Covalent interactions between lignin and hemicelluloses in plant secondary cell walls.

The plant secondary cell wall is a complex structure composed of polysaccharides and lignin, and is a key evolutionary innovation of vascular land plants. Although cell wall composition is well understood, the cross-linking of the different polymers is only now yielding to investigation. Cross-linking between hemicelluloses and lignin occurs via two different mechanisms: incorporation into lignin by radical coupling of ferulate substitutions on xylan in commelinid monocots, and incorporation of hemicellulosic glycosyl residues by re-aromatisation of lignification intermediates. Recent genetic evidence indicates that hemicellulose:lignin cross-linking has a substantial impact on plant cell wall recalcitrance. Engineering plant biomass with modified frequencies of cross-links will have significant impacts on biomass utilisation.O.M.T was a recipient of an iCASE studentship from the BBSRC and Novozymes (Reference BB/M015432/1). P.D. was supported by the Leverhulme Trust Centre for Natural Material Innovation and the OpenPlant Synthetic Biology Research Centre BB/L014130/1

Molecular architecture of softwood revealed by solid-state NMR

Economically important softwood from conifers is mainly composed of the polysaccharides cellulose, galactoglucomannan and xylan, and the phenolic polymer, lignin. The interactions between these polymers lead to wood mechanical strength and must be overcome in biorefining. Here, we use 13C multidimensional solid-state NMR to analyse the polymer interactions in never-dried cell walls of the softwood, spruce. In contrast to some earlier softwood cell wall models, most of the xylan binds to cellulose in the two-fold screw conformation. Moreover, galactoglucomannan alters its conformation by intimately binding to the surface of cellulose microfibrils in a semi-crystalline fashion. Some galactoglucomannan and xylan bind to the same cellulose microfibrils, and lignin is associated with both of these cellulose-bound polysaccharides. We propose a model of softwood molecular architecture which explains the origin of the different cellulose environments observed in the NMR experiments. Our model will assist strategies for improving wood usage in a sustainable bioeconomy

Structural Imaging of Native Cryo-Preserved Secondary Cell Walls Reveals the Presence of Macrofibrils and Their Formation Requires Normal Cellulose, Lignin and Xylan Biosynthesis

The woody secondary cell walls of plants are the largest repository of renewable carbon biopolymers on the planet. These walls are made principally from cellulose and hemicelluloses and are impregnated with lignin. Despite their importance as the main load bearing structure for plant growth, as well as their industrial importance as both a material and energy source, the precise arrangement of these constituents within the cell wall is not yet fully understood. We have adapted low temperature scanning electron microscopy (cryo-SEM) for imaging the nanoscale architecture of angiosperm and gymnosperm cell walls in their native hydrated state. Our work confirms that cell wall macrofibrils, cylindrical structures with a diameter exceeding 10 nm, are a common feature of the native hardwood and softwood samples. We have observed these same structures in Arabidopsis thaliana secondary cell walls, enabling macrofibrils to be compared between mutant lines that are perturbed in cellulose, hemicellulose, and lignin formation. Our analysis indicates that the macrofibrils in Arabidopsis cell walls are dependent upon the proper biosynthesis, or composed, of cellulose, xylan, and lignin. This study establishes that cryo-SEM is a useful additional approach for investigating the native nanoscale architecture and composition of hardwood and softwood secondary cell walls and demonstrates the applicability of Arabidopsis genetic resources to relate fibril structure with wall composition and biosynthesis.Peer reviewe

Removal of glucuronic acid from xylan is a strategy to improve the conversion of plant biomass to sugars for bioenergy

BACKGROUND: Plant lignocellulosic biomass can be a source of fermentable sugars for the production of second generation biofuels and biochemicals. The recalcitrance of this plant material is one of the major obstacles in its conversion into sugars. Biomass is primarily composed of secondary cell walls, which is made of cellulose, hemicelluloses and lignin. Xylan, a hemicellulose, binds to the cellulose microfibril and is hypothesised to form an interface between lignin and cellulose. Both softwood and hardwood xylan carry glucuronic acid side branches. As xylan branching may be important for biomass recalcitrance and softwood is an abundant, non-food competing, source of biomass it is important to investigate how conifer xylan is synthesised. RESULTS: Here, we show using Arabidopsis gux mutant biomass that removal of glucuronosyl substitutions of xylan can allow 30% more glucose and over 700% more xylose to be released during saccharification. Ethanol yields obtained through enzymatic saccharification and fermentation of gux biomass were double those obtained for non-mutant material. Our analysis of additional xylan branching mutants demonstrates that absence of GlcA is unique in conferring the reduced recalcitrance phenotype. As in hardwoods, conifer xylan is branched with GlcA. We use transcriptomic analysis to identify conifer enzymes that might be responsible for addition of GlcA branches onto xylan in industrially important softwood. Using a combination of in vitro and in vivo activity assays, we demonstrate that a white spruce (Picea glauca) gene, PgGUX, encodes an active glucuronosyl transferase. Glucuronic acid introduced by PgGUX reduces the sugar release of Arabidopsis gux mutant biomass to wild-type levels indicating that it can fulfil the same biological function as native glucuronosylation. CONCLUSION: Removal of glucuronic acid from xylan results in the largest increase in release of fermentable sugars from Arabidopsis plants that grow to the wild-type size. Additionally, plant material used in this work did not undergo any chemical pretreatment, and thus increased monosaccharide release from gux biomass can be achieved without the use of environmentally hazardous chemical pretreatment procedures. Therefore, the identification of a gymnosperm enzyme, likely to be responsible for softwood xylan glucuronosylation, provides a mutagenesis target for genetically improved forestry trees.This work was supported by the Leverhulme Trust Centre for Natural Material Innovation and the OpenPlant Synthetic Biology Research Centre. J.J.L. was in receipt of a studentship from the Biotechnology and Biological Sciences Research Council (BBSRC) of the UK as part of the Cambridge BBSRC-DTP Programme (Reference BB/J014540/1). O.M.T was a recipient of an iCASE studentship from the BBSRC (Reference BB/M015432/1)

An engineered GH1 β-glucosidase displays enhanced glucose tolerance and increased sugar release from lignocellulosic materials.

β-glucosidases play a critical role among the enzymes in enzymatic cocktails designed for plant biomass deconstruction. By catalysing the breakdown of β-1, 4-glycosidic linkages, β-glucosidases produce free fermentable glucose and alleviate the inhibition of other cellulases by cellobiose during saccharification. Despite this benefit, most characterised fungal β-glucosidases show weak activity at high glucose concentrations, limiting enzymatic hydrolysis of plant biomass in industrial settings. In this study, structural analyses combined with site-directed mutagenesis efficiently improved the functional properties of a GH1 β-glucosidase highly expressed by Trichoderma harzianum (ThBgl) under biomass degradation conditions. The tailored enzyme displayed high glucose tolerance levels, confirming that glucose tolerance can be achieved by the substitution of two amino acids that act as gatekeepers, changing active-site accessibility and preventing product inhibition. Furthermore, the enhanced efficiency of the engineered enzyme in terms of the amount of glucose released and ethanol yield was confirmed by saccharification and simultaneous saccharification and fermentation experiments using a wide range of plant biomass feedstocks. Our results not only experimentally confirm the structural basis of glucose tolerance in GH1 β-glucosidases but also demonstrate a strategy to improve technologies for bioethanol production based on enzymatic hydrolysis

Ectopic callose deposition into woody biomass modulates the nano-architecture of macrofibrils

Plant biomass plays an increasingly important role in the circular bioeconomy, replacing non-renewable fossil resources. Genetic engineering of this lignocellulosic biomass could benefit biorefinery transformation chains by lowering economic and technological barriers to industrial processing. However, previous efforts have mostly targeted the major constituents of woody biomass: cellulose, hemicellulose and lignin. Here we report the engineering of wood structure through the introduction of callose, a polysaccharide novel to most secondary cell walls. Our multiscale analysis of genetically engineered poplar trees shows that callose deposition modulates cell wall porosity, water and lignin contents and increases the lignin-cellulose distance, ultimately resulting in substantially decreased biomass recalcitrance. We provide a model of the wood cell wall nano-architecture engineered to accommodate the hydrated callose inclusions. Ectopic polymer introduction into biomass manifests in new physico-chemical properties and offers new avenues when considering lignocellulose engineering.Bourdon et al. demonstrate the possibility to ectopically synthesize callose, a polymer restricted to primary cell walls, into Arabidopsis and aspen secondary cell walls to manipulate their ultrastructure and ultimately reduce their recalcitrance

Two conifer GUX clades are responsible for distinct glucuronic acid patterns on xylan

Wood of coniferous trees (softwood), is a globally significant carbon sink and an important source of biomass. Despite that, little is known about the genetic basis of softwood cell wall biosynthesis. Branching of xylan, one of the main hemicelluloses in softwood secondary cell walls, with glucuronic acid (GlcA) is critical for biomass recalcitrance. Here, we investigate the decoration patterns of xylan by conifer GlucUronic acid substitution of Xylan (GUX) enzymes. Through molecular phylogenetics we identify two distinct conifer GUX clades. Using transcriptional profiling we show that the genes are preferentially expressed in secondary cell wall forming tissues. With in vitro and in planta assays we demonstrate that conifer GUX enzymes from both clades are active glucuronyltransferases. Conifer GUX enzymes from each clade have different specific activities. While members of clade one add evenly spaced GlcA branches, the members of clade two are also capable of glucuronidating two consecutive xyloses. Importantly, these types of xylan patterning are present in softwood. As xylan patterning might modulate xylan-cellulose and xylan-lignin interactions, our results further the understanding of softwood cell wall biosynthesis and provide breeding or genetic engineering targets that can be used to modify softwood properties

Structural Imaging of Native Cryo-Preserved Secondary Cell Walls Reveals the Presence of Macrofibrils and Their Formation Requires Normal Cellulose, Lignin and Xylan Biosynthesis.

The woody secondary cell walls of plants are the largest repository of renewable carbon biopolymers on the planet. These walls are made principally from cellulose and hemicelluloses and are impregnated with lignin. Despite their importance as the main load bearing structure for plant growth, as well as their industrial importance as both a material and energy source, the precise arrangement of these constituents within the cell wall is not yet fully understood. We have adapted low temperature scanning electron microscopy (cryo-SEM) for imaging the nanoscale architecture of angiosperm and gymnosperm cell walls in their native hydrated state. Our work confirms that cell wall macrofibrils, cylindrical structures with a diameter exceeding 10 nm, are a common feature of the native hardwood and softwood samples. We have observed these same structures in Arabidopsis thaliana secondary cell walls, enabling macrofibrils to be compared between mutant lines that are perturbed in cellulose, hemicellulose, and lignin formation. Our analysis indicates that the macrofibrils in Arabidopsis cell walls are dependent upon the proper biosynthesis, or composed, of cellulose, xylan, and lignin. This study establishes that cryo-SEM is a useful additional approach for investigating the native nanoscale architecture and composition of hardwood and softwood secondary cell walls and demonstrates the applicability of Arabidopsis genetic resources to relate fibril structure with wall composition and biosynthesis