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.

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.

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.

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.

Spent mushroom substrates for ethanol production - Effect of chemical and structural factors on enzymatic saccharification and ethanolic fermentation of Lentinula edodes-pretreated hardwood.

Spent mushroom substrates (SMS) from cultivation of shiitake (Lentinula edodes) on three hardwood species were investigated regarding their potential for cellulose saccharification and for ethanolic fermentation of the produced hydrolysates. High glucan digestibility was achieved during enzymatic saccharification of the SMSs, which was related to the low mass fractions of lignin and xylan, and it was neither affected by the relative content of lignin guaiacyl units nor the substrate crystallinity. The high nitrogen content in SMS hydrolysates, which was a consequence of the fungal pretreatment, was positive for the fermentation, and it ensured ethanol yields corresponding to 84-87% of the theoretical value in fermentations without nutrient supplementation.

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.

A multi-omics approach reveals function of Secretory Carrier-Associated Membrane Proteins in wood formation of​ ​​Populus​​ ​trees.

Background: Secretory Carrier-Associated Membrane Proteins (SCAMPs) are highly conserved 32-38 kDa proteins that are involved in membrane trafficking. A systems approach was taken to elucidate function of SCAMPs in wood formation of Populus trees. Phenotypic and multi-omics analyses were performed in woody tissues of transgenic Populus trees carrying an RNAi construct for Populus tremula x tremuloides SCAMP3 (PttSCAMP3; Potri.

A collection of genetically engineered Populus trees reveals wood biomass traits that predict glucose yield from enzymatic hydrolysis.

Wood represents a promising source of sugars to produce bio-based renewables, including biofuels. However, breaking down lignocellulose requires costly pretreatments because lignocellulose is recalcitrant to enzymatic saccharification. Increasing saccharification potential would greatly contribute to make wood a competitive alternative to petroleum, but this requires improving wood properties.

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.

Expression of a fungal glucuronoyl esterase in Populus: effects on wood properties and saccharification efficiency.

The secondary walls of angiosperms contain large amounts of glucuronoxylan that is thought to be covalently linked to lignin via ester bonds between 4-O-methyl-α-D-glucuronic acid (4-O-Me-GlcA) moieties in glucuronoxylan and alcohol groups in lignin. This linkage is proposed to be hydrolysed by glucuronoyl esterases (GCEs) secreted by wood-degrading fungi. We report effects of overexpression of a GCE from the white-rot basidiomycete Phanerochaete carnosa, PcGCE, in hybrid aspen (Populus tremula L.

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.