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Strong reduction of acetylation, especially in xylan, obtained by knocking out components of a xylan acetylation machinery typically causes dwarfism, reduced mechanical strength of the stem, collapsed vessels, and stunted plant growth (Lee et al., 2011b; Manabe et al., 2013; Yuan et al., 2013, 2016). Plants with severely reduced acetylation may therefore not necessarily exhibit increased sugar yield after enzymatic saccharification (Lee et al., 2011b; Xiong et al., 2013; Yuan et al., 2013). However, moderate decrease of xylan acetylation in hybrid aspen was not only well-supported by plants but also lead to better saccharification (Pawar et al., 2017b). On the other hand, excess xylan acetylation in rice, while providing some beneficial effects on saccharification, disrupted the structure of the secondary cell wall and lead to growth defects (Zhang et al., 2017).
Microbial enzymes with acetyl xylan esterase (AXE) activity could be used in planta to reduce xylan acetylation. 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, 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). These results encourage further trials with microbial enzymes capable of deacetylation of xylan in cell walls.
The acetylation was further investigated using 2D HSQC NMR spectroscopic analysis of DMSO-extracted xylan (Supplementary Data Sheets S2, S3), which revealed the presence of acetylated and non-acetylated Xylp residues (Figure 3B). The signals shown in green were used to obtain the relative content of acetylated Xylp units whereas the signals shown in blue represent different non-acetylated Xylp. For the WT, 49% of the total Xylp units were acetylated. That included 22% monoacetylation at position C-2 (X2), 16% monoacetylation at position C-3 (X3), 6% di-acetylation at positions C-2 and C-3 (X23), and 5% acetylation at C-3 and meGlcA (X3G2) (Figure 3C). Transgenic lines exhibited reductions in signals from all acetylated Xylp, by 16% for X23, 14% for X2, 5% for X3G2, and 11% for X3. In total, the content of acetylated Xylp units was reduced by 12% in transgenic lines compared to the WT (Figure 3C). Moreover, there was a 44% increase in the content of non-acetylated Xylp units preceeding either non-acetylated or C-2 acetylated units (X-X/X2). The results suggest that the CE5 enzyme acted on Xylp positions 2 and 3, and could also deacetylate 2,3-double-acetylated Xylp as well as position 3 in glucuronosylated Xylp units of aspen wood xylan.
A 27% increase in glucose yield and 11% increase in xylose yield in enzymatic saccharification of non-pretreated wood, as well as a 3% increase in glucose yield after acid pretreatment and enzymatic saccharification were observed in WP:CE5 lines (Figure 4). Similarly increased sugar yield of enzymatic saccharification without pretreatment was reported for transgenic aspen and Arabidopsis with reduced xylan acetylation by either supressing native RWA genes or by overexpressing CE1 AXE (Pawar et al., 2016, 2017a,b). Similarly, the positive influence was smaller after acid pretreatment (Pawar et al., 2017a, b). The observed reductions in recalcitrance could be a direct consequence of increased porosity and accessibility due to xylan deacetylation by CE5 AXE acting in close proximity to cellulose. 041b061a72