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At least 19 records
All sourcesNASA NTRSDOE OSTI
DOE OSTI2024

Biochemical Characterization of Rice Xylan Biosynthetic Enzymes in Determining Xylan Chain Elongation and Substitutions

Abstract Grass xylan consists of a linear chain of β-1,4-linked xylosyl residues that often form domains substituted only with either arabinofuranose (Araf) or glucuronic acid (GlcA)/methylglucuronic acid (MeGlcA) residues, and it lacks the unique reducing end tetrasaccharide sequence found in dicot xylan. The mechanism of how grass xylan backbone elongation is initiated and how its distinctive substitution pattern is determined remains elusive. Here, we performed biochemical characterization of rice xylan biosynthetic enzymes, including xylan synthases, glucuronyltransferases and methyltransferases. Activity assays of rice xylan synthases demonstrated that they required short xylooligomers as acceptors for their activities. While rice xylan glucuronyltransferases effectively glucuronidated unsubstituted xylohexaose acceptors, they transferred little GlcA residues onto (Araf)-substituted xylohexaoses and rice xylan 3-O-arabinosyltransferase could not arabinosylate GlcA-substituted xylohexaoses, indicating that their intrinsic biochemical properties may contribute to the distinctive substitution patterns of rice xylan. In addition, we found that rice xylan methyltransferase exhibited a low substrate binding affinity, which may explain the partial GlcA methylation in rice xylan. Furthermore, immunolocalization of xylan in xylem cells of both rice and Arabidopsis showed that it was deposited together with cellulose in secondary walls without forming xylan-rich nanodomains. Together, our findings provide new insights into the biochemical mechanisms underlying xylan backbone elongation and substitutions in grass species.

Cell Biology↗
DOE OSTI2022

Identification of xylan arabinosyl 2- O -xylosyltransferases catalyzing the addition of 2- O -xylosyl residue onto arabinosyl side chains of xylan in grass species

Grass xylan, the major hemicellulose in both primary and secondary cell walls, is heavily decorated with α-1,3-linked arabinofuranosyl (Araf) residues that may be further substituted at O-2 with xylosyl (Xyl) or Araf residues. Although xylan 3-O-arabinosyltransferases (XATs) catalyzing 3-O-Araf addition onto xylan have been characterized, glycosyltransferases responsible for the transfer of 2-O-Xyl or 2-O-Araf onto 3-O-Araf residues of xylan to produce the Xyl-Araf and Araf-Araf disaccharide side chains remain to be identified. In this report, we showed that a rice GT61 member, named OsXAXT1 (xylan arabinosyl 2-O-xylosyltransferase 1) herein, was able to mediate the addition of Xyl-Araf disaccharide side chains onto xylan when heterologously co-expressed with OsXAT2 in the Arabidopsis gux1/2/3 (glucuronic acid substitution of xylan 1/2/3) triple mutant that lacks any glycosyl substitutions. Recombinant OsXAXT1 protein expressed in human embryonic kidney 293 cells exhibited a xylosyltransferase activity catalyzing the addition of Xyl from UDP-Xyl onto arabinosylated xylooligomers. Further, consistent with its function as a xylan arabinosyl 2-O-xylosyltransferase, CRISPR-Cas9-mediated mutations of the OsXAXT1 gene in transgenic rice plants resulted in a reduction in the level of Xyl-Araf disaccharide side chains in xylan. Furthermore, we revealed that XAXT1 close homologs from several other grass species, including switchgrass, maize, and Brachypodium, possessed the same functions as OsXAXT1, indicating functional conservation of XAXTs in grass species. Together, our findings establish that grass XAXTs are xylosyltransferases catalyzing Xyl transfer onto O-2 of Araf residues of xylan to form the Xyl-Araf disaccharide side chains, which furthers our understanding of genes involved in xylan biosynthesis.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Structural features of xylan dictate reactivity and functionalization potential for bio-based materials

Plant-based materials have the potential to replace some petroleum-based products, offering compostability and biodegradability as critical advantages. Xylan-rich biomass sources are gaining recognition due to their abundance and underutilization in current industrial applications. Research of potential xylan applications has been complicated by the complex and heterogeneous structure that varies for different xylan feedstocks. Acylation is a broadly used reaction in functionalization of polysaccharides at an industrial scale. However, the efficiency of this reaction varies with the xylan source. To optimize xylan valorization, a systematic understanding of structure–reactivity relationships is essential. This study explores, characterizes, and compares various xylan feedstocks in the acylation process. Xylan feedstocks were analyzed for their chemical composition, degree of polymerization, branching, solubility, and presence of impurities. These features were correlated with xylan glycotypes’ reactivity toward functionalization with succinic anhydride in an optimized DMSO/KOH condition, achieving carboxyl contents of up to 1.46. We used principal component analysis and hierarchical clustering to identify key structural features of xylan that promote its reactivity. Our findings reveal that xylans with higher xylose content and lower degrees of branching exhibit enhanced reactivity, achieving higher carboxyl content and yields. Structural analyses confirmed successful modification, and light scattering analyses showed dramatic changes in the solution properties. Succinylation improves the solubility and film-forming properties of native xylans. This study shows key structure–reactivity relationships in xylan succinylation, establishing that low branching, high xylose content, and reduced lignin impurity enhance chemical functionalization. The results offer a framework for selecting optimal biomass feedstocks and support future efforts in genetic and synthetic biology to design plants with tunable xylan architectures. These findings advance the hemicellulose valorization for applications in coatings and packaging.

Acylation↗
DOE OSTI2022

Outstanding questions on xylan biosynthesis

Xylan is the second most abundant polysaccharide in plant biomass. It is a crucial component of cell wall structure as well as a significant factor contributing to biomass recalcitrance. Xylan consists of a linear chain of β-1,4-linked xylosyl residues that are often substituted with glycosyl side chains, such as glucuronosyl/methylglucuronosyl and arabinofuranosyl residues, and acetylated at O-2 and/or O-3. Xylan from gymnosperms and dicots contains a unique reducing end tetrasaccharide sequence that is not detected in xylan from grasses, bryophytes and seedless vascular plants. Grass xylan is heavily decorated at O-3 with arabinofuranosyl residues that are frequently esterified with hydroxycinnamates. Further, genetic and biochemical studies have uncovered a number of genes involved in xylan backbone elongation and acetylation, xylan glycosyl substitutions and their modifications, and the synthesis of the unique xylan reducing end tetrasaccharide sequence, but some outstanding issues on the biosynthesis of xylan still remain unanswered. Here, we provide a brief overview of xylan structure and focus on discussion of the current understanding and open questions on xylan biosynthesis. Further elucidation of the biochemical mechanisms underlying xylan biosynthesis will not only shed new insights into cell wall biology but also provide molecular tools for genetic modification of biomass composition tailored for diverse end uses.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Grass xylan structural variation suggests functional specialization and distinctive interaction with cellulose and lignin

SUMMARY Xylan is the most abundant non‐cellulosic polysaccharide in grass cell walls, and it has important structural roles. The name glucuronoarabinoxylan (GAX) is used to describe this variable hemicellulose. It has a linear backbone of β‐1,4‐xylose (Xyl) residues that may be substituted with α‐1,2‐linked (4‐ O ‐methyl)‐glucuronic acid (GlcA), α‐1,3‐linked arabinofuranose (Ara f ), and sometimes acetylation at the O ‐2 and/or O ‐3 positions. The role of these substitutions remains unclear, although there is increasing evidence that they affect the way xylan interacts with other cell wall components, particularly cellulose and lignin. Here, we used substitution‐dependent endo‐xylanase enzymes to investigate the variability of xylan substitution in grass culm cell walls. We show that there are at least three different types of xylan: (i) an arabinoxylan with evenly distributed Ara f substitutions without GlcA (AXe); (ii) a glucuronoarabinoxylan with clustered GlcA modifications (GAXc); and (iii) a highly substituted glucuronoarabinoxylan (hsGAX). Immunolocalization of AXe and GAXc in Brachypodium distachyon culms revealed that these xylan types are not restricted to a few cell types but are instead widely detected in Brachypodium cell walls. We hypothesize that there are functionally specialized xylan types within the grass cell wall. The even substitutions of AXe may permit folding and binding on the surface of cellulose fibrils, whereas the more complex substitutions of the other xylans may support a role in the matrix and interaction with other cell wall components.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2024

Identification of glycosyltransferases mediating 2‐ O ‐arabinopyranosyl and 2‐ O ‐galactosyl substitutions of glucuronosyl side chains of xylan

SUMMARY Xylan is one of the major hemicelluloses in plant cell walls and its xylosyl backbone is often decorated at O ‐2 with glucuronic acid (GlcA) and/or methylglucuronic acid (MeGlcA) residues. The GlcA/MeGlcA side chains may be further substituted with 2‐ O ‐arabinopyranose (Ara p ) or 2‐ O ‐galactopyranose (Gal) residues in some plant species, but the enzymes responsible for these substitutions remain unknown. During our endeavor to investigate the enzymatic activities of Arabidopsis MUR3‐clade members of the GT47 glycosyltransferase family, we found that one of them was able to transfer Ara p from UDP‐Ara p onto O ‐2 of GlcA side chains of xylan, and thus it was named xylan 2‐ O ‐arabinopyranosyltransferase 1 (AtXAPT1). The function of AtXAPT1 was verified in planta by its T‐DNA knockout mutation showing a loss of the Ara p substitution on xylan GlcA side chains. Further biochemical characterization of XAPT close homologs from other plant species demonstrated that while the poplar ones had the same catalytic activity as AtXAPT1, those from Eucalyptus , lemon‐scented gum, sea apple, 'Ohi'a lehua, duckweed and purple yam were capable of catalyzing both 2‐ O ‐Ara p and 2‐ O ‐Gal substitutions of xylan GlcA side chains albeit with differential activities. Sequential reactions with XAPTs and glucuronoxylan methyltransferase 3 (GXM3) showed that XAPTs acted poorly on MeGlcA side chains, whereas GXM3 could efficiently methylate arabinosylated or galactosylated GlcA side chains of xylan. Furthermore, molecular docking and site‐directed mutagenesis analyses of Eucalyptus XAPT1 revealed critical roles of several amino acid residues at the putative active site in its activity. Together, these findings establish that XAPTs residing in the MUR3 clade of family GT47 are responsible for 2‐ O ‐arabinopyranosylation and 2‐ O ‐galactosylation of GlcA side chains of xylan.

Zhong, Ruiqin↗
DOE OSTI2023

Patterns in interactions of variably acetylated xylans with hydrophobic cellulose surfaces

The recalcitrance of plant cell wall lignocellulosic biomass to deconstruction is a major hurdle to sustainable biofuel/bioproduct economy. A multitude of interactions stabilize lignocellulosic biomass structure. Among these, tight packing of hemicellulose-cellulose is partly responsible for biomass recalcitrance. Here, unrestrained molecular dynamics simulations are employed to understand the influence of the nature and pattern of naturally-occuring acetyl decorations of the xylan backbone on interactions with the (100) hydrophobic cellulose surface. Periodically O2-acetylated xylan (2AcX) assume twofold helical screw conformations that are stabilized by a combination of multiple hydrophobic contacts and hydrogen bonds with the hydrophobic cellulose surface. In contrast, acetylation at the O3 position in xylan obstructs interactions, thereby adopting threefold helical screw conformations that potentially preferably interact with lignin rather than cellulose. Fully acetylated xylan desorbs from the surface implying a minimum number of unsubstituted residues on the xylan backbone is required for interaction with the surface. Here, the substituted residues must form ~ 20% fewer contacts than the unsubstituted residues to sustain stable twofold helical screw xylan conformations on the cellulose surface. Thus, specific roles of macromolecular conformations of cellulose and hemicellulose in influencing the supramolecular interactions and function of plant cell walls have been determined.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Chemo-Enzymatic Synthesis of Long-Chain Oligosaccharides for Studying Xylan-Modifying Enzymes

Plant research is hampered in several aspects by a lack of pure oligosaccharide samples that closely represent structural features of cell wall glycans. An alternative to purely chemical synthesis to access these oligosaccharides is chemo-enzymatic synthesis using glycosynthases. These enzymes enable the ligation of oligosaccharide donors, when activated for example as α-glycosyl fluorides, with suitable acceptor oligosaccharides. Herein, the synthesis of xylan oligosaccharides up to dodecasaccharides is reported, with glycosynthase-mediated coupling reactions as key steps. The xylo-oligosaccharide donors were protected at the non-reducing end with a 4-O-tetrahydropyranyl (THP) group to prevent polymerization. Installation of an unnatural 3-O-methylether substituent at the reducing end xylose of the oligosaccharides ensured good water solubility. Biochemical assays demonstrated enzymatic activity for the xylan acetyltransferase XOAT1 from Arabidopsis thaliana, xylan arabinofuranosyl-transferase XAT3 enzymes from rice and switchgrass, and the xylan glucuronosyltransferase GUX3 from Arabidopsis thaliana. In case of the glucuronosyltransferase GUX3, MALDI-MS/MS analysis of the reaction product suggested that a single glucuronosyl substituent was installed primarily at the central xylose residues of the dodecasaccharide acceptor, demonstrating the value of long-chain acceptors for assaying biosynthetic glycosyltransferases.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2023

Overexpression of REDUCED WALL ACETYLATION C increases xylan acetylation and biomass recalcitrance in Populus

Abstract Plant lignocellulosic biomass, i.e. secondary cell walls of plants, is a vital alternative source for bioenergy. However, the acetylation of xylan in secondary cell walls impedes the conversion of biomass to biofuels. Previous studies have shown that REDUCED WALL ACETYLATION (RWA) proteins are directly involved in the acetylation of xylan but the regulatory mechanism of RWAs is not fully understood. In this study, we demonstrate that overexpression of a Populus trichocarpa PtRWA-C gene increases the level of xylan acetylation and increases the lignin content and S/G ratio, ultimately yielding poplar woody biomass with reduced saccharification efficiency. Furthermore, through gene coexpression network and expression quantitative trait loci (eQTL) analysis, we found that PtRWA-C was regulated not only by the secondary cell wall hierarchical regulatory network but also by an AP2 family transcription factor HARDY (HRD). Specifically, HRD activates PtRWA-C expression by directly binding to the PtRWA-C promoter, which is also the cis-eQTL for PtRWA-C. Taken together, our findings provide insights into the functional roles of PtRWA-C in xylan acetylation and consequently saccharification and shed light on synthetic biology approaches to manipulate this gene and alter cell wall properties. These findings have substantial implications for genetic engineering of woody species, which could be used as a sustainable source of biofuels, valuable biochemicals, and biomaterials.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Xylan-cellulose core structure of oat water-extractable β-glucan macromolecule: Insight into interactions and organization of the cell wall complex

Water-extractable β-glucan with high molar mass (HM) determines health benefits of oat food. Oat β-glucan was extracted by a standardized in vitro digestion method and co-existing water-extractable polysaccharide (WEP) fraction and its HM-arabinoxylan (HM-AX) subfraction were isolated to identify their highly acid-resistant subunit and investigate molecular interactions between constituent polymers. The WEP and HM-AX samples consisted of arabinoxylans (AXs) (74 and 76 %, respectively), however, cellulose constituted the secondary component (6.6 and 12.8 %, respectively). Multi-detection HPSEC along with specific enzymatic hydrolysis of AXs revealed the presence of the HM-xylan domain (16 and 34 %, respectively) built of numerous single- and multi-component populations with random coil and rod-like conformations, which were embedded in a xylan matrix with spherical conformation and controlled the macromolecular shape. Unlike single-component populations, the multi-component ones were resistant to hydrolytic action of AX-hydrolyzing enzymes and represented the subunits that anchor matrix polysaccharides onto cellulose surface. These results indicate that water-extractable β-glucan macromolecule comprises as integral element a cellulose core with two linking populations, HM-xylan and low molar mass glucomannan, which are surrounded by a feruloylated AX-arabinan-arabinogalactan composite and next laminated by β-glucan matrix. Here, the stiff cellulose-xylan backbone is the basis of HM β-glucan organization, controlled by its cellulose-like segments.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Pectin-associated mannans and xylans play distinct roles in cell-cell adhesion in pine and poplar wood

Wood particles of pine required longer treatments of acidic chlorite and dilute alkali to achieve similar proportions of cell separation as of poplar. Chemical extraction and enzymatic digestion studies indicated that rhamnogalacturonan-I (RG-I), mannan and xylan contribute to cell-cell adhesion in pine wood. Mannan associated with lignin in an acidic chlorite fraction and with xylan in a dilute alkali fraction. RG-I and xylan were extracted in both chlorite and dilute alkali in poplar. Scanning electron microscopy and a probe diffusion study using Fluorescence Recovery After Photobleaching showed that lignin, RG-I and mannan initiated cell separation at cell corners and dilute alkali extraction of xylan was required for complete cell separation in pine. Cell-cell separation increased enzymatic saccharification within 24 h by 2-fold in poplar and 3.4-fold in pine. Finally, our results inform a strategy to reduce particle size in woody biomass and provide insights into different molecular bases of cell-cell adhesion.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS1992

Xylan - A potential contaminant for lunar samples and Antarctic meteorites

The possibility that lunar samples have been contaminated by the proprietary lubricant paint known as Xylan, which has been applied to screw threads in dry-N sample processing cabinets at NASA JSC, is considered. From a sample analysis using sealed-tube and stepped combustion, it is argued that the unexpectedly high concentration of organic materials found in EET A79001 is not due to Xylan contamination. It is considered unlikely that previous C and N analyses of lunar samples have been affected by the introduction of Xylan.

Wright, I. P.↗
DOE OSTI2023

Development of sulfonated carbon-based solid-acid catalysts derived from biorefinery residues and biomass ash for xylan hydrolysis

In this study, biorefinery residues and biomass ash were used to develop sulfonated carbon-based solid-acid catalysts to enhance xylan hydrolysis. Here, metals extracted from the biomass ash were impregnated into a carbon support prepared from biorefinery residues to synthesize sulfonated ash-impregnated carbon-based catalyst. Metals present in the ash most probably are responsible for the increased acid density in the catalyst. Using this catalyst, 11.4 % xylose yield was produced, an increase of 171 % over the yield obtained from a catalyst without ash impregnation. Using a xylan-to-catalyst ratio of 1:2, 72.9 % xylose yield was obtained, comparable to using 0.5 wt% sulfuric acid under the same reaction condition. Moreover, this catalyst significantly inhibited xylose dehydration, only forming 11.3 % furfural compared to that generated using dilute acid. Hence, these results show the potential for using biomass ash as a metal source to increase the number of acid sites in solid-acid catalysts used for biomass pretreatment.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2025

Hempseed cell wall polysaccharides are dominated by linear xylans and cellulose: Comprehensive structural profiling of ten cultivars of industrial hemp, Cannabis sativa L .

Hempseed is a rich source of dietary fiber; however, there has been limited research on the variability of carbohydrate composition in hempseed cell walls. The primary aim of this study was to conduct a comprehensive chemical and structural analysis of the cell wall polysaccharides in ten hempseed cultivars. Water-soluble polysaccharides (WSP) and water-insoluble residues (WIR) were isolated and subsequently analyzed for their monosaccharide composition using HPAEC-PAD, glycosyl linkage analysis using GC–MS, and structural characterization via NMR spectroscopy. All hempseed cultivars contained a high proportion of insoluble fibers and smaller amounts of soluble polysaccharides. Glucose and xylose were the most abundant components of the WIR fractions, while the WSP fractions contained abundant amounts of galactose, galacturonic acid, arabinose, rhamnose, and mannose. The results of linkage and spectroscopic analysis were consistent with the compositional analysis, identifying cellulose and acetylated linear xylans as primary components of WIR, and arabinogalactans, rhamnogalacturonans, heteromannans, xyloglucans, and arabinan as predominant in WSP. Altogether, the study revealed a comparable cell wall structure among the analyzed hemp seed varieties. The high fiber content of whole hempseed-based ingredients presents significant potential for food manufacturers seeking to develop products with enhanced dietary fiber content, offering both functional and nutritional benefits for consumers.

59 BASIC BIOLOGICAL SCIENCES↗