Phylogenetic tree of GAUT Protein Family and gene model, RNAi construct, and relative transcript abundance of GAUT4 in switchgrass, rice and poplar knockdown (KD) lines.

bio engineered↗

DOE OSTI2026

Glycosyl linkage analysis of fractionated cell walls from switchgrass WT and PvGAUT4-KD lines

bio engineered↗

DOE OSTI2026

Glycosyl linkage analysis of fractionated cell walls from P.deltoides WT and PdGAUT4-KD lines.

bio engineered↗

DOE OSTI2024

Putative rhamnogalacturonan-II glycosyltransferase identified through callus gene editing bypasses embryo lethality

Rhamnogalacturonan II (RG-II) is a structurally complex and conserved domain of the pectin present in the primary cell walls of vascular plants. Borate crosslinking of RG-II is required for plants to grow and develop normally. Mutations that alter RG-II structure also affect crosslinking and are lethal or severely impair growth. Thus, few genes involved in RG-II synthesis have been identified. Here we developed a method to generate viable loss-of-function Arabidopsis (Arabidopsis thaliana) mutants in callus tissue via CRISPR/Cas9-mediated gene editing. We combined this with a candidate gene approach to characterize the male gametophyte defective 2 (MPG2) gene that encodes a putative family GT29 glycosyltransferase. Plants homozygous for this mutation do not survive. We showed that in the callus mutant cell walls, RG-II does not crosslink normally because it lacks 3-deoxy-D-manno-octulosonic acid (Kdo) and thus cannot form the α-L-Rhap-(1→5)-α-D-kdop-(1→ sidechain. We suggest that MGP2 encodes an inverting RG-II CMP-β-Kdo transferase (RCKT1). Our discovery provides further insight into the role of sidechains in RG-II dimerization. Our method also provides a viable strategy for further identifying proteins involved in the biosynthesis of RG-II.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2023

Acetylation in Ionic Liquids Dramatically Increases Yield in the Glycosyl Composition and Linkage Analysis of Insoluble and Acidic Polysaccharides

Not Available

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

DOE OSTI2023

Involvement of the Streptococcus mutans PgfE and GalE 4-epimerases in protein glycosylation, carbon metabolism, and cell division

Streptococcus mutans is a key pathogen associated with dental caries and is often implicated in infective endocarditis. This organism forms robust biofilms on tooth surfaces and can use collagen-binding proteins (CBPs) to efficiently colonize collagenous substrates, including dentin and heart valves. One of the best characterized CBPs of S. mutans is Cnm, which contributes to adhesion and invasion of oral epithelial and heart endothelial cells. These virulence properties were subsequently linked to post-translational modification (PTM) of the Cnm threonine-rich repeat region by the Pgf glycosylation machinery, which consists of 4 enzymes: PgfS, PgfM1, PgfE, and PgfM2. Inactivation of the S. mutans pgf genes leads to decreased collagen binding, reduced invasion of human coronary artery endothelial cells, and attenuated virulence in the Galleria mellonella invertebrate model. The present study aimed to better understand Cnm glycosylation and characterize the predicted 4-epimerase, PgfE. Using a truncated Cnm variant containing only 2 threonine-rich repeats, mass spectrometric analysis revealed extensive glycosylation with HexNAc2. Compositional analysis, complemented with lectin blotting, identified the HexNAc2 moieties as GlcNAc and GalNAc. Comparison of PgfE with the other S. mutans 4-epimerase GalE through structural modeling, nuclear magnetic resonance, and capillary electrophoresis demonstrated that GalE is a UDP-Glc-4-epimerase, while PgfE is a GlcNAc-4-epimerase. While PgfE exclusively participates in protein O-glycosylation, we found that GalE affects galactose metabolism and cell division. Furthermore, this study further emphasizes the importance of O-linked protein glycosylation and carbohydrate metabolism in S. mutans and identifies the PTM modifications of the key CBP, Cnm.

4-epimerases↗

DOE OSTI2022

Composition and yield of non-cellulosic and cellulosic sugars in soluble and particulate fractions during consolidated bioprocessing of poplar biomass by Clostridium thermocellum

Abstract Background Terrestrial plant biomass is the primary renewable carbon feedstock for enabling transition to a sustainable bioeconomy. Consolidated bioprocessing (CBP) by the cellulolytic thermophile Clostridium thermocellum offers a single step microbial platform for production of biofuels and biochemicals via simultaneous solubilization of carbohydrates from lignocellulosic biomass and conversion to products. Here, solubilization of cell wall cellulosic, hemicellulosic, and pectic polysaccharides in the liquor and solid residues generated during CBP of poplar biomass by C. thermocellum was analyzed. Results The total amount of biomass solubilized in the C. thermocellum DSM1313 fermentation platform was 5.8, 10.3, and 13.7% of milled non-pretreated poplar after 24, 48, and 120 h, respectively. These results demonstrate solubilization of 24% cellulose and 17% non-cellulosic sugars after 120 h, consistent with prior reports. The net solubilization of non-cellulosic sugars by C. thermocellum (after correcting for the uninoculated control fermentations) was 13 to 36% of arabinose (Ara), xylose (Xyl), galactose (Gal), mannose (Man), and glucose (Glc); and 15% and 3% of fucose and glucuronic acid, respectively. No rhamnose was solubilized and 71% of the galacturonic acid (GalA) was solubilized. These results indicate that C. thermocellum may be selective for the types and/or rate of solubilization of the non-cellulosic wall polymers. Xyl, Man, and Glc were found to accumulate in the fermentation liquor at levels greater than in uninoculated control fermentations, whereas Ara and Gal did not accumulate, suggesting that C. thermocellum solubilizes both hemicelluloses and pectins but utilizes them differently. After five days of fermentation, the relative amount of Rha in the solid residues increased 21% indicating that the Rha-containing polymer rhamnogalacturonan I (RG-I) was not effectively solubilized by C. thermocellum CBP, a result confirmed by immunoassays. Comparison of the sugars in the liquor versus solid residue showed that C. thermocellum solubilized hemicellulosic xylan and mannan, but did not fully utilize them, solubilized and appeared to utilize pectic homogalacturonan, and did not solubilize RG-I. Conclusions The significant relative increase in RG-I in poplar solid residues following CBP indicates that C. thermocellum did not solubilize RG-I. These results support the hypothesis that this pectic glycan may be one barrier for efficient solubilization of poplar by C. thermocellum .

09 BIOMASS FUELS↗

DOE OSTI2021

Structure of Lipopolysaccharide from Liberibacter crescens Is Low Molecular Weight and Offers Insight into Candidatus Liberibacter Biology

Huanglongbing (HLB) disease, also known as citrus greening disease, was first reported in the US in 2005. Since then, the disease has decimated the citrus industry in Florida, resulting in billions of dollars in crop losses and the destruction of thousands of acres of citrus groves. The causative agent of citrus greening disease is the phloem limited pathogen Candidatus Liberibacter asiaticus. As it has not been cultured, very little is known about the structural biology of the organism. Liberibacter are part of the Rhizobiaceae family, which includes nitrogen-fixing symbionts of legumes as well as the Agrobacterium plant pathogens. To better understand the Liberibacter genus, a closely related culturable bacterium (Liberibacter crescens or Lcr) has attracted attention as a model organism for structural and functional genomics of Liberibacters. Given that the structure of lipopolysaccharides (LPS) from Gram-negative bacteria plays a crucial role in mediating host-pathogen interactions, we sought to characterize the LPS from Lcr. We found that the major lipid A component of the LPS consisted of a pentaacylated molecule with a β-6-GlcN disaccharide backbone lacking phosphate. The polysaccharide portion of the LPS was unusual compared to previously described members of the Rhizobiaceae family in that it contained ribofuranosyl residues. The LPS structure presented here allows us to extrapolate known LPS structure/function relationships to members of the Liberibacter genus which cannot yet be cultured. It also offers insights into the biology of the organism and how they manage to effectively attack citrus trees.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2021

Lipopolysaccharide associated with β-2,6 fructan mediates TLR4-dependent immunomodulatory activity in vitro

Levan, a β-2,6 fructofuranose polymer produced by microbial species, has been reported for its immunomodulatory properties via interaction with toll-like receptor 4 (TLR4) which recognises lipopolysaccharide (LPS). However, the molecular mechanisms underlying these interactions remain elusive. Here, we investigated the immunomodulatory properties of levan using thoroughly-purified and characterised samples from Erwinia herbicola and other sources. E. herbicola levan was purified by gel-permeation chromatography and LPS was removed from the levan following a novel alkali treatment developed in this study. E. herbicola levan was then characterised by gas chromatography–mass spectrometry and NMR. We found that levan containing LPS, but not LPS-depleted levan, induced TLR4-mediated cytokine production by bone marrow-derived dendritic cells and/or activated TLR4 reporter cells. These data indicated that the immunomodulatory properties of the levan toward TLR4-expressing immune cells were mediated by the LPS. This work also demonstrates the importance of LPS removal when assessing the immunomodulatory activity of polysaccharides.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2021

Protocols for isolating and characterizing polysaccharides from plant cell walls: a case study using rhamnogalacturonan-II

Abstract Background In plants, a large diversity of polysaccharides comprise the cell wall. Each major type of plant cell wall polysaccharide, including cellulose, hemicellulose, and pectin, has distinct structures and functions that contribute to wall mechanics and influence plant morphogenesis. In recent years, pectin valorization has attracted much attention due to its expanding roles in biomass deconstruction, food and material science, and environmental remediation. However, pectin utilization has been limited by our incomplete knowledge of its structure. Herein, we present a workflow of principles relevant for the characterization of polysaccharide primary structure using nature’s most complex polysaccharide, rhamnogalacturonan-II (RG-II), as a model. Results We outline how to isolate RG-II from celery and duckweed cell walls and from red wine using chemical or enzymatic treatments coupled with size-exclusion chromatography. From there, we applied mass spectrometry (MS)-based techniques to determine the glycosyl residue and linkage compositions of the intact RG-II and derived oligosaccharides including special considerations for labile monosaccharides. In doing so, we demonstrated that in the duckweed Wolffiella repanda the arabinopyranosyl (Ara p ) residue of side chain B is substituted at O- 2 with rhamnose. We used electrospray-MS techniques to identify non-glycosyl modifications including methyl-ethers, methyl-esters, and acetyl-esters on RG-II-derived oligosaccharides. We then showed the utility of proton nuclear magnetic resonance spectroscopy ( 1 H-NMR) to investigate the structure of intact RG-II and to complement the RG-II dimerization studies performed using size-exclusion chromatography. Conclusions The complexity of pectic polysaccharide structures has hampered efforts aimed at their valorization. In this work, we used RG-II as a model to demonstrate the steps necessary to isolate and characterize polysaccharides using chromatographic, MS, and NMR techniques. The principles can be applied to the characterization of other saccharide structures and will help inform researchers on how saccharide structure relates to functional properties in the future.

09 BIOMASS FUELS↗

DOE OSTI2021

A t FUT4 and A t FUT6 Are Arabinofuranose-Specific Fucosyltransferases

The bulk of plant biomass is comprised of plant cell walls, which are complex polymeric networks, composed of diverse polysaccharides, proteins, polyphenolics, and hydroxyproline-rich glycoproteins (HRGPs). Glycosyltransferases (GTs) work together to synthesize the saccharide components of the plant cell wall. The Arabidopsis thaliana fucosyltransferases (FUTs), At FUT4, and At FUT6, are members of the plant-specific GT family 37 (GT37). At FUT4 and At FUT6 transfer fucose (Fuc) onto arabinose (Ara) residues of arabinogalactan (AG) proteins (AGPs) and have been postulated to be non-redundant AGP-specific FUTs. At FUT4 and At FUT6 were recombinantly expressed in mammalian HEK293 cells and purified for biochemical analysis. We report an updated understanding on the specificities of At FUT4 and At FUT6 that are involved in the synthesis of wall localized AGPs. Our findings suggest that they are selective enzymes that can utilize various arabinogalactan (AG)-like and non-AG-like oligosaccharide acceptors, and only require a free, terminal arabinofuranose. We also report with GUS promoter-reporter gene studies that AtFUT4 and AtFUT6 gene expression is sub-localized in different parts of developing A. thaliana roots.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2018

Sugar release and growth of biofuel crops are improved by downregulation of pectin biosynthesis

Cell walls in crops and trees have been engineered for production of biofuels and commodity chemicals, but engineered varieties often fail multi-year field trials and are not commercialized. In this paper, we engineered reduced expression of a pectin biosynthesis gene (Galacturonosyltransferase 4, GAUT4) in switchgrass and poplar, and find that this improves biomass yields and sugar release from biomass processing. Both traits were maintained in a 3-year field trial of GAUT4-knockdown switchgrass, with up to sevenfold increased saccharification and ethanol production and sixfold increased biomass yield compared with control plants. We show that GAUT4 is an α-1,4-galacturonosyltransferase that synthesizes homogalacturonan (HG). Downregulation of GAUT4 reduces HG and rhamnogalacturonan II (RGII), reduces wall calcium and boron, and increases extractability of cell wall sugars. Finally, decreased recalcitrance in biomass processing and increased growth are likely due to reduced HG and RGII cross-linking in the cell wall.

09 BIOMASS FUELS↗