Sun-Li Chong

Autohydrolysis of birch wood.

Abstract Autohydrolysis of wood as a pretreatment stage before alkaline pulping is seen as an opportunity to approach the ideals of a forest biorefinery. In autohydrolysis the degradation products of primarily hemicelluloses and some other wood components are mainly dissolved (liquid phase) and they also escape partly as gas. In the present paper, birch was autohydrolysed at two intensity levels (characterised by P-factors of 200 and 1000) and the mass balances of autohydrolysis including solid, liquid, and gaseous phases were established. Approximately 10% and 25% of the total wood substance was transferred to the liquid phase at the two autohydrolysis intensities, respectively. In both cases about 50% of the degraded wood substance consisted of xylose-based material. The share of monomeric xylose was about 6% and 50% in the two samples, respectively. From the residual oligosaccharide fractions, 13% and 90% accounted for the DP2 to DP4 components in the P200 and P1000 samples, respectively. As expected, the weight-average molar mass of the components in the P1000 hydrolysate was substantially lower (0.4 kDa) than those in the corresponding P200 hydrolysate (2.1 kDa). Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) measurements revealed that relative to xylotriose the P1000 sample contained significantly fewer acidic pentosan fractions than the P200 sample. Xylooligosaccharides dissolved at both intensity levels remained highly acetylated, and some of these also carried 4-O-methylglucopyranosyluronic acid substituents.

Reduced Wall Acetylation Proteins Play Vital and Distinct Roles in Cell Wall O-Acetylation in Arabidopsis

Four proteins in Arabidopsis are essential for plant development and play overlapping but different roles in acetylation of cell wall polysaccharides. The Reduced Wall Acetylation (RWA) proteins are involved in cell wall acetylation in plants. Previously, we described a single mutant, rwa2, which has about 20% lower level of O-acetylation in leaf cell walls and no obvious growth or developmental phenotype. In this study, we generated double, triple, and quadruple loss-of-function mutants of all four members of the RWA family in Arabidopsis (Arabidopsis thaliana). In contrast to rwa2, the triple and quadruple rwa mutants display severe growth phenotypes revealing the importance of wall acetylation for plant growth and development. The quadruple rwa mutant can be completely complemented with the RWA2 protein expressed under 35S promoter, indicating the functional redundancy of the RWA proteins. Nevertheless, the degree of acetylation of xylan, (gluco)mannan, and xyloglucan as well as overall cell wall acetylation is affected differently in different combinations of triple mutants, suggesting their diversity in substrate preference. The overall degree of wall acetylation in the rwa quadruple mutant was reduced by 63% compared with the wild type, and histochemical analysis of the rwa quadruple mutant stem indicates defects in cell differentiation of cell types with secondary cell walls.

Expression of fungal acetyl xylan esterase in Arabidopsis thaliana improves saccharification of stem lignocellulose

Cell wall hemicelluloses and pectins are O-acetylated at specific positions, but the significance of these substitutions is poorly understood. Using a transgenic approach, we investigated how reducing the extent of O-acetylation in xylan affects cell wall chemistry, plant performance and the recalcitrance of lignocellulose to saccharification. The Aspergillus niger acetyl xylan esterase AnAXE1 was expressed in Arabidopsis under the control of either the constitutively expressed 35S CAMV promoter or a woody-tissue-specific GT43B aspen promoter, and the protein was targeted to the apoplast by its native signal peptide, resulting in elevated acetyl esterase activity in soluble and wall-bound protein extracts and reduced xylan acetylation. No significant alterations in cell wall composition were observed in the transgenic lines, but their xylans were more easily digested by a β-1,4-endoxylanase, and more readily extracted by hot water, acids or alkali. Enzymatic saccharification of lignocellulose after hot water and alkali pretreatments produced up to 20% more reducing sugars in several lines. Fermentation by Trametes versicolor of tissue hydrolysates from the line with a 30% reduction in acetyl content yielded ~70% more ethanol compared with wild type. Plants expressing 35S:AnAXE1 and pGT43B:AnAXE1 developed normally and showed increased resistance to the biotrophic pathogen Hyaloperonospora arabidopsidis, probably due to constitutive activation of defence pathways. However, unintended changes in xyloglucan and pectin acetylation were only observed in 35S:AnAXE1-expressing plants. This study demonstrates that postsynthetic xylan deacetylation in woody tissues is a promising strategy for optimizing lignocellulosic biomass for biofuel production.

The α-glucuronidase Agu1 from Schizophyllum commune is a member of a novel glycoside hydrolase family (GH115)

Schizophyllum commune produces an α-glucuronidase that is active on polymeric xylan, while the ascomycete α-glucuronidases are only active on xylan oligomers. In this study, we have identified the gene (agu1) encoding this enzyme and confirmed the functionality by overexpression of the gene in S. commune and degradation of aldopentauronic acids, (MeGlcA)3-Xyl4, in the cultivation medium of the transformants. Expression analysis demonstrated that agu1 is not co-regulated with the predominant xylanase-encoding gene (xynA) of S. commune. The detailed sequence analysis of Agu1 demonstrated that this gene belongs to a novel glycoside hydrolase family (GH115) that also contains candidate genes from ascomycete fungi and bacteria. Phylogenetic analysis showed that the fungal GH115 α-glucuronidases are distinctly separate from the prokaryotic clade and distributed over three branches. The identification of putative genes encoding this enzyme in industrial fungi, such as Aspergillus oryzae and Hypocrea jecorina, will provide a starting point for further analysis of the importance of this enzyme for the hydrolysis of plant biomass.

O-Acetylation of glucuronoxylan in Arabidopsis thaliana wild type and its change in xylan biosynthesis mutants

O-Acetylglucuronoxylans (AcGX) in Arabidopsis thaliana carry acetyl residues on the 2-O and/or 3-O positions of the xylopyranosyl (Xylp) units, but the distribution of different O-acetylated Xylp units is partly unclear. We studied a possible correlation of xylan acetylation and the activities of different glycosyltransferases involved in xylan biosynthesis by analyzing the distribution of O-acetyl substituents on AcGX from Arabidopsis wild-type and mutants irx7, irx9-1, irx10, irx14 and gux1gux2. The relative contents of the Xylp structural units were determined with quantitative two-dimensional heteronuclear single quantum coherence nuclear magnetic resonance spectroscopy. In the wild type, the degree of acetylation (DA) was 60%. Mono- and diacetylated Xylp units constituted 44 and 6% of the AcGX backbone, respectively; while (4-O-methyl)-glucopyranosyluronic acid (1 → 2)-linked Xylp units, most of which also carry 3-O-acetylation, represented 13%. The DA was decreased in irx7, irx9-1 and irx14 due to the decrease in monoacetylation (2-O and 3-O), indicating a relationship between acetylation and other AcGX biosynthetic processes. The possible interactions that could lead to such changes have been discussed. No change in DA was observed in irx10 and gux1gux2, but monoacetylation was nonetheless elevated in gux1gux2. This indicates that acetylation occurs after addition of GlcpA to the xylan backbone. Mass fragmentation analysis suggests that the prevalent acetylation pattern is the acetyl group added on every other Xylp unit.

Quantitation of 4-O-methylglucuronic acid from plant cell walls.

The accuracy of commercial α-D-glucopyranosyl uronic acid (GlcA) as a calibration standard for the determination of the 4-O-methyl-α-D-glucopyranosyl uronic acid (meGlcA) content in plant materials was studied. A batch of meGlcA standard was purified from commercial birch xylan and quantified using nuclear magnetic resonance spectroscopy. Both commercial GlcA and the purified meGlcA were used as standards for the quantitation of meGlcA in Arabidopsis thaliana stems, as well as wood and wheat straw samples using acid methanolysis and gas chromatography (GC). The GlcA standard was partially lactonized during acid methanolysis, thus yielding six glycoside peaks in GC. If all six GlcA-derived peaks were included in the GlcA calibration curve, the calculated meGlcA content was underestimated by 30% compared with that obtained using the purified meGlcA as a standard. The meGlcA content was best estimated by including either the two main GlcA peaks or only peaks corresponding to pyranosides and furanosides of GlcA in the calibration curve.

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

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.ResultsAnAXE1-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.

Functional comparison of versatile carbohydrate esterases from families CE1, CE6 and CE16 on acetyl-4-O-methylglucuronoxylan and acetyl-galactoglucomannan

BACKGROUND The backbone structure of many hemicelluloses is acetylated, which presents a challenge when the objective is to convert corresponding polysaccharides to fermentable sugars or else recover hemicelluloses for biomaterial applications. Carbohydrate esterases (CE) can be harnessed to overcome these challenges. METHODS Enzymes from different CE families, AnAcXE (CE1), OsAcXE (CE6), and MtAcE (CE16) were compared based on action and position preference towards acetyl-4-O-methylglucuronoxylan (MGX) and acetyl-galactoglucomannan (GGM). To determine corresponding positional preferences, the relative rate of acetyl group released by each enzyme was analyzed by real time 1H NMR. RESULTS AnAcXE (CE1) showed lowest specific activity towards MGX, where OsAcXE (CE6) and MtAcE were approximately four times more active than AnAcXE (CE1). MtAcE (CE16) was further distinguished by demonstrating 100 times higher activity on GGM compared to AnAcXE (CE1) and OsAcXE (CE6), and five times higher activity on GGM than MGX. Following 24h incubation, all enzymes removed between 78 and 93% of total acetyl content from MGX and GGM, where MtAcE performed best on both substrates. MAJOR CONCLUSIONS Considering action on MGX, all esterases showed preference for doubly substituted xylopyranosyl residues (2,3-O-acetyl-Xylp). Considering action on GGM, OsAcXE (CE6) preferentially targeted 2-O-acetyl-mannopyranosyl residues (2-O-acetyl-Manp) whereas AnAcXE (CE1) demonstrated highest activity towards 3-O-acetyl-Manp positions; regiopreference of MtAcE (CE16) on GGM was less clear. GENERAL SIGNIFICANCE The current comparative analysis identifies options to control the position of acetyl group release at initial stages of reaction, and enzyme combinations likely to accelerate deacetylation of major hemicellulose sources.

Active fungal GH115 α-glucuronidase produced in Arabidopsis thaliana affects only the UX1-reactive glucuronate decorations on native glucuronoxylans

BackgroundExpressing microbial polysaccharide-modifying enzymes in plants is an attractive approach to custom tailor plant lignocellulose and to study the importance of wall structures to plant development. Expression of α-glucuronidases in plants to modify the structures of glucuronoxylans has not been yet attempted. Glycoside hydrolase (GH) family 115 α-glucuronidases cleave the internal α-D-(4-O-methyl)glucopyranosyluronic acid ((Me)GlcA) from xylans or xylooligosaccharides. In this work, a GH115 α-glucuronidase from Schizophyllum commune, ScAGU115, was expressed in Arabidopsis thaliana and targeted to apoplast. The transgene effects on native xylans’ structures, plant development, and lignocellulose saccharification were evaluated and compared to those of knocked out glucuronyltransferases AtGUX1 and AtGUX2.ResultsThe ScAGU115 extracted from cell walls of Arabidopsis was active on the internally substituted aldopentaouronic acid (XUXX). The transgenic plants did not show any change in growth or in lignocellulose saccharification. The cell wall (Me)GlcA and other non-cellulosic sugars, as well as the lignin content, remained unchanged. In contrast, the gux1gux2 double mutant showed a 70% decrease in (Me)GlcA to xylose molar ratio, and, interestingly, a 60% increase in the xylose content. Whereas ScAGU115-expressing plants exhibited a decreased signal in native secondary walls from the monoclonal antibody UX1 that recognizes (Me)GlcA on non-acetylated xylan, the signal was not affected after wall deacetylation. In contrast, gux1gux2 mutant was lacking UX1 signals in both native and deacetylated cell walls. This indicates that acetyl substitution on the xylopyranosyl residue carrying (Me)GlcA or on the neighboring xylopyranosyl residues may restrict post-synthetic modification of xylans by ScAGU115 in planta.ConclusionsActive GH115 α-glucuronidase has been produced for the first time in plants. The cell wall–targeted ScAGU115 was shown to affect those glucuronate substitutions of xylan, which are accessible to UX1 antibody and constitute a small fraction in Arabidopsis, whereas majority of (Me)GlcA substitutions were resistant, most likely due to the shielding by acetyl groups. Plants expressing ScAGU115 did not show any defects under laboratory conditions indicating that the UX1 epitope of xylan is not essential under these conditions. Moreover the removal of the UX1 xylan epitope does not affect lignocellulose saccharification.

Glucuronic acid in Arabidopsis thaliana xylans carries a novel pentose substituent

Glucuronic acids in Arabidopsis thaliana xylans exist in 4-O-methylated (MeGlcA) and non-methylated (GlcA) forms at a ratio of about 3:2. The matrix-assisted laser desorption/ionization mass spectrometry analysis of the endoxylanase liberated acidic oligosaccharides from the Arabidopsis inflorescence stem showed that two peaks with GlcA (GlcA-Xyl4Ac1 and GlcA-Xyl5Ac2) had abnormally high intensities, as well as different tandem mass spectra, than their 4-O-methylated counterparts. These peaks were interestingly enriched in the xylan biosynthesis mutant irx7 and irx9-1. Multi-stages fragmentation analysis using negative ion electrospray-ion trap mass spectrometry indicated that this GlcA was further carrying a pentose residue in the glucuronoxylan-derived oligosaccharide from irx9-1. The structure was also identified in Arabidopsis wild type. The results prove evidence of a new pentose substitution on the GlcA residue of Arabidopsis GX, which is likely present in the primary walls.