In muro deacetylation of xylan affects lignin properties and improves saccharification of aspen wood

Abstract

BackgroundLignocellulose from fast growing hardwood species is a preferred source of polysaccharides for advanced biofuels and “green” chemicals. However, the extensive acetylation of hardwood xylan hinders lignocellulose saccharification by obstructing enzymatic xylan hydrolysis and causing inhibitory acetic acid concentrations during microbial sugar fermentation. To optimize lignocellulose for cost-effective saccharification and biofuel production, an acetyl xylan esterase AnAXE1 from Aspergillus niger was introduced into aspen and targeted to cell walls.Results AnAXE1-expressing plants exhibited reduced xylan acetylation and grew normally. Without pretreatment, their lignocellulose yielded over 25% more glucose per unit mass of wood (dry weight) than wild-type plants. Glucose yields were less improved (+7%) after acid pretreatment, which hydrolyses xylan. The results indicate that AnAXE1 expression also reduced the molecular weight of xylan, and xylan–lignin complexes and/or lignin co-extracted with xylan, increased cellulose crystallinity, altered the lignin composition, reducing its syringyl to guaiacyl ratio, and increased lignin solubility in dioxane and hot water. Lignin-associated carbohydrates became enriched in xylose residues, indicating a higher content of xylo-oligosaccharides.ConclusionsThis work revealed several changes in plant cell walls caused by deacetylation of xylan. We propose that deacetylated xylan is partially hydrolyzed in the cell walls, liberating xylo-oligosaccharides and their associated lignin oligomers from the cell wall network. Deacetylating xylan thus not only increases its susceptibility to hydrolytic enzymes during saccharification but also changes the cell wall architecture, increasing the extractability of lignin and xylan and facilitating saccharification.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-017-0782-4) contains supplementary material, which is available to authorized users.

Hybrid Aspen Expressing a Carbohydrate Esterase Family 5 Acetyl Xylan Esterase Under Control of a Wood-Specific Promoter Shows Improved Saccharification

“…Diluted cDNA (20-30 times) was used for transcript analysis of transgenic plants. The expression was normalized to ubiquitin (Potri.005G198700) and tubulin (Potri.001G464400), and presented relative to the levels in the lowest-expressing line as previously described (Pawar et al, 2017a). The primers of reference and target genes are provided in Supplementary Table S1.…”

“…These enzymes are grouped in at least eight Carbohydrate Esterase (CE) families that differ with regard to protein structure and other properties (Biely, 2012;Pawar et al, 2013). Previous studies have shown that introduction of an Aspergillus niger AXE1 (AnAXE1) from CE1 in Arabidopsis or in hybrid aspen and targeting the enzyme to the cell wall for post-synthetic xylan deacetylation significantly improved the cellulose digestibility without changing the growth properties of these plants (Pawar et al, 2016(Pawar et al, , 2017a. Post-synthetic xylan deacetylation was considered as a more promising strategy than synthetic xylan deacetylation in the Golgi, since the latter could induce excess glucuronidation (Donev et al, 2018) caused by the promiscuous activity of glucuronyl transferases GUX1 and GUX2 (Grantham et al, 2017).…”

Engineering of Bioenergy Crops: Dominant Genetic Approaches to Improve Polysaccharide Properties and Composition in Biomass

“…To engineer a reduction in xylan acetylation, an acetyl xylan esterase from the lignocellulosedegrading fungus Aspergillus niger (AnAXE1) was overexpressed in Arabidopsis (Figure 3) (Pawar et al, 2016). After observing beneficial effects, the same gene was used to generate transgenic overexpressors in hybrid aspen (Populus tremula L. × tremuloides Michx) (Pawar et al, 2017). The transgenic trees developed normally with approximately 10% less xylan acetylation.…”

Analytical Enzymatic Saccharification of Lignocellulosic Biomass for Conversion to Biofuels and Bio-Based Chemicals

“…Non-pressurized systems include water baths [64,66] and heat blocks [62,63], and typical temperatures are in the range 80-90 • C ( Table 2). Pressurized systems include autoclaves [61,67], ASE 350 Accelerated Solvent Extractor (Dionex, Sunnyvale, CA, USA) [68], Parr reactors (Parr Instrument Company, Moline, IL, USA) [65,69,70], single-mode microwave systems [54][55][56][57][58][59] (Table 2), and partially or fully automated robotic systems for 96-well plate format [46][47][48][49][50] (Table 2). One investigation has been based on transparent plastic microtiter plates for solvation of biomass using ionic liquids where biomass dissolution was determined by measuring scattered light [74].…”

“…The approaches differ with regard to potential separation of the solid and liquid phases after pretreatment (Figure 1, Table 2). In some studies, the solids have been collected by centrifugation and thoroughly washed and neutralized with water and buffer [43,47,48,[54][55][56][57][58][59][60]71,73], while in other studies there has only been a neutralization step using concentrated buffer to adjust the pH prior to the enzymatic digestion step ( Table 2). Separation and washing is laborious, but the advantage is that more detailed information can be gathered from the experiment.…”