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 RWA genes encoding acetyl-CoA transporters using the 35S promoter, or constitutively and wood-specifically (using the WP promoter) expressed fungal acetyl xylan esterases of families CE1 (AnAXE1) and CE5 (HjAXE), to reduce acetylation in hybrid aspen. All these transformations improved the saccharification of wood from greenhouse-grown trees. Here, we describe the chemical properties and saccharification potential of the resulting lines grown in a five-year field trial, and one type of them (WP:AnAXE1) in greenhouse conditions. Chemically, the lignocellulose of the field- and greenhouse-field-grown plants slightly differed, but the reductions in acetylation and saccharification improvement of engineered trees were largely maintained in the field. The main novel phenotypic observation in the field was higher lignification in lines with the WP promoter than those with the 35S promoter. Following growth in the field, saccharification glucose yields were higher from most transformed lines than from wild-type (WT) plants with no pretreatment, but there was no improvement in saccharification with acid pretreatment. Thus, acid pretreatment removes most recalcitrance caused by acetylation. We found a complex relationship between acetylation and glucose yields in saccharification without pretreatment, suggesting that other variables, for example, the acetylation pattern, affect recalcitrance. Bigger gains in glucose yields were observed in lines with the 35S promoter than in those with the WP promoter, possibly due to their lower lignin content. However, better lignocellulose saccharification of these lines was offset by a growth penalty and their glucose yield per tree was lower. In a comparison of the best lines with each construct, WP:AnAXE1 provided the highest glucose yield per tree from saccharification, with and without pretreatment, WP:HjAXE yields were similar to those of WT plants, and yields of lines with other constructs were lower. These results show that lignocellulose properties of field-grown trees can be improved by reducing cell wall acetylation using various approaches, but some affect productivity in the field. Thus, better understanding of molecular and physiological consequences of deacetylation is needed to obtain quantitatively better results

Sequential extraction of hemicelluloses by subcritical water improves saccharification of hybrid aspen wood grown in greenhouse and field conditions

Fast growing hardwoods are one of the major renewable resources available to produce bio-based materials, platform chemicals and biofuels. However, the industrial processing of lignocellulosic biomass is hindered by the complex molecular structure of the cell wall components and their supramolecular organization. This highlights the necessity of improving green processing strategies to enhance biomass conversion to valuable products from industrial wood production species. In the present study, we implemented a hydrothermal step by sequential subcritical water (SW) in aspen wood prior to saccharification and validated the process for trees grown in greenhouse and field conditions. Subcritical water enables extraction of non-cellulosic cell wall polysaccharides in native polymeric form. A major part of the pectic fraction was easily extracted within the first 10 min, while acetylated xylan was enriched in the subsequent extracts after 20- and 30-min rounds. Prolonged extraction (above 60 min) resulted in partial deacetylation and a reduction of the molar mass of xylan. The analysis of the residues enriched with cellulose and lignin showed several micromorphological changes caused by subcritical water treatment, such as an increased porosity, a loosening of the fibre matrix and a decrease in the macrofibrillar dimensions. These morphological and molecular changes in the organization of cell wall polymers after SW treatment significantly enhanced saccharification yields compared to those of non-treated aspen wood chips from both field and greenhouse conditions. Our study demonstrates that SW can be implemented as pretreatment prior to saccharification reducing the requirements for chemical acid pretreatments. This process enables the extraction of native non-cellulosic cell wall polymers for potential material applications and promotes the subsequent biochemical conversion of the residual biomass into fermentable sugars and platform chemicals in future biorefineries

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

Fast-growing broad-leaf tree species can serve as feedstocks for production of bio-based chemicals and fuels through biochemical conversion of wood to monosaccharides. This conversion is hampered by the xylan acetylation pattern. To reduce xylan acetylation in the wood, the Hypocrea jecorina acetyl xylan esterase (HjAXE) from carbohydrate esterase (CE) family 5 was expressed in hybrid aspen under the control of the wood-specific PtGT43B promoter and targeted to the secretory pathway. The enzyme was predicted to deacetylate polymeric xylan in the vicinity of cellulose due to the presence of a cellulose-binding module. Cell-wall-bound protein fractions from developing wood of transgenic plants were capable of releasing acetyl from finely ground wood powder, indicative of active AXE present in cell walls of these plants, whereas no such activity was detected in wild-type plants. The transgenic lines grew in height and diameter as well as wild-type trees, whereas their internodes were slightly shorter, indicating higher leaf production. The average acetyl content in the wood of these lines was reduced by 13%, mainly due to reductions in di-acetylated xylose units, and in C-2 and C-3 mono-acetylated xylose units. Analysis of soluble cell wall polysaccharides revealed a 4% reduction in the fraction of xylose units and an 18% increase in the fraction of glucose units, whereas the contents of cellulose and lignin were not affected. Enzymatic saccharification of wood from transgenic plants resulted in 27% higher glucose yield than for wild-type plants. Brunauer-Emmett-Teller (BET) analysis and Simons' staining pointed toward larger surface area and improved cellulose accessibility for wood from transgenic plants compared to wood from wild-type plants, which could be achieved by HjAXE deacetylating xylan bound to cellulose. The results show that CE5 family can serve as a source of enzymes for in planta reduction of recalcitrance to saccharification.Peer reviewe

Aspen Tension Wood Fibers Contain β-(1→4)-Galactans and Acidic Arabinogalactans Retained by Cellulose Microfibrils in Gelatinous Walls

Contractile cell walls are found in various plant organs and tissues such as tendrils, contractile roots, and tension wood. The tension-generating mechanism is not known but is thought to involve special cell wall architecture. We previously postulated that tension could result from the entrapment of certain matrix polymers within cellulose microfibrils. As reported here, this hypothesis was corroborated by sequential extraction and analysis of cell wall polymers that are retained by cellulose microfibrils in tension wood and normal wood of hybrid aspen (Populus tremula × Populus tremuloides). β-(1→4)-Galactan and type II arabinogalactan were the main large matrix polymers retained by cellulose microfibrils that were specifically found in tension wood. Xyloglucan was detected mostly in oligomeric form in the alkali-labile fraction and was enriched in tension wood. β-(1→4)-Galactan and rhamnogalacturonan I backbone epitopes were localized in the gelatinous cell wall layer. Type II arabinogalactans retained by cellulose microfibrils had a higher content of (methyl)glucuronic acid and galactose in tension wood than in normal wood. Thus, β-(1→4)-galactan and a specialized form of type II arabinogalactan are trapped by cellulose microfibrils specifically in tension wood and, thus, are the main candidate polymers for the generation of tensional stresses by the entrapment mechanism. We also found high β-galactosidase activity accompanying tension wood differentiation and propose a testable hypothesis that such activity might regulate galactan entrapment and, thus, mechanical properties of cell walls in tension wood.This work was supported by the Swedish Governmental Agency for Innovation Systems, the Swedish Research Council, the Russian Foundation for Basic Research (grant nos. 15–04–02560 and 15–04–05721), and the Biotechnology and Biological Sciences Research Council (grant no. BB/G016240/1 and funds from the Sustainable Energy Centre Cell Wall Sugars Programme).This is the author accepted manuscript. The final version is available from the American Society of Plant Biologists via http://dx.doi.org/10.​1104/​pp.​15.​0069

Aspen Tension Wood Fibers Contain β-(1---> 4)-Galactans and Acidic Arabinogalactans Retained by Cellulose Microfibrils in Gelatinous Walls.

Contractile cell walls are found in various plant organs and tissues such as tendrils, contractile roots, and tension wood. The tension-generating mechanism is not known but is thought to involve special cell wall architecture. We previously postulated that tension could result from the entrapment of certain matrix polymers within cellulose microfibrils. As reported here, this hypothesis was corroborated by sequential extraction and analysis of cell wall polymers that are retained by cellulose microfibrils in tension wood and normal wood of hybrid aspen (Populus tremula × Populus tremuloides). β-(1→4)-Galactan and type II arabinogalactan were the main large matrix polymers retained by cellulose microfibrils that were specifically found in tension wood. Xyloglucan was detected mostly in oligomeric form in the alkali-labile fraction and was enriched in tension wood. β-(1→4)-Galactan and rhamnogalacturonan I backbone epitopes were localized in the gelatinous cell wall layer. Type II arabinogalactans retained by cellulose microfibrils had a higher content of (methyl)glucuronic acid and galactose in tension wood than in normal wood. Thus, β-(1→4)-galactan and a specialized form of type II arabinogalactan are trapped by cellulose microfibrils specifically in tension wood and, thus, are the main candidate polymers for the generation of tensional stresses by the entrapment mechanism. We also found high β-galactosidase activity accompanying tension wood differentiation and propose a testable hypothesis that such activity might regulate galactan entrapment and, thus, mechanical properties of cell walls in tension wood.This work was supported by the Swedish Governmental Agency for Innovation Systems, the Swedish Research Council, the Russian Foundation for Basic Research (grant nos. 15–04–02560 and 15–04–05721), and the Biotechnology and Biological Sciences Research Council (grant no. BB/G016240/1 and funds from the Sustainable Energy Centre Cell Wall Sugars Programme).This is the author accepted manuscript. The final version is available from the American Society of Plant Biologists via http://dx.doi.org/10.​1104/​pp.​15.​0069

The Sample Handling System for the Mars Icebreaker Life Mission: from Dirt to Data

The Mars icebreaker life mission will search for subsurface life on mars. It consists of three payload elements: a drill to retrieve soil samples from approx. 1 meter below the surface, a robotic sample handling system to deliver the sample from the drill to the instruments, and the instruments themselves. This paper will discuss the robotic sample handling system

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. All four genes are expressed in developing wood but only RWA-A and -B are activated by master switches of the secondary cell wall PtNST1 and PtMYB21. Histochemical analysis of promoter:: GUS lines in hybrid aspen (Populus tremula x tremuloides) showed activation of RWA-A and -B promoters in the secondary wall formation zone, while RWA-C and -D promoter activity was diffuse. Ectopic downregulation of either clade reduced wood xylan and xyloglucan acetylation. Suppressing both clades simultaneously using the wood-specific promoter reduced wood acetylation by 25% and decreased acetylation at position 2 of Xylp in the dimethyl sulfoxide-extracted xylan. This did not affect plant growth but decreased xylose and increased glucose contents in the noncellulosic monosaccharide fraction, and increased glucose and xylose yields of wood enzymatic hydrolysis without pretreatment. Both RWA clades regulate wood xylan acetylation in aspen and are promising targets to improve wood saccharification.Peer reviewe

Suppression of xylan endotransglycosylase PtxtXyn10A affects cellulose microfibril angle in secondary wall in aspen wood.

Certain xylanases from family GH10 are highly expressed during secondary wall deposition, but their function is unknown. We carried out functional analyses of the secondary-wall specific PtxtXyn10A in hybrid aspen (Populus tremula × tremuloides). PtxtXyn10A function was analysed by expression studies, overexpression in Arabidopsis protoplasts and by downregulation in aspen. PtxtXyn10A overexpression in Arabidopsis protoplasts resulted in increased xylan endotransglycosylation rather than hydrolysis. In aspen, the enzyme was found to be proteolytically processed to a 68 kDa peptide and residing in cell walls. Its downregulation resulted in a corresponding decrease in xylan endotransglycosylase activity and no change in xylanase activity. This did not alter xylan molecular weight or its branching pattern but affected the cellulose-microfibril angle in wood fibres, increased primary growth (stem elongation, leaf formation and enlargement) and reduced the tendency to form tension wood. Transcriptomes of transgenic plants showed downregulation of tension wood related genes and changes in stress-responsive genes. The data indicate that PtxtXyn10A acts as a xylan endotransglycosylase and its main function is to release tensional stresses arising during secondary wall deposition. Furthermore, they suggest that regulation of stresses in secondary walls plays a vital role in plant development.Formas (including HemiPop and FuncFiber), Swedish Research Council (VR), Swedish Governmental Agency for Innovation Systems (VINNOVA), Swedish Center for Biomimetic Fiber Engineering (funded by the Knut & Alice Wallenberg Foundation and the Foundation for Strategic Research), European projects EDEN (QLK5-CT-2001-00443) and RENEWALL, FORE, Bio4Energy, Wood Ultrastructure Research Centre, SamNordisk Skogsforskning (project no. 107), Japan Society for the Promotion of Science (JSPS KAKENHI Grant Number 24580243) and the Academy of Finland (1127759).This is the accepted manuscript. The final version is available at http://onlinelibrary.wiley.com/doi/10.1111/nph.13099/abstract