Modification of xylan in secondary walls alters cell wall biosynthesis and wood formation programs and improves saccharification.

Wood of broad-leaf tree species is a valued source of renewable biomass for biorefinery and a target for genetic improvement efforts to reduce its recalcitrance. Glucuronoxylan (GX) plays a key role in recalcitrance through its interactions with cellulose and lignin. To reduce recalcitrance, we modified wood GX by expressing GH10 and GH11 endoxylanases from Aspergillus nidulans in hybrid aspen (Populus tremula L.

Unraveling the unique structural motifs of glucuronoxylan from hybrid aspen wood.

Xylan is a fundamental structural polysaccharide in plant secondary cell walls and a valuable resource for biorefinery applications. Deciphering the molecular motifs of xylans that mediate their interaction with cellulose and lignin is fundamental to understand the structural integrity of plant cell walls and to design lignocellulosic materials. In the present study, we investigated the pattern of acetylation and glucuronidation substitution in hardwood glucuronoxylan (GX) extracted from aspen wood using subcritical water and alkaline conditions.

The effect of nitrogen source and levels on hybrid aspen tree physiology and wood formation.

Nitrogen can be taken up by trees in the form of nitrate, ammonium and amino acids, but the influence of the different forms on tree growth and development is poorly understood in angiosperm species like Populus. We studied the effects of both organic and inorganic forms of nitrogen on growth and wood formation of hybrid aspen trees in experimental conditions that allowed growth under four distinct steady-state nitrogen levels. Increased nitrogen availability had a positive influence on biomass accumulation and the radial dimensions of both xylem vessels and fibers, and a negative influence on wood density.

In vitro evaluation of the application of an optimized xylanase cocktail for improved monogastric feed digestibility.

Xylanases from glycoside hydrolase (GH) families 10 and 11 are common feed additives for broiler chicken diets due to their catalytic activity on the nonstarch polysaccharide xylan. This study investigated the potential of an optimized binary GH10 and GH11 xylanase cocktail to mitigate the antinutritional effects of xylan on the digestibility of locally sourced chicken feed. Immunofluorescence visualization of the activity of the xylanase cocktail on xylan in the yellow corn of the feed showed a substantial collapse in the morphology of cell walls.

LPMO-supported saccharification of biomass: effects of continuous aeration of reaction mixtures with variable fractions of water-insoluble solids and cellulolytic enzymes.

Background: High substrate concentrations and high sugar yields are important aspects of enzymatic saccharification of lignocellulosic substrates. The benefit of supporting the catalytic action of lytic polysaccharide monooxygenase (LPMO) through continuous aeration of slurries of pretreated softwood was weighed against problems associated with increasing substrate content (quantitated as WIS, water-insoluble solids, in the range 12.5-17.

Flexure wood formation via growth reprogramming in hybrid aspen involves jasmonates and polyamines and transcriptional changes resembling tension wood development.

Stem bending in trees induces flexure wood but its properties and development are poorly understood. Here, we investigated the effects of low-intensity multidirectional stem flexing on growth and wood properties of hybrid aspen, and on its transcriptomic and hormonal responses. Glasshouse-grown trees were either kept stationary or subjected to several daily shakes for 5 wk, after which the transcriptomes and hormones were analyzed in the cambial region and developing wood tissues, and the wood properties were analyzed by physical, chemical and microscopy techniques.

Impact of xylan on field productivity and wood saccharification properties in aspen.

Xylan that comprises roughly 25% of hardwood biomass is undesirable in biorefinery applications involving saccharification and fermentation. Efforts to reduce xylan levels have therefore been made in many species, usually resulting in improved saccharification. However, such modified plants have not yet been tested under field conditions.

Genetic markers and tree properties predicting wood biorefining potential in aspen (Populus tremula) bioenergy feedstock.

Background: Wood represents the majority of the biomass on land and constitutes a renewable source of biofuels and other bioproducts. However, wood is recalcitrant to bioconversion, raising a need for feedstock improvement in production of, for instance, biofuels. We investigated the properties of wood that affect bioconversion, as well as the underlying genetics, to help identify superior tree feedstocks for biorefining.

Field testing of transgenic aspen from large greenhouse screening identifies unexpected winners.

Trees constitute promising renewable feedstocks for biorefinery using biochemical conversion, but their recalcitrance restricts their attractiveness for the industry. To obtain trees with reduced recalcitrance, large-scale genetic engineering experiments were performed in hybrid aspen blindly targeting genes expressed during wood formation and 32 lines representing seven constructs were selected for characterization in the field. Here we report phenotypes of five-year old trees considering 49 traits related to growth and wood properties.

Xylan glucuronic acid side chains fix suberin-like aliphatic compounds to wood cell walls.

Wood is the most important repository of assimilated carbon in the biosphere, in the form of large polymers (cellulose, hemicelluloses including glucuronoxylan, and lignin) that interactively form a composite, together with soluble extractives including phenolic and aliphatic compounds. Molecular interactions among these compounds are not fully understood. We have targeted the expression of a fungal α-glucuronidase to the wood cell wall of aspen (Populus tremula L.

Comparison of solid and liquid fractions of pretreated Norway spruce as reductants in LPMO-supported saccharification of cellulose.

The role of lignin in enzymatic saccharification of cellulose involving lytic polysaccharide monooxygenase (LPMO) was investigated in experiments with the solid and liquid fractions of pretreated Norway spruce from a biorefinery demonstration plant using hydrothermal pretreatment and impregnation with sulfur dioxide. Pretreated biomass before and after enzymatic saccharification was characterized using HPAEC, HPLC, -GC/MS, 2D-HSQC NMR, FTIR, and SEM. Chemical characterization indicated that relatively harsh pretreatment conditions resulted in that the solid phase contained no or very little hemicellulose but considerable amounts of pseudo-lignin, and that the liquid phase contained a relatively high concentration (∼5 g/L) of lignin-derived phenolics.

Saccharification Potential of Transgenic Greenhouse- and Field-Grown Aspen Engineered for Reduced Xylan Acetylation.

High acetylation of xylan in hardwoods decreases their value as biorefinery feedstocks. To counter this problem, we have constitutively suppressed genes encoding acetyl-CoA transporters using the promoter, or constitutively and wood-specifically (using the promoter) expressed fungal acetyl xylan esterases of families CE1 () and CE5 (), to reduce acetylation in hybrid aspen. All these transformations improved the saccharification of wood from greenhouse-grown trees.

Overexpression of vesicle-associated membrane protein PttVAP27-17 as a tool to improve biomass production and the overall saccharification yields in Populus trees.

Background: Bioconversion of wood into bioproducts and biofuels is hindered by the recalcitrance of woody raw material to bioprocesses such as enzymatic saccharification. Targeted modification of the chemical composition of the feedstock can improve saccharification but this gain is often abrogated by concomitant reduction in tree growth.

Results: In this study, we report on transgenic hybrid aspen (Populus tremula × tremuloides) lines that showed potential to increase biomass production both in the greenhouse and after 5 years of growth in the field.

Engineering Non-cellulosic Polysaccharides of Wood for the Biorefinery.

Non-cellulosic polysaccharides constitute approximately one third of usable woody biomass for human exploitation. In contrast to cellulose, these substances are composed of several different types of unit monosaccharides and their backbones are substituted by various groups. Their structural diversity and recent examples of their modification in transgenic plants and mutants suggest they can be targeted for improving wood-processing properties, thereby facilitating conversion of wood in a biorefinery setting.

deacetylation of xylan affects lignin properties and improves saccharification of aspen wood.

Background: Lignocellulose 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 AXE1 from was introduced into aspen and targeted to cell walls.

Downregulation of RWA genes in hybrid aspen affects xylan acetylation and wood saccharification.

High acetylation of angiosperm wood hinders its conversion to sugars by glycoside hydrolases, subsequent ethanol fermentation and (hence) its use for biofuel production. We studied the REDUCED WALL ACETYLATION (RWA) gene family of the hardwood model Populus to evaluate its potential for improving saccharification. The family has two clades, AB and CD, containing two genes each.

Cytosolic invertase contributes to the supply of substrate for cellulose biosynthesis in developing wood.

Carbon for cellulose biosynthesis is derived from sucrose. Cellulose is synthesized from uridine 5'-diphosphoglucose (UDP-glucose), but the enzyme(s) responsible for the initial sucrose cleavage and the source of UDP-glucose for cellulose biosynthesis in developing wood have not been defined. We investigated the role of CYTOSOLIC INVERTASEs (CINs) during wood formation in hybrid aspen (Populus tremula × tremuloides) and characterized transgenic lines with reduced CIN activity during secondary cell wall biosynthesis.

Aspen pectate lyase PtxtPL1-27 mobilizes matrix polysaccharides from woody tissues and improves saccharification yield.

Background: Wood cell walls are rich in cellulose, hemicellulose and lignin. Hence, they are important sources of renewable biomass for producing energy and green chemicals. However, extracting desired constituents from wood efficiently poses significant challenges because these polymers are highly cross-linked in cell walls and are not easily accessible to enzymes and chemicals.