Effect of cellulose reducing ends and primary hydroxyl groups modifications on cellulose-cellulase interactions and cellulose hydrolysis.

Cellulose reducing ends are believed to play a vital role in the cellulose recalcitrance to enzymatic conversion. However, their role in insoluble cellulose accessibility and hydrolysis is not clear. Thus, in this study, reducing ends of insoluble cellulose derived from various sources were modified by applying reducing and/or oxidizing agents.

Proteomic and metabolic disturbances in lignin-modified Brachypodium distachyon.

Lignin biosynthesis begins with the deamination of phenylalanine and tyrosine (Tyr) as a key branch point between primary and secondary metabolism in land plants. Here, we used a systems biology approach to investigate the global metabolic responses to lignin pathway perturbations in the model grass Brachypodium distachyon. We identified the lignin biosynthetic protein families and found that ammonia-lyases (ALs) are among the most abundant proteins in lignifying tissues in grasses.

THF co-solvent pretreatment prevents lignin redeposition from interfering with enzymes yielding prolonged cellulase activity.

Background: Conventional aqueous dilute sulfuric acid (DSA) pretreatment of lignocellulosic biomass facilitates hemicellulose solubilization and can improve subsequent enzymatic digestibility of cellulose to fermentable glucose. However, much of the lignin after DSA pretreatment either remains intact within the cell wall or readily redeposits back onto the biomass surface. This redeposited lignin has been shown to reduce enzyme activity and contribute to rapid enzyme deactivation, thus, necessitating significantly higher enzyme loadings than deemed economical for biofuel production from biomass.

Targeting hydroxycinnamoyl CoA: shikimate hydroxycinnamoyl transferase for lignin modification in Brachypodium distachyon.

Background: Hydroxycinnamoyl CoA: shikimate hydroxycinnamoyl transferase (HCT) is a central enzyme of the so-called "esters" pathway to monolignols. As originally envisioned, HCT functions twice in this pathway, to form coumaroyl shikimate and then, in the "reverse" direction, to convert caffeoyl shikimate to caffeoyl CoA. The discovery of a caffeoyl shikimate esterase (CSE) that forms caffeic acid directly from caffeoyl shikimate calls into question the need for the reverse HCT reaction in lignin biosynthesis.

4-Coumarate 3-hydroxylase in the lignin biosynthesis pathway is a cytosolic ascorbate peroxidase.

Lignin biosynthesis is evolutionarily conserved among higher plants and features a critical 3-hydroxylation reaction involving phenolic esters. However, increasing evidence questions the involvement of a single pathway to lignin formation in vascular plants. Here we describe an enzyme catalyzing the direct 3-hydroxylation of 4-coumarate to caffeate in lignin biosynthesis as a bifunctional peroxidase that oxidizes both ascorbate and 4-coumarate at comparable rates.

SPL7 and SPL8 represent a novel flowering regulation mechanism in switchgrass.

The aging pathway in flowering regulation is controlled mainly by microRNA156 (miR156). Studies in Arabidopsis thaliana reveal that nine miR156-targeted SQUAMOSA PROMOTER BINDING-LIKE (SPL) genes are involved in the control of flowering. However, the roles of SPLs in flowering remain elusive in grasses.

Characterization of novel glycosyl hydrolases discovered by cell wall glycan directed monoclonal antibody screening and metagenome analysis of maize aerial root mucilage.

An indigenous maize landrace from the Sierra Mixe region of Oaxaca, Mexico exhibits extensive formation of aerial roots which exude large volumes of a polysaccharide-rich gel matrix or "mucilage" that harbors diazotrophic microbiota. We hypothesize that the mucilage associated microbial community carries out multiple functions, including disassembly of the mucilage polysaccharide. In situ, hydrolytic assay of the mucilage revealed endogenous arabinofuranosidase, galactosidase, fucosidase, mannosidase and xylanase activities.

Gene regulatory networks for lignin biosynthesis in switchgrass (Panicum virgatum).

Cell wall recalcitrance is the major challenge to improving saccharification efficiency in converting lignocellulose into biofuels. However, information regarding the transcriptional regulation of secondary cell wall biogenesis remains poor in switchgrass (Panicum virgatum), which has been selected as a biofuel crop in the United States. In this study, we present a combination of computational and experimental approaches to develop gene regulatory networks for lignin formation in switchgrass.

Elicitors and defense gene induction in plants with altered lignin compositions.

A reduction in the lignin content in transgenic plants induces the ectopic expression of defense genes, but the importance of altered lignin composition in such phenomena remains unclear. Two Arabidopsis lines with similar lignin contents, but strikingly different lignin compositions, exhibited different quantitative and qualitative transcriptional responses. Plants with lignin composed primarily of guaiacyl units overexpressed genes responsive to oomycete and bacterial pathogen attack, whereas plants with lignin composed primarily of syringyl units expressed a far greater number of defense genes, including some associated with cis-jasmone-mediated responses to aphids; these plants exhibited altered responsiveness to bacterial and aphid inoculation.

Sugar yield and composition of tubers from Jerusalem Artichoke (Helianthus tuberosus) irrigated with saline waters.

Currently, major biofuel crops are also food crops that demand fertile soils and good-quality water. Jerusalem artichoke (Helianthus tuberosus, Asteraceae) produces high tonnage of tubers that are rich in sugars, mainly in the form of inulin. In this study, plants of the cultivar "White Fuseau" grown under five salinity levels were evaluated for tuber yield.

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.

Working towards recalcitrance mechanisms: increased xylan and homogalacturonan production by overexpression of () causes increased recalcitrance and decreased growth in .

Background: The development of fast-growing hardwood trees as a source of lignocellulosic biomass for biofuel and biomaterial production requires a thorough understanding of the plant cell wall structure and function that underlie the inherent recalcitrance properties of woody biomass. Downregulation of in was recently reported to result in improved biomass saccharification, plant growth, and biomass yield. To further understand function in biomass recalcitrance and plant growth, here we report the effects of overexpression in .

Deactivation of Cellulase at the Air-Liquid Interface Is the Main Cause of Incomplete Cellulose Conversion at Low Enzyme Loadings.

Amphiphilic additives such as bovine serum albumin (BSA) and Tween have been used to improve cellulose hydrolysis by cellulases. However, there has been a lack of clarity to explain their mechanism of action in enzymatic hydrolysis of pure or low-lignin cellulosic substrates. In this work, a commercial Trichoderma reesei enzyme preparation and the amphiphilic additives BSA and Tween 20 were applied for hydrolysis of pure Avicel cellulose.

Development and use of a switchgrass ( L.) transformation pipeline by the BioEnergy Science Center to evaluate plants for reduced cell wall recalcitrance.

Background: The mission of the BioEnergy Science Center (BESC) was to enable efficient lignocellulosic-based biofuel production. One BESC goal was to decrease poplar and switchgrass biomass recalcitrance to biofuel conversion while not affecting plant growth. A transformation pipeline (TP), to express transgenes or transgene fragments (constructs) in these feedstocks with the goal of understanding and decreasing recalcitrance, was considered essential for this goal.

Dynamic changes in transcriptome and cell wall composition underlying brassinosteroid-mediated lignification of switchgrass suspension cells.

Background: Plant cell walls contribute the majority of plant biomass that can be used to produce transportation fuels. However, the complexity and variability in composition and structure of cell walls, particularly the presence of lignin, negatively impacts their deconstruction for bioenergy. Metabolic and genetic changes associated with secondary wall development in the biofuel crop switchgrass () have yet to be reported.

Visualizing chemical functionality in plant cell walls.

Understanding plant cell wall cross-linking chemistry and polymeric architecture is key to the efficient utilization of biomass in all prospects from rational genetic modification to downstream chemical and biological conversion to produce fuels and value chemicals. In fact, the bulk properties of cell wall recalcitrance are collectively determined by its chemical features over a wide range of length scales from tissue, cellular to polymeric architectures. Microscopic visualization of cell walls from the nanometer to the micrometer scale offers an in situ approach to study their chemical functionality considering its spatial and chemical complexity, particularly the capabilities of characterizing biomass non-destructively and in real-time during conversion processes.

Adding tetrahydrofuran to dilute acid pretreatment provides new insights into substrate changes that greatly enhance biomass deconstruction by and fungal enzymes.

Background: Consolidated bioprocessing (CBP) by anaerobes, such as which combine enzyme production, hydrolysis, and fermentation are promising alternatives to historical economic challenges of using fungal enzymes for biological conversion of lignocellulosic biomass. However, limited research has integrated CBP with real pretreated biomass, and understanding how pretreatment impacts subsequent deconstruction by CBP vs. fungal enzymes can provide valuable insights into CBP and suggest other novel biomass deconstruction strategies.

The effect of liquid hot water pretreatment on the chemical-structural alteration and the reduced recalcitrance in poplar.

Background: Hydrothermal pretreatment using liquid hot water (LHW) is capable of substantially reducing the cell wall recalcitrance of lignocellulosic biomass. It enhances the saccharification of polysaccharides, particularly cellulose, into glucose with relatively low capital required. Due to the close association with biomass recalcitrance, the structural change of the components of lignocellulosic materials during the pretreatment is crucial to understand pretreatment chemistry and advance the bio-economy.

Overcoming factors limiting high-solids fermentation of lignocellulosic biomass to ethanol.

Simultaneous saccharification and fermentation (SSF) of solid biomass can reduce the complexity and improve the economics of lignocellulosic ethanol production by consolidating process steps and reducing end-product inhibition of enzymes compared with separate hydrolysis and fermentation (SHF). However, a long-standing limitation of SSF has been too low ethanol yields at the high-solids loading of biomass needed during fermentation to realize sufficiently high ethanol titers favorable for more economical ethanol recovery. Here, we illustrate how competing factors that limit ethanol yields during high-solids fermentations are overcome by integrating newly developed cosolvent-enhanced lignocellulosic fractionation (CELF) pretreatment with SSF.

Enhanced guide-RNA design and targeting analysis for precise CRISPR genome editing of single and consortia of industrially relevant and non-model organisms.

Motivation: Genetic diversity of non-model organisms offers a repertoire of unique phenotypic features for exploration and cultivation for synthetic biology and metabolic engineering applications. To realize this enormous potential, it is critical to have an efficient genome editing tool for rapid strain engineering of these organisms to perform novel programmed functions.

Results: To accommodate the use of CRISPR/Cas systems for genome editing across organisms, we have developed a novel method, named CRISPR Associated Software for Pathway Engineering and Research (CASPER), for identifying on- and off-targets with enhanced predictability coupled with an analysis of non-unique (repeated) targets to assist in editing any organism with various endonucleases.

From model to crop: functional characterization of SPL8 in M. truncatula led to genetic improvement of biomass yield and abiotic stress tolerance in alfalfa.

Biomass yield, salt tolerance and drought tolerance are important targets for alfalfa (Medicago sativa L.) improvement. Medicago truncatula has been developed into a model plant for alfalfa and other legumes.

The miR156-SPL4 module predominantly regulates aerial axillary bud formation and controls shoot architecture.

Grasses possess basal and aerial axillary buds. Previous studies have largely focused on basal bud (tiller) formation but scarcely touched on aerial buds, which may lead to aerial branch development. Genotypes with and without aerial buds were identified in switchgrass (Panicum virgatum), a dedicated bioenergy crop.

Comparison of four glycosyl residue composition methods for effectiveness in detecting sugars from cell walls of dicot and grass tissues.

Background: The effective use of plant biomass for biofuel and bioproduct production requires a comprehensive glycosyl residue composition analysis to understand the different cell wall polysaccharides present in the different biomass sources. Here we compared four methods side-by-side for their ability to measure the neutral and acidic sugar composition of cell walls from herbaceous, grass, and woody model plants and bioenergy feedstocks.

Results: Arabidopsis, , rice, and switchgrass leaf cell walls, as well as cell walls from wood, rice stems, and switchgrass tillers, were analyzed by (1) gas chromatography-mass spectrometry (GC-MS) of alditol acetates combined with a total uronic acid assay; (2) carbodiimide reduction of uronic acids followed by GC-MS of alditol acetates; (3) GC-MS of trimethylsilyl (TMS) derivatives; and (4) high-pressure, anion-exchange chromatography (HPAEC).

Understanding the Changes to Biomass Surface Characteristics after Ammonia and Organosolv Pretreatments by Using Time-of-Flight Secondary-Ion Mass Spectrometry (TOF-SIMS).

Surface characteristic changes to poplar after ammonia and organosolv pretreatments were investigated by means of time-of-flight secondary-ion mass spectrometry (TOF-SIMS) analysis. Whereas normalized total polysaccharides and lignin contents on the surface differed from bulk chemical compositions, the surface cellulose ions detected by TOF-SIMS showed the same value trend as the cellulose content in the biomass. In addition, the lignin syringyl/guaiacyl ratio according to TOF-SIMS results showed the same trend as the ratio measured by means of NMR spectroscopic analysis, even though the ratio scales for each method were different.