Ectopic Production of 3,4-Dihydroxybenzoate in Planta Affects Cellulose Structure and Organization.

Lignocellulosic biomass is a highly sustainable and largely carbon dioxide neutral feedstock for the production of biofuels and advanced biomaterials. Although thermochemical pretreatment is typically used to increase the efficiency of cell wall deconstruction, genetic engineering of the major plant cell wall polymers, especially lignin, has shown promise as an alternative approach to reduce biomass recalcitrance. Poplar trees with reduced lignin content and altered composition were previously developed by overexpressing bacterial 3-dehydroshikimate dehydratase (QsuB) enzyme to divert carbon flux from the shikimate pathway. In this work, three transgenic poplar lines with increasing QsuB expression levels and different lignin contents were studied using small-angle neutron scattering (SANS) and wide-angle X-ray scattering (WAXS). SANS showed that although the cellulose microfibril cross-sectional dimension remained unchanged, the ordered organization of the microfibrils progressively decreased with increased QsuB expression. This was correlated with decreasing total lignin content in the QsuB lines. WAXS showed that the crystallite dimensions of cellulose microfibrils transverse to the growth direction were not affected by the QsuB expression, but the crystallite dimensions parallel to the growth direction were decreased by ∼20%. Cellulose crystallinity was also decreased with increased QsuB expression, which could be related to high levels of 3,4-dihydroxybenzoate, the product of QsuB expression, disrupting microfibril crystallization. In addition, the cellulose microfibril orientation angle showed a bimodal distribution at higher QsuB expression levels. Overall, this study provides new structural insights into the impact of ectopic synthesis of small-molecule metabolites on cellulose organization and structure that can be used for future efforts aimed at reducing biomass recalcitrance.

Economic and Sustainability Impacts of Yield and Composition Variation in Bioenergy Crops: Switchgrass (Panicum virgatum L.).

Economically viable production of biobased products and fuels requires high-yielding, high-quality, sustainable process-advantaged crops, developed using bioengineering or advanced breeding approaches. Understanding which crop phenotypic traits have the largest impact on biofuel economics and sustainability outcomes is important for the targeted feedstock crop development. Here, we evaluated biomass yield and cell-wall composition traits across a large natural variant population of switchgrass (Panicum virgatum L.) grown across three common garden sites. Samples from 331 switchgrass genotypes were collected and analyzed for carbohydrate and lignin components. Considering plant survival and biomass after multiple years of growth, we found that 84 of the genotypes analyzed may be suited for commercial production in the southeastern U.S. These genotypes show a range of growth and compositional traits across the population that are apparently independent of each other. We used these data to conduct techno-economic analyses and life cycle assessments evaluating the performance of each switchgrass genotype under a standard cellulosic ethanol process model with pretreatment, added enzymes, and fermentation. We find that switchgrass yield per area is the largest economic driver of the minimum fuel selling price (MSFP), ethanol yield per hectare, global warming potential (GWP), and cumulative energy demand (CED). At any yield, the carbohydrate content is significant but of secondary importance. Water use follows similar trends but has more variability due to an increased dependence on the biorefinery model. Analyses presented here highlight the primary importance of plant yield and the secondary importance of carbohydrate content when selecting a feedstock that is both economical and sustainable.

Applications of biomass-derived solvents in biomass pretreatment - Strategies, challenges, and prospects.

Biomass pretreatment is considered a key step in the 2nd generation biofuel production from lignocellulosic biomass. Research on conventional biomass pretreatment solvents has mainly been focused on carbohydrate conversion efficiency, while their hazardousness and/or carbon intensity were not comprehensively considered. Recent sustainability issues request further consideration for eco-friendly and sustainable alternatives like biomass-derived solvents. Carbohydrate and lignin-derived solvents have been proposed and investigated as green alternatives in many biomass processes. In this review, the applications of different types of biomass pretreatment solvents, including organic, ionic liquid, and deep eutectic solvents, are thoroughly discussed. The role of water as a co-solvent in these pretreatment processes is also reviewed. Finally, current research challenges and prospects of utilizing biomass-derived pretreatment solvents for pretreatment are discussed. Given bioethanol's market potential and increasing public awareness about environmental concerns, it will be a priority adopting sustainable and green biomass pretreatment solvents in biorefinery.

Improved chemical and isotopic labeling of biomembranes in Bacillus subtilis by leveraging CRISPRi inhibition of beta-ketoacyl-ACP synthase (fabF).

Assessing the structure of living microbial cell membranes is a challenging analytical goal. The cell membrane is defined by its transverse structure, an approximately 5 nm-thick selectively permeable bilayer that serves many important cellular functions. Compositionally complex, dynamic, and organized in both the transverse and lateral dimensions, understanding the cell membrane structure-and the role that structure plays in cellular function, communication, and environmental sensing is an active scientific effort. Previously, we have devised a novel isotopic labeling approach for membrane lipids to enable direct in vivo structural studies of the cell membrane in the Gram-positive bacterium, Bacillus subtilis, using small-angle neutron scattering. This was accomplished through a genetic inhibition of fatty acid (FA) degradation (ΔfadN) and a chemical inhibition of FA biosynthesis using cerulenin, an irreversible inhibitor of type II fatty acid synthases. Here, we improve upon the previous system by introducing a dCas9/sgRNA-fabF complex that blocks transcription of the essential fabF gene when under xylose induction. This leads to greater sensitivity to cerulenin in the mutant strain (JEBS102) and more robust cell growth when supplementary FAs are introduced to the culture medium. A subtle change in FA uptake is noted when compared to the prior labeling strategy. This is seen in the gas chromatography/mass spectrometry (GC/MS) data as a higher ratio of n16:0 to a15:0, and manifests in an apparent increase in the membrane thickness determined via neutron scattering. This represents an improved method of isotopic labeling for the cell membrane of Bacillus subtilis; enabling improved investigations of cellular uptake and utilization of FAs, cell membrane structure and organization as a phenotypic response to metabolic and environmental changes.

Increased nitrogen use efficiency in crop production can provide economic and environmental benefits.

Potential economic and environmental benefits of increasing nitrogen-use efficiency (NUE) are widely recognized but scarcely quantified. This study quantifies the effects of increased NUE on 1) the national agricultural economy using a simulation model of US agriculture and 2) regional water quality effects using a biogeochemical model for the Arkansas-White-Red river basin. National economic effects are reported for NUE improvement scenarios of 10%, 20%, 50%, and 100%, whereas regional water quality effects are estimated for a 20% NUE improvement scenario in the Arkansas-White-Red river basin. Simulating a 20% increase in NUE in row crops is shown to reduce N requirements by 1.4 million tonnes y-1 and increase farmer net profits by 1.6% ($743 million) per year by 2026 over the baseline simulation for the same period. For each 10% increase in NUE, annual farm revenues for commodity crops increased over the baseline by approximately $350 million per year by 2026. Changes in crop prices and land-use relative to the baseline were less than 2%. This suggests a net benefit even though fertilizer cost savings can result in increased cultivation of land, i.e., 'Jevon's paradox'. Results from the biogeochemical model of the Arkansas-White-Red river basin suggest that a 20% increase in NUE corresponds to a 5.72% reduction in nitrate loadings to freshwaters, with higher reductions in agricultural watersheds. The value of these reductions was estimated as $43 ha-1, for a total of $15.3 to 136.7 million yr-1 in avoided water treatment costs. After estimating the social value of increased NUE, we conclude with a discussion of potential strategies to increase efficiency and the research needed to achieve this goal. These include perennialization of the agricultural landscape, genetic crop improvement, targeted fertilizer application, and manipulation of the plant-root microbiome.

Implementation of a self-consistent slab model of bilayer structure in the SasView suite.

Slab models are simple and useful structural descriptions which have long been used to describe lyotropic lamellar phases, such as lipid bilayers. Typically, slab models assume a midline symmetry and break a bilayer structure into three pieces, a central solvent-free core and two symmetric outer layers composed of the soluble portion of the amphiphile and associated solvent. This breakdown matches reasonably well to the distribution of neutron scattering length density and therefore is a convenient and common approach for the treatment of small-angle scattering data. Here, an implementation of this model within the SasView software suite is reported. The implementation is intended to provide physical consistency through the area per amphiphile molecule and number of solvent molecules included within the solvent-exposed outer layer. The proper use of this model requires knowledge of (or good estimates for) the amphiphile and solvent molecule volume and atomic composition, ultimately providing a self-consistent data treatment with only two free parameters: the lateral area per amphiphile molecule and the number of solvent molecules included in the outer region per amphiphile molecule. The use of this code is demonstrated in the fitting of standard lipid bilayer data sets, obtaining structural parameters consistent with prior literature and illustrating the typical and ideal cases of fitting for neutron scattering data obtained using single or multiple contrast conditions. While demonstrated here for lipid bilayers, this model is intended for general application to block copolymers, surfactants, and other lyotropic lamellar phase structures for which a slab model is able to reasonably estimate the neutron scattering length density/electron-density profile of inner and outer layers of the lamellae.

Deconstruction of biomass enabled by local demixing of cosolvents at cellulose and lignin surfaces.

A particularly promising approach to deconstructing and fractionating lignocellulosic biomass to produce green renewable fuels and high-value chemicals pretreats the biomass with organic solvents in aqueous solution. Here, neutron scattering and molecular-dynamics simulations reveal the temperature-dependent morphological changes in poplar wood biomass during tetrahydrofuran (THF):water pretreatment and provide a mechanism by which the solvent components drive efficient biomass breakdown. Whereas lignin dissociates over a wide temperature range (>25 °C) cellulose disruption occurs only above 150 °C. Neutron scattering with contrast variation provides direct evidence for the formation of THF-rich nanoclusters (Rg ∼ 0.5 nm) on the nonpolar cellulose surfaces and on hydrophobic lignin, and equivalent water-rich nanoclusters on polar cellulose surfaces. The disassembly of the amphiphilic biomass is thus enabled through the local demixing of highly functional cosolvents, THF and water, which preferentially solvate specific biomass surfaces so as to match the local solute polarity. A multiscale description of the efficiency of THF:water pretreatment is provided: matching polarity at the atomic scale prevents lignin aggregation and disrupts cellulose, leading to improvements in deconstruction at the macroscopic scale.

Silencing Folylpolyglutamate Synthetase1 (FPGS1) in Switchgrass (Panicum virgatum L.) Improves Lignocellulosic Biofuel Production.

Switchgrass (Panicum virgatum L.) is a lignocellulosic perennial grass with great potential in bioenergy field. Lignocellulosic bioenergy crops are mostly resistant to cell wall deconstruction, and therefore yield suboptimal levels of biofuel. The one-carbon pathway (also known as C1 metabolism) is critical for polymer methylation, including that of lignin and hemicelluloses in cell walls. Folylpolyglutamate synthetase (FPGS) catalyzes a biochemical reaction that leads to the formation of folylpolyglutamate, an important cofactor for many enzymes in the C1 pathway. In this study, the putatively novel switchgrass PvFPGS1 gene was identified and its functional role in cell wall composition and biofuel production was examined by RNAi knockdown analysis. The PvFPGS1-downregulated plants were analyzed in the field over three growing seasons. Transgenic plants with the highest reduction in PvFPGS1 expression grew slower and produced lower end-of-season biomass. Transgenic plants with low-to-moderate reduction in PvFPGS1 transcript levels produced equivalent biomass as controls. There were no significant differences observed for lignin content and syringyl/guaiacyl lignin monomer ratio in the low-to-moderately reduced PvFPGS1 transgenic lines compared with the controls. Similarly, sugar release efficiency was also not significantly different in these transgenic lines compared with the control lines. However, transgenic plants produced up to 18% more ethanol while maintaining congruent growth and biomass as non-transgenic controls. Severity of rust disease among transgenic and control lines were not different during the time course of the field experiments. Altogether, the unchanged lignin content and composition in the low-to-moderate PvFPGS1-downregulated lines may suggest that partial downregulation of PvFPGS1 expression did not impact lignin biosynthesis in switchgrass. In conclusion, the manipulation of PvFPGS1 expression in bioenergy crops may be useful to increase biofuel potential with no growth penalty or increased susceptibility to rust in feedstock.

Gene targets for engineering osmotolerance in Caldicellulosiruptor bescii.

BACKGROUND: Caldicellulosiruptor bescii, a promising biocatalyst being developed for use in consolidated bioprocessing of lignocellulosic materials to ethanol, grows poorly and has reduced conversion at elevated medium osmolarities. Increasing tolerance to elevated fermentation osmolarities is desired to enable performance necessary of a consolidated bioprocessing (CBP) biocatalyst. RESULTS: Two strains of C. bescii showing growth phenotypes in elevated osmolarity conditions were identified. The first strain, ORCB001, carried a deletion of the FapR fatty acid biosynthesis and malonyl-CoA metabolism repressor and had a severe growth defect when grown in high-osmolarity conditions-introduced as the addition of either ethanol, NaCl, glycerol, or glucose to growth media. The second strain, ORCB002, displayed a growth rate over three times higher than its genetic parent when grown in high-osmolarity medium. Unexpectedly, a genetic complement ORCB002 exhibited improved growth, failing to revert the observed phenotype, and suggesting that mutations other than the deleted transcription factor (the fruR/cra gene) are responsible for the growth phenotype observed in ORCB002. Genome resequencing identified several other genomic alterations (three deleted regions, three substitution mutations, one silent mutation, and one frameshift mutation), which may be responsible for the observed increase in osmolarity tolerance in the fruR/cra-deficient strain, including a substitution mutation in dnaK, a gene previously implicated in osmoresistance in bacteria. Differential expression analysis and transcription factor binding site inference indicates that FapR negatively regulates malonyl-CoA and fatty acid biosynthesis, as it does in many other bacteria. FruR/Cra regulates neighboring fructose metabolism genes, as well as other genes in global manner. CONCLUSIONS: Two systems able to effect tolerance to elevated osmolarities in C. bescii are identified. The first is fatty acid biosynthesis. The other is likely the result of one or more unintended, secondary mutations present in another transcription factor deletion strain. Though the locus/loci and mechanism(s) responsible remain unknown, candidate mutations are identified, including a mutation in the dnaK chaperone coding sequence. These results illustrate both the promise of targeted regulatory manipulation for osmotolerance (in the case of fapR) and the challenges (in the case of fruR/cra).

Nanomechanics and Raman Spectroscopy of in Situ Native Carbohydrate Storage Granules for Enhancing Starch Quality and Lignocellulosic Biomass Production.

Alternative energy strategies based on plant biomass-derived bioenergy and biofuels rely on understanding and optimization of plant structure, chemistry, and performance. Starch, a constitutive element of all green plants, is important to food, biofuels, and industrial applications. Models of carbohydrate storage granules are highly heterogeneous in representing morphology and structure, though a deeper understanding of the role of structure in functional behavior is emerging. A better understanding of the in situ nanoscale properties of native granules is needed to help improve the starch quality in food crops as well as optimize lignocellulosic biomass production in perennial nonfood crops. Here, we present a new technique called soft mechanical nano-ablation (sMNA) for accessing the interior of the granules without compromising the inner nanostructure. We then explore the nanomechanics of granules within the ray parenchyma cells of Populus xylem, a desirable woody biofuel feedstock. The employed soft outer layer nanoablation and atomic force microscopy reveal that the inner structure comprises 156 nm blocklets arranged in a semicrystalline organization. The nanomechanical properties of the inner and outer structures of a single starch granule are measured and found to exhibit large variations, changing by a factor of 3 in Young's modulus and a factor of 2 in viscoplastic index. These findings demonstrate how the introduced approach facilitates studies of structure-function relationships among starch granules and more complex secondary cell wall features as they relate to plant performance.

Technoeconomic and life-cycle analysis of single-step catalytic conversion of wet ethanol into fungible fuel blendstocks.

Technoeconomic and life-cycle analyses are presented for catalytic conversion of ethanol to fungible hydrocarbon fuel blendstocks, informed by advances in catalyst and process development. Whereas prior work toward this end focused on 3-step processes featuring dehydration, oligomerization, and hydrogenation, the consolidated alcohol dehydration and oligomerization (CADO) approach described here results in 1-step conversion of wet ethanol vapor (40 wt% in water) to hydrocarbons and water over a metal-modified zeolite catalyst. A development project increased liquid hydrocarbon yields from 36% of theoretical to >80%, reduced catalyst cost by an order of magnitude, scaled up the process by 300-fold, and reduced projected costs of ethanol conversion 12-fold. Current CADO products conform most closely to gasoline blendstocks, but can be blended with jet fuel at low levels today, and could potentially be blended at higher levels in the future. Operating plus annualized capital costs for conversion of wet ethanol to fungible blendstocks are estimated at $2.00/GJ for CADO today and $1.44/GJ in the future, similar to the unit energy cost of producing anhydrous ethanol from wet ethanol ($1.46/GJ). Including the cost of ethanol from either corn or future cellulosic biomass but not production incentives, projected minimum selling prices for fungible blendstocks produced via CADO are competitive with conventional jet fuel when oil is $100 per barrel but not at $60 per barrel. However, with existing production incentives, the projected minimum blendstock selling price is competitive with oil at $60 per barrel. Life-cycle greenhouse gas emission reductions for CADO-derived hydrocarbon blendstocks closely follow those for the ethanol feedstock.

Correction to: Multiple levers for overcoming the recalcitrance of lignocellulosic biomass.

[This corrects the article DOI: 10.1186/s13068-019-1353-7.].

Production of deuterated biomass by cultivation of Lemna minor (duckweed) in D2O.

MAIN CONCLUSION: Common duckweed Lemna minor was cultivated in 50% D2O to produce biomass with 50-60% deuterium incorporation containing cellulose with degree of polymerization close (85%) to that of H2O-grown controls. The small aquatic plant duckweed, particularly the genus Lemna, widely used for toxicity testing, has been proposed as a potential source of biomass for conversion into biofuels as well as a platform for production of pharmaceuticals and specialty chemicals. Ability to produce deuterium-substituted duckweed can potentially extend the range of useful products as well as assist process improvement. Cultivation of these plants under deuterating conditions was previously been reported to require addition of kinetin to induce growth and was hampered by anomalies in cellular morphology and protein metabolism. Here, we report the production of biomass with 50-60% deuterium incorporation by long-term photoheterotrophic growth of common duckweed Lemna minor in 50% D2O with 0.5% glucose. L. minor grown in 50% D2O without addition of kinetin exhibited a lag phase twice that of H2O-grown controls, before start of log phase growth at 40% of control rates. Compared to continuous white fluorescent light, growth rates increased fivefold for H2O and twofold for 50% D2O when plants were illuminated at higher intensity with a metal halide lamp and a diurnal cycle of 12-h light/12-h dark. Deuterium incorporation was determined by a combination of 1H and 2H nuclear magnetic resonance (NMR) to be 40-60%. The cellulose from the deuterated plants had an average-number degree of polymerization (DPn) and polydispersity index (PDI) close to that of H2O-grown controls, while Klason lignin content was reduced. The only major gross morphological change noted was root inhibition.

Multiple levers for overcoming the recalcitrance of lignocellulosic biomass.

Background: The recalcitrance of cellulosic biomass is widely recognized as a key barrier to cost-effective biological processing to fuels and chemicals, but the relative impacts of physical, chemical and genetic interventions to improve biomass processing singly and in combination have yet to be evaluated systematically. Solubilization of plant cell walls can be enhanced by non-biological augmentation including physical cotreatment and thermochemical pretreatment, the choice of biocatalyst, the choice of plant feedstock, genetic engineering of plants, and choosing feedstocks that are less recalcitrant natural variants. A two-tiered combinatoric investigation of lignocellulosic biomass deconstruction was undertaken with three biocatalysts (Clostridium thermocellum, Caldicellulosiruptor bescii, Novozymes Cellic® Ctec2 and Htec2), three transgenic switchgrass plant lines (COMT, MYB4, GAUT4) and their respective nontransgenic controls, two Populus natural variants, and augmentation of biological attack using either mechanical cotreatment or cosolvent-enhanced lignocellulosic fractionation (CELF) pretreatment. Results: In the absence of augmentation and under the conditions tested, increased total carbohydrate solubilization (TCS) was observed for 8 of the 9 combinations of switchgrass modifications and biocatalysts tested, and statistically significant for five of the combinations. Our results indicate that recalcitrance is not a trait determined by the feedstock only, but instead is coequally determined by the choice of biocatalyst. TCS with C. thermocellum was significantly higher than with the other two biocatalysts. Both CELF pretreatment and cotreatment via continuous ball milling enabled TCS in excess of 90%. Conclusion: Based on our results as well as literature studies, it appears that some form of non-biological augmentation will likely be necessary for the foreseeable future to achieve high TCS for most cellulosic feedstocks. However, our results show that this need not necessarily involve thermochemical processing, and need not necessarily occur prior to biological conversion. Under the conditions tested, the relative magnitude of TCS increase was augmentation > biocatalyst choice > plant choice > plant modification > plant natural variants. In the presence of augmentation, plant modification, plant natural variation, and plant choice exhibited a small, statistically non-significant impact on TCS.

Rex in Caldicellulosiruptor bescii: Novel regulon members and its effect on the production of ethanol and overflow metabolites.

Rex is a global redox-sensing transcription factor that senses and responds to the intracellular [NADH]/[NAD+ ] ratio to regulate genes for central metabolism, and a variety of metabolic processes in Gram-positive bacteria. We decipher and validate four new members of the Rex regulon in Caldicellulosiruptor bescii; a gene encoding a class V aminotransferase, the HydG FeFe Hydrogenase maturation protein, an oxidoreductase, and a gene encoding a hypothetical protein. Structural genes for the NiFe and FeFe hydrogenases, pyruvate:ferredoxin oxidoreductase, as well as the rex gene itself are also members of this regulon, as has been predicted previously in different organisms. A C. bescii rex deletion strain constructed in an ethanol-producing strain made 54% more ethanol (0.16 mmol/L) than its genetic parent after 36 hr of fermentation, though only under nitrogen limited conditions. Metabolomic interrogation shows this rex-deficient ethanol-producing strain synthesizes other reduced overflow metabolism products likely in response to more reduced intracellular redox conditions and the accumulation of pyruvate. These results suggest ethanol production is strongly dependent on the native intracellular redox state in C. bescii, and highlight the combined promise of using this gene and manipulation of culture conditions to yield strains capable of producing ethanol at higher yields and final titer.

Hemicellulose-Cellulose Composites Reveal Differences in Cellulose Organization after Dilute Acid Pretreatment.

Model hemicellulose-cellulose composites that mimic plant cell wall polymer interactions were prepared by synthesizing deuterated bacterial cellulose in the presence of glucomannan or xyloglucan. Dilute acid pretreatment (DAP) of these materials was studied using small-angle neutron scattering, X-ray diffraction, and sum frequency generation spectroscopy. The macrofibril dimensions of the pretreated cellulose alone were smaller but with similar entanglement of macrofibrillar network as native cellulose. In addition, the crystallite size dimension along the (010) plane increased. Glucomannan-cellulose underwent similar changes to cellulose, except that the macrofibrillar network was more entangled after DAP. Conversely, in xyloglucan-cellulose the macrofibril dimensions and macrofibrillar network were relatively unchanged after pretreatment, but the cellulose Iß content was increased. Our results point to a tight interaction of xyloglucan with microfibrils while glucomannan only interacts with macrofibril surfaces. This study provides insight into roles of different hemicellulose-cellulose interactions and may help in improving pretreatment processes or engineering plants with decreased recalcitrance.

Ultrastructure and Enzymatic Hydrolysis of Deuterated Switchgrass.

Neutron scattering of deuterated plants can provide fundamental insight into the structure of lignocellulosics in plant cell walls and its deconstruction by pretreatment and enzymes. Such plants need to be characterized for any alterations to lignocellulosic structure caused by growth in deuterated media. Here we show that glucose yields from enzymatic hydrolysis at lower enzyme loading were 35% and 30% for untreated deuterated and protiated switchgrass, respectively. Lignin content was 4% higher in deuterated switchgrass but there were no significant lignin structural differences. Transmission electron microscopy showed differences in lignin distribution and packing of fibers in the cell walls that apparently increased surface area of cellulose in deuterated switchgrass, increasing cellulose accessibility and lowering its recalcitrance. These differences in lignification were likely caused by abiotic stress due to growth in deuterated media.

Hemicellulose characterization of deuterated switchgrass.

This work describes the structural characterization of hemicellulose isolated from hydroponically grown switchgrass in H2O medium (protiated) or 50% D2O medium (deuterated) through compositional analysis, GPC, FTIR, 13C and 1H/13C HSQC NMR. 4-O-methyl glucuronoarabinoxylan (GAX), the major hemicellulose in switchgrass isolated from deuterated switchgrass, had structural properties similar to hemicellulose isolated from protiated switchgrass. Both had comparable arabinose to xylose ratio (0.25) and molecular weight (47-50 kDa). Structural similarities show that deuterated switchgrass hemicellulose can be used as a model carbohydrate polymer in neutron scattering, or pharmaceutical studies due to their immunomodulatory activity and gastroprotective effects.

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. 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. Decreased recalcitrance in biomass processing and increased growth are likely due to reduced HG and RGII cross-linking in the cell wall.