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Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

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At least 163 records · Page 9
All sourcesNASA NTRSDOE OSTI
DOE OSTI2023

Galactosylation of xyloglucan is essential for the stabilization of the actin cytoskeleton and endomembrane system through the proper assembly of cell walls

Abstract Xyloglucan, a major hemicellulose, interacts with cellulose and pectin to assemble primary cell walls in plants. Loss of the xyloglucan galactosyltransferase MURUS3 (MUR3) leads to the deficiency of galactosylated xyloglucan and perturbs plant growth. However, it is unclear whether defects in xyloglucan galactosylation influence the synthesis of other wall polysaccharides, cell wall integrity, cytoskeleton behaviour, and endomembrane homeostasis. Here, we found that in mur3-7 etiolated seedlings cellulose was reduced, CELLULOSE SYNTHASE (CESA) genes were down-regulated, the density and mobility of cellulose synthase complexes (CSCs) were decreased, and cellulose microfibrils become discontinuous. Pectin, rhamnogalacturonan II (RGII), and boron contents were reduced in mur3-7 plants, and B-RGII cross-linking was abnormal. Wall porosity and thickness were significantly increased in mur3-7 seedlings. Endomembrane aggregation was also apparent in the mur3-7 mutant. Furthermore, mutant seedlings and their actin filaments were more sensitive to Latrunculin A (LatA) treatment. However, all defects in mur3-7 mutants were substantially restored by exogenous boric acid application. Our study reveals the importance of MUR3-mediated xyloglucan galactosylation for cell wall structural assembly and homeostasis, which is required for the stabilization of the actin cytoskeleton and the endomembrane system.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

A Group of O-Acetyltransferases Catalyze Xyloglucan Backbone Acetylation and Can Alter Xyloglucan Xylosylation Pattern and Plant Growth When Expressed in Arabidopsis

Abstract Xyloglucan is a major hemicellulose in plant cell walls and exists in two distinct types, XXXG and XXGG. While the XXXG-type xyloglucan from dicot species only contains O-acetyl groups on side-chain galactose (Gal) residues, the XXGG-type xyloglucan from Poaceae (grasses) and Solanaceae bears O-acetyl groups on backbone glucosyl (Glc) residues. Although O-acetyltransferases responsible for xyloglucan Gal acetylation have been characterized, the biochemical mechanism underlying xyloglucan backbone acetylation remains to be elucidated. In this study, we showed that recombinant proteins of a group of DUF231 members from rice and tomato were capable of transferring acetyl groups onto O-6 of Glc residues in cello-oligomer acceptors, indicating that they are xyloglucan backbone 6-O-acetyltransferases (XyBATs). We further demonstrated that XyBAT-acetylated cellohexaose oligomers could be readily xylosylated by AtXXT1 (Arabidopsis xyloglucan xylosyltransferase 1) to generate acetylated, xylosylated cello-oligomers, whereas AtXXT1-xylosylated cellohexaose oligomers were much less effectively acetylated by XyBATs. Heterologous expression of a rice XyBAT in Arabidopsis led to a severe reduction in cell expansion and plant growth and a drastic alteration in xyloglucan xylosylation pattern with the formation of acetylated XXGG-type units, including XGG, XGGG, XXGG, XXGG,XXGGG and XXGGG (G denotes acetylated Glc). In addition, recombinant proteins of two Arabidopsis XyBAT homologs also exhibited O-acetyltransferase activity toward cellohexaose, suggesting their possible role in mediating xyloglucan backbone acetylation in vivo. Our findings provide new insights into the biochemical mechanism underlying xyloglucan backbone acetylation and indicate the importance of maintaining the regular xyloglucan xylosylation pattern in cell wall function.

Cell Biology↗
DOE OSTI2021

A Single Xyloglucan Xylosyltransferase Is Sufficient for Generation of the XXXG Xylosylation Pattern of Xyloglucan

ABSTRACT Xyloglucan is the most abundant hemicellulose in the primary cell walls of dicots. Dicot xyloglucan is the XXXG type consisting of repeating units of three consecutive xylosylated Glc residues followed by one unsubstituted Glc. Its xylosylation is catalyzed by xyloglucan 6-xylosyltransferases (XXTs) and there exist five XXTs (AtXXT1-5) in Arabidopsis. While AtXXT1 and AtXXT2 have been shown to add the first two Xyl residues in the XXXG repeat, which XXTs are responsible for the addition of the third Xyl residue remains elusive although AtXXT5 was a proposed candidate. In this report, we generated recombinant proteins of all five Arabidopsis XXTs and one rice XXT (OsXXT1) in the mammalian HEK293 cells and investigated their ability to sequentially xylosylate Glc residues to generate the XXXG xylosylation pattern. We found that like AtXXT1/2, AtXXT4 and OsXXT1 could efficiently xylosylate the cellohexaose (G6) acceptor to produce mono- and di-xylosylated G6, whereas AtXXT5 was only barely capable of adding one Xyl onto G6. When AtXXT1-catalyzed products were used as acceptors, AtXXT1/2/4 and OsXXT1, but not AtXXT5, were able to xylosylate additional Glc residues to generate tri- and tetra-xylosylated G6. Further characterization of the tri- and tetra-xylosylated G6 revealed that they had the sequence of GXXXGG and GXXXXG with three and four consecutive xylosylated Glc residues, respectively. In addition, we have found that although tri-xylosylation occurred on G6, cello-oligomers with a degree of polymerization of 3 to 5 could only be mono- and di-xylosylated. Together, these results indicate that each of AtXXT1/2/4 and OsXXT1 is capable of sequentially adding Xyl onto three contiguous Glc residues to generate the XXXG xylosylation pattern and these findings provide new insight into the biochemical mechanism underlying xyloglucan biosynthesis.

Cell Biology↗
DOE OSTI2025

The effects of expression of a hyperthermophilic endoglucanase and nutrient conditions on poplar growth and physiology

Abstract Efficient production and processing of poplar biomass feedstock requires costly pretreatments and enzyme additives. Transgenic alterations of poplar can reduce the need for these inputs by increasing biomass, improving lignocellulose quality and enhancing nutrient uptake. Previously, a transgenic line of poplar expressing a bacterial hyperthermophilic endoglucanase (TnCelB) in Populus alba × grandidentata (P39) was developed and characterized. This study reports the effects on the TnCelB transgenic poplar line under a reduced nutrient treatment. Overall, the nutrient treatment was the source of more observed significant differences than the genotype. Wild type and TnCelB poplar had similar responses in biomass allocation and net photosynthesis. TnCelB trees had a wrinkled leaf phenotype and relative to wild type, had reduced total biomass, reduced water-use efficiency, and a decreased proportion of cellulose to hemicellulose and lignin. In low nutrient conditions, TnCelB trees had increased structural carbohydrates with stable lignin values. The TnCelB line presents a viable option for poplar biomass feedstock, offering biomass comparable to wild type poplar and more efficient processing, with only mild negative phenotypes.

Natalie, Bethanie M. (ORCID:0009000843856585)↗
DOE OSTI2022

Revealing oxidative pentose metabolism in new Pseudomonas putida isolates

Abstract The Pseudomonas putida group in the Gammaproteobacteria has been intensively studied for bioremediation and plant growth promotion. Members of this group have recently emerged as promising hosts to convert intermediates derived from plant biomass to biofuels and biochemicals. However, most strains of P. putida cannot metabolize pentose sugars derived from hemicellulose. Here, we describe three isolates that provide a broader view of the pentose sugar catabolism in the P. putida group. One of these isolates clusters with the well‐characterized P. alloputida KT2440 (Strain BP6); the second isolate clustered with plant growth‐promoting strain P. putida W619 (Strain M2), while the third isolate represents a new species in the group (Strain BP8). Each of these isolates possessed homologous genes for oxidative xylose catabolism ( xylDXA ) and a potential xylonate transporter. Strain M2 grew on arabinose and had genes for oxidative arabinose catabolism ( araDXA ). A CRISPR interference (CRISPRi) system was developed for strain M2 and identified conditionally essential genes for xylose growth. A glucose dehydrogenase was found to be responsible for initial oxidation of xylose and arabinose in strain M2. These isolates have illuminated inherent diversity in pentose catabolism in the P. putida group and may provide alternative hosts for biomass conversion.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Oxic–anoxic cycling promotes coupling between complex carbon metabolism and denitrification in woodchip bioreactors

Denitrifying woodchip bioreactors (WBRs) are increasingly used to manage the release of non-point source nitrogen (N) by stimulating microbial denitrification. Woodchips serve as a renewable organic carbon (C) source, yet the recalcitrance of organic C in lignocellulosic biomass causes many WBRs to be C-limited. Prior studies have observed that oxic–anoxic cycling increased the mobilization of organic C, increased nitrate (NO 3 – ) removal rates, and attenuated production of nitrous oxide (N 2 O). Here, in this work, we use multi-omics approaches and amplicon sequencing of fungal 5.8S-ITS2 and prokaryotic 16S rRNA genes to elucidate the microbial drivers for enhanced NO 3 – removal and attenuated N 2 O production under redox-dynamic conditions. Transient oxic periods stimulated the expression of fungal ligninolytic enzymes, increasing the bioavailability of woodchip-derived C and stimulating the expression of denitrification genes. Nitrous oxide reductase (nosZ) genes were primarily clade II, and the ratio of clade II/clade I nosZ transcripts during the oxic–anoxic transition was strongly correlated with the N 2 O yield. Analysis of metagenome-assembled genomes revealed that many of the denitrifying microorganisms also have a genotypic ability to degrade complex polysaccharides like cellulose and hemicellulose, highlighting the adaptation of the WBR microbiome to the ecophysiological niche of the woodchip matrix.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Galactomannan utilization by Cellvibrio japonicus relies on a single essential α‐galactosidase encoded by the aga27A gene

Plant mannans are a component of lignocellulose that can have diverse compositions in terms of its backbone and side-chain substitutions. Consequently, the degradation of mannan substrates requires a cadre of enzymes for complete reduction to substituent monosaccharides that can include mannose, galactose, and/or glucose. One bacterium that possesses this suite of enzymes is the Gram-negative saprophyte Cellvibrio japonicus, which has 10 predicted mannanases from the Glycoside Hydrolase (GH) families 5, 26, and 27. Here we describe a systems biology approach to identify and characterize the essential mannan-degrading components in this bacterium. The transcriptomic analysis uncovered significant changes in gene expression for most mannanases, as well as many genes that encode carbohydrate active enzymes (CAZymes) when mannan was actively being degraded. A comprehensive mutational analysis characterized 54 CAZyme-encoding genes in the context of mannan utilization. Growth analysis of the mutant strains found that the man26C, aga27A, and man5D genes, which encode a mannobiohydrolase, α-galactosidase, and mannosidase, respectively, were important for the deconstruction of galactomannan, with Aga27A being essential. Our updated model of mannan degradation in C. japonicus proposes that the removal of galactose sidechains from substituted mannans constitutes a crucial step for the complete degradation of this hemicellulose.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Altering the substitution and cross‐linking of glucuronoarabinoxylans affects cell wall architecture in Brachypodium distachyon

Summary The Poaceae family of plants provides cereal crops that are critical for human and animal nutrition, and also, they are an important source of biomass. Interacting plant cell wall components give rise to recalcitrance to digestion; thus, understanding the wall molecular architecture is important to improve biomass properties. Xylan is the main hemicellulose in grass cell walls. Recently, we reported structural variation in grass xylans, suggesting functional specialisation and distinct interactions with cellulose and lignin. Here, we investigated the functions of these xylans by perturbing the biosynthesis of specific xylan types. We generated CRISPR/Cas9 knockout mutants in Brachypodium distachyon XAX1 and GUX2 genes involved in xylan substitution. Using carbohydrate gel electrophoresis, we identified biochemical changes in different xylan types. Saccharification, cryo‐SEM, subcritical water extraction and ssNMR were used to study wall architecture. Bd XAX1A and Bd GUX2 enzymes modify different types of grass xylan. Brachypodium mutant walls are likely more porous, suggesting the xylan substitutions directed by both Bd XAX1A and GUX2 enzymes influence xylan‐xylan and/or xylan–lignin interactions. Since xylan substitutions influence wall architecture and digestibility, our findings open new avenues to improve cereals for food and to use grass biomass for feed and the production of bioenergy and biomaterials.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Mesophyll conductance and cell wall composition: insights from a meta‐analysis across species

Mesophyll conductance (g m ) plays a critical role in plant photosynthesis by regulating the diffusion of CO 2 from substomatal cavities to the site of carbon fixation within the chloroplasts. Despite its importance, our understanding of the factors influencing g m , particularly the role of cell wall properties, remains incomplete. In this issue of New Phytologist , Roig-Oliver et al. ( 2025 ; pp. 2384–2391) address this knowledge gap through a comprehensive meta-analysis across multiple species from the major clades of land plants. By analyzing trends in g m and two other key physiological parameters – the ratio of pectin to cellulose and hemicellulose in the cell wall by weight (P/(C + H)), and cell wall thickness (T cw ) – their findings reveal strong correlations of gm with the combination of P/(C + H) and T cw , where g m is directly proportional to P/(C + H) and inversely proportional to T cw . This work not only underscores the influence of cell wall composition on CO 2 diffusion but also provides a valuable framework for further experimental and modeling studies. By shedding light on the dynamic properties of cell walls, shaped by both composition and environmental regulation, this study opens new frontiers for understanding and improving photosynthesis.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Engineering 2‐Pyrone‐4,6‐Dicarboxylic Acid Production Reveals Metabolic Plasticity of Poplar

Woody biomass is a promising source of fermentable sugars for biofuels and bio-based chemicals, but its industrial use is limited by the costly biorefinery process. A viable strategy to reduce costs involves enhancing both biomass processability and the generation of high-value co-products. Here, we report the implementation of a synthetic metabolic pathway in Populus tremula × P. alba to produce 2-pyrone-4,6-dicarboxylic acid (PDC), a key building block for biodegradable plastics and high-performance materials. This artificial pathway—comprising microbial genes AroG, QsuB, PmdA, PmdB, and PmdC—enabled de novo PDC production in the stems of transgenic poplar. Pathway expression also induced substantial metabolic reprogramming and altered cell wall composition. These include the hyperaccumulation of simple phenolics like protocatechuic acid (PCA) and vanillic acid (VA), alongside reduced levels of p-hydroxybenzoic acid. A large portion of VA was ester-linked to cell wall lignin, while PCA was incorporated into the lignin backbone, forming novel benzodioxane units; concurrently, lignin in transgenic plants exhibited a drastic reduction in guaiacyl- and syringyl-units, with a notable increase in p-hydroxyphenyl-units. Hemicellulose content, particularly xylan, was also significantly increased. Moreover, expression of the PDC-pathway led to the formation of novel VA-derived suberin aromatics, enhancing suberization in bark and roots and improving salt stress tolerance. These changes led to improved saccharification efficiency, with up to 25% more glucose and 2.5 times xylose released from woody biomass. These results demonstrate the metabolic flexibility of poplar and highlight its potential for engineering cost-effective, stress-resilient bioenergy crops with enhanced biorefinery traits.

2-pyrone-4↗
DOE OSTI2026

Simultaneous Overexpression of FERULOYL‐CoA 6′‐HYDROXYLASE 1 and COUMARIN SYNTHASE Leads to Coumarin‐Enriched Lignin and Improved Saccharification in Greenhouse‐ and Field‐Grown Poplar

ABSTRACT The urgent need for renewable resources has increased the interest in woody biomass to manufacture bio‐based products. However, lignin recalcitrance limits the enzymatic conversion of wood into fermentable sugars, posing a major challenge for biomass deconstruction. To address this problem, we aimed at incorporating the coumarin scopoletin into the lignin polymer of poplar ( Populus tremula × P . alba ) by expressing FERULOYL‐CoA 6′‐HYDROXYLASE 1 ( F6′H1 ) and COUMARIN SYNTHASE ( COSY ) in lignifying cells. Three constructs were evaluated: two bicistronic constructs, SCOP1 ( COSY followed by F6′H1 ) and SCOP2 ( F6′H1 followed by COSY ), and one monocistronic, SCOP3 (only F6′H1 ). SCOP1 poplars produced most free scopoletin without altering overall lignin, cellulose or hemicellulose content. SCOP2 poplars were overall less efficient in scopoletin production and most of these lines showed a severe biomass yield penalty, whereas SCOP3 caused plant lethality. NMR and metabolic analyses confirmed that scopoletin cross‐coupled with G and S monomers during lignification in SCOP1 lines. In addition to scopoletin, the detection of benzodioxane structures revealed the incorporation of dihydroxycoumarins. Overall coumarin incorporation in lignin amounted up to 2.3%. After alkaline pretreatment, wood from greenhouse‐grown SCOP1 poplars released up to 29% more glucose compared to the wild type upon limited saccharification. Field‐testing of three SCOP1 lines showed a 6 to 11% increase in saccharification efficiency, with the line containing the lowest scopoletin levels maintaining normal growth. These results demonstrate that engineering lignin composition in poplar can improve saccharification, and emphasize the importance of construct design, translational research and field validation.

alternative lignin monomers↗
DOE OSTI2020

Rewired phenolic metabolism and improved saccharification efficiency of a Zea mays cinnamyl alcohol dehydrogenase 2 (zmcad2) mutant

Lignocellulosic biomass is an abundant byproduct from cereal crops that can potentially be valorized as a feedstock to produce biomaterials. Zea mays CINNAMYL ALCOHOL DEHYDROGENASE 2 (ZmCAD2) is involved in lignification, and is a promising target to improve the cellulose–to–glucose conversion of maize stover. Here, we analyzed a field–grown zmcad2 Mutator transposon insertional mutant. Zmcad2 mutant plants had an 18% lower Klason lignin content, whereas their cellulose content was similar to that of control lines. The lignin in zmcad2 mutants contained increased levels of hydroxycinnamaldehydes, i.e. the substrates of ZmCAD2, ferulic acid and tricin. Ferulates decorating hemicelluloses were not altered. Phenolic profiling further revealed that hydroxycinnamaldehydes are partly converted into (dihydro)ferulic acid and sinapic acid and their derivatives in zmcad2 mutants. Syringyl lactic acid hexoside, a metabolic sink in CAD–deficient dicot trees, appeared not to be a sink in zmcad2 maize. The enzymatic cellulose–to–glucose conversion efficiency was determined after 10 different thermochemical pre–treatments. Zmcad2 yielded significantly higher conversions compared with controls for almost every pre–treatment. However, the relative increase in glucose yields after alkaline pre–treatment was not higher than the relative increase when no pre–treatment was applied, suggesting that the positive effect of the incorporation of hydroxycinnamaldehydes was leveled off by the negative effect of reduced p–coumarate levels in the cell wall. Here, taken together, our results reveal how phenolic metabolism is affected in CAD–deficient maize, and further support mutating CAD genes in cereal crops as a promising strategy to improve lignocellulosic biomass for sugar–platform biorefineries.

59 BASIC BIOLOGICAL SCIENCES↗
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 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 OSTI2025

Rheological Properties of Enzymatically Hydrolyzed Corn Stover Pretreated via Deacetylation and Mechanical Refining

Lignocellulosic biomass is a feedstock for fuels and chemicals that does not compete with food resources and has less contaminants than refuse-derived biomass feedstocks. To convert lignocellulosics to biofuels or value-added products, multiple processing steps are typically necessary. One method of producing biofuels from lignocellulosic biomass utilizes a deacetylation and mechanical refining pretreatment and an enzymatic hydrolysis reaction to produce fermentable sugars from cellulose and hemicellulose. The rheological properties of biomass, such as yield stress and plastic viscosity, change during enzymatic hydrolysis and alter the energy requirements for pumping and mixing, an important consideration for the design of processing equipment. The dynamic changes in rheological properties that occur in a corn stover feedstock undergoing enzymatic hydrolysis are characterized in this work, and the influence on pressure losses in piping systems is estimated. Two rheometer geometries were fabricated with stereolithography 3D printing to reduce wall slip and sample ejection. The slurries have complex rheological behaviors that include shear-thinning behavior. Shear stress ramps were performed on samples at 20 and 50 degrees C using the custom geometries, and the Herschel-Bulkley model was fit to the data. The dynamic nature of the rheological properties is correlated with changes in the average fiber length at various extents of reaction, and the influence of solids concentration on the observed rheology and piping pressure losses is discussed.

09 BIOMASS FUELS↗
DOE OSTI2024

Atomistic, macromolecular model of the Populus secondary cell wall informed by solid-state NMR

Plant secondary cell walls (SCWs) are composed of a heterogeneous interplay of three major biopolymers: cellulose, hemicelluloses, and lignin. Details regarding specific intermolecular interactions and higher-order architecture of the SCW superstructure remain ambiguous. Here, we use solid-state nuclear magnetic resonance (ssNMR) measurements to infer refined details about the structural configuration, intermolecular interactions, and relative proximity of all three major biopolymers within air-dried Populus wood. To enhance the utility of these findings and enable evaluation of hypotheses in a physics-based environment in silico, the NMR observables are articulated into an atomistic, macromolecular model for biopolymer assemblies within the plant SCW. Through molecular dynamics simulation, we quantitatively evaluate several variations of atomistic models to determine structural details that are corroborated by ssNMR measurements.

09 BIOMASS FUELS↗
DOE OSTI2024

Metabolic engineering of Caldicellulosiruptor bescii for 2,3-butanediol production from unpretreated lignocellulosic biomass and metabolic strategies for improving yields and titers

ABSTRACT The platform chemical 2,3-butanediol (2,3-BDO) is used to derive products, such as 1,3-butadiene and methyl ethyl ketone, for the chemical and fuel production industries. Efficient microbial 2,3-BDO production at industrial scales has not been achieved yet for various reasons, including product inhibition to host organisms, mixed stereospecificity in product formation, and dependence on expensive substrates (i.e., glucose). In this study, we explore engineering of a 2,3-BDO pathway inCaldicellulosiruptor bescii, an extremely thermophilic (optimal growth temperature = 78°C) and anaerobic bacterium that can break down crystalline cellulose and hemicellulose into fermentable C 5 and C 6 sugars. In addition, C. besciigrows on unpretreated plant biomass, such as switchgrass. Biosynthesis of 2,3-BDO involves three steps: two molecules of pyruvate are condensed into acetolactate; acetolactate is decarboxylated to acetoin, and finally, acetoin is reduced to 2,3-BDO.C. besciinatively produces acetoin; therefore, in order to complete the 2,3-BDO biosynthetic pathway,C. besciiwas engineered to produce a secondary alcohol dehydrogenase (sADH) to catalyze the final step. Two previously characterized, thermostable sADH enzymes with high affinity for acetoin, one from a bacterium and one from an archaeon, were tested independently. When either sADH was present inC. bescii,the recombinant strains were able to produce up to 2.5-mM 2,3-BDO from crystalline cellulose and xylan and 0.2-mM 2,3-BDO directly from unpretreated switchgrass. This serves as the basis for higher yields and productivities, and to this end, limiting factors and potential genetic targets for further optimization were assessed using the genome-scale metabolic model ofC. bescii. IMPORTANCE Lignocellulosic plant biomass as the substrate for microbial synthesis of 2,3-butanediol is one of the major keys toward cost-effective bio-based production of this chemical at an industrial scale. However, deconstruction of biomass to release the sugars for microbial growth currently requires expensive thermochemical and enzymatic pretreatments. In this study, the thermo-cellulolytic bacteriumCaldicellulosiruptor besciiwas successfully engineered to produce 2,3-butanediol from cellulose, xylan, and directly from unpretreated switchgrass. Genome-scale metabolic modeling ofC. besciiwas applied to adjust carbon and redox fluxes to maximize productivity of 2,3-butanediol, thereby revealing bottlenecks that require genetic modifications.

Biotechnology & Applied Microbiology↗