in industrially important softwood. Using a combination of in vitro and in vivo activity assays, we demonstrate that a white spruce (Picea glauca) gene, PgGUX, encodes an active glucuronosyl transferase. Glucuronic acid introduced by PgGUX reduces the sugar release of Arabidopsis gux mutant biomass to wild-type levels indicating that it can fulfil the same biological function as native glucuronosylation.ConclusionRemoval of glucuronic acid from xylan results in the largest increase in release of fermentable sugars from Arabidopsis plants that grow to the wild-type size. Additionally, plant material used in this work did not undergo any chemical pretreatment, and thus increased monosaccharide release from gux biomass can be achieved without the use of environmentally hazardous chemical pretreatment procedures. Therefore, the identification of a gymnosperm enzyme, likely to be responsible for softwood xylan glucuronosylation, provides a mutagenesis target for genetically improved forestry trees.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-017-0902-1) contains supplementary material, which is available to authorized users.

Covalent interactions between lignin and hemicelluloses in plant secondary cell walls

“…Additional indirect evidence for glucuronic acid-mediated xylan:lignin cross-links is the finding that glucuronic acid is important for biomass recalcitrance in Arabidopsis [51]. Enzymatic saccharification of the gux1 gux2 mutant, which lacks glucuronic acid on secondary cell wall xylan, shows that up to twice as much glucose is released and five times more xylose in limited-saccharification studies.…”

Secondary cell wall biosynthesis

“…Simultaneous mutations of three Arabidopsis GUX genes (GUX1, GUX2 and GUX3) result in a complete loss of GlcA residues in xylan but only a mild alteration in secondary wall thickening and stem strength (Lee et al, 2012a). Microsomes from tobacco leaves or BY2 cells expressing GUXs from Arabidopsis and spruce have been shown to exhibit xylan glucuronyltransferase activities (Lee et al, 2012a;Rennie et al, 2012;Lyczakowski et al, 2017).…”

Jan J. Lyczakowski

“…Part of our iGEM project looked at improving the process of biofuel production (Lewicka et al ., ) and the progress we made in this endeavour helped decide my interest in contributing to the development of sustainable energy and materials. Later on, during my PhD studies with Prof. Paul Dupree at the University of Cambridge, I was fortunate again to work with a great team of people and we were able to discover a possible route towards generating a superior plant feedstock for biofuel production (Lyczakowski et al ., ).…”