Solid State NMR data
Applying multi-dimensional solid state NMR to explore the nanoarchitecture of native and engineered plant cell walls
59 BASIC BIOLOGICAL SCIENCES↗
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Deconstruction by C. thermocellum —from microbe mediated to dynamic redistribution of cellulosomes
Clostridium thermocellum is one of the most efficient microorganisms for the deconstruction of cellulosic biomass. To achieve this high level of cellulolytic activity, C. thermocellum uses large multienzyme complexes known as cellulosomes to break down complex polysaccharides, notably cellulose, found in plant cell walls. The attachment of bacterial cells to the nearby substrate via the cellulosome has been hypothesized to be the reason for this high efficiency. The region lying between the cell and the substrate has shown great variation and dynamics that are affected by the growth stage of cells and the substrate used for growth. Here, we used both super-resolution imaging and machine-learning approaches to study the distribution of C. thermocellum cellulosomes at different stages of growth. We show that C. thermocellum initially retains its cellulosomes primarily on the cell surface but then relocates large cellulosome clusters to the interface with biomass, therefore depleting its cell surface of cellulosomes. These results indicate dynamic redistribution of cellulosomes during growth, with a functional shift toward substrate-associated degradation later during growth on biomass.
09 BIOMASS FUELS↗
Supercharged cellulases show superior thermal stability and enhanced activity towards pretreated biomass and cellulose
Non-productive binding of cellulolytic enzymes to various plant cell wall components, such as lignin and cellulose, necessitates high enzyme loadings to achieve efficient conversion of pretreated lignocellulosic biomass to fermentable sugars. Protein supercharging was previously employed as one of the strategies to reduce non-productive binding to biomass. However, various questions remain unanswered regarding the hydrolysis kinetics of supercharged enzymes towards pretreated biomass substrates and the role played by enzyme interactions with individual cell wall polymers such as cellulose and xylan. In this study, CBM2a (from Thermobifida fusca ) fused with endocellulase Cel5A (from T. fusca ) was used as the model wild-type enzyme and CBM2a was supercharged using Rosetta, to obtain eight variants with net charges spanning -14 to +6. These enzymes were recombinantly expressed in E. coli , purified from cell lysates, and their hydrolytic activities were tested against pretreated biomass substrates (AFEX and EA treated corn stover). Although the wild-type enzyme showed greater activity compared to both negatively and positively supercharged enzymes towards pretreated biomass, thermal denaturation assays identified two negatively supercharged constructs that perform better than the wild-type enzyme (~3 to 4-fold difference in activity) upon thermal deactivation at higher temperatures. To better understand the causal factor of reduced supercharged enzyme activity towards AFEX corn stover, we performed hydrolysis assays on cellulose-I/xylan/pNPC, lignin inhibition assays, and thermal stability assays. Altogether, these assays showed that the negatively supercharged mutants were highly impacted by reduced activity towards xylan whereas the positively supercharged mutants showed dramatically reduced activity towards cellulose and xylan. It was identified that a combination of impaired cellulose binding and lower thermal stability was the cause of reduced hydrolytic activity of positively supercharged enzyme sub-group. Overall, this study demonstrated a systematic approach to investigate the behavior of supercharged enzymes and identified supercharged enzyme constructs that show superior activity at elevated temperatures. Future work will address the impact of parameters such as pH, salt concentration, and assay temperature on the hydrolytic activity and thermal stability of supercharged enzymes.
09 BIOMASS FUELS↗
Towards Elucidating Structure–Spectra Relationships in Rhamnogalacturonan II: Computational Protocols for Accurate 13C and 1H Shifts for Apiose and Its Borate Esters
Apiose is a naturally occurring, uncommon branched-chain pentose found in plant cell walls as part of the complex polysaccharide Rhamnogalacturonan II (RG-II). The structural elucidation of the three-dimensional structure of RG-II by nuclear magnetic resonance (NMR) spectroscopy is significantly complicated by the ability of apiose to cross-link via borate ester linkages to form RG-II dimers. Here, we developed a computational approach to gain insight into the structure–spectra relationships of apio–borate complexes in an effort to complement experimental assignments of NMR signals in RG-II. Our protocol involved structure optimizations using density functional theory (DFT) followed by isotropic magnetic shielding constant calculations using the gauge-invariant atomic orbital (GIAO) approach to predict chemical shifts. We evaluated the accuracy of 23 different functional–basis set (FBS) combinations with and without implicit solvation for predicting the experimental 1 H and 13 C shifts of a methyl apioside and its three borate derivatives. The computed NMR predictions were evaluated on the basis of the overall shift accuracy, relative shift ordering, and the ability to distinguish between dimers and monomers. We demonstrate that the consideration of implicit solvation during geometry optimizations in addition to the magnetic shielding constant calculations greatly increases the accuracy of NMR chemical shift predictions and can correctly reproduce the ordering of the 13 C shifts and yield predictions that are, on average, within 1.50 ppm for 13 C and 0.12 ppm for 1 H shifts for apio–borate compounds.
BASIC BIOLOGICAL SCIENCES,BIOMASS FUELS↗
Isolation of Hybridomas for Golgi-associated Proteins and a Plant Calmodulin
The demonstration of a role for calcium in the mechanism of the gravitropic response indicates a role for calmodulin. Localization studies indicate that plant cell walls have a high content of calmodulin which suggests a regulatory role for CaM in both gravitropic curvature and auxin-induced growth. Auxin regulation of cell wall loosening and elongation is the basis for most models of this phenomenon. Auxin treatment of pea stem tissue rapidly increases the ctivity of Golgi-localized B-1,4-glucan synthase (GS), an enzyme involved in biosynthesis of wall xyloglucan which apparently constitutes the substrate for the wall loosening process. In order to determine whether auxin stimulates GS activity either by modulation of existing enzyme or induces de novo formation of Golgi glucan synthase, a study was undertaken to isolate and quantitate glucan synthase. This enzyme appears to be an integral protein of the Golgi membrane and has resisted isolation with retention of activity. The production of monoclonal antibody for glucan synthase was undertaken due to the inability to isolate GS by standard detergent/liposome techniques.
Kuzmanoff, K. M.↗
Exoproteomics Reveals Outer Membrane Vesicle Mediated Catabolism of Lignin-Derived Aromatics by P. putida KT2440
Lignin is an abundant component of plant cell walls which is heterogeneous and highly resistant to degradation. Biological funneling of lignin via microbes represents an efficient approach for reducing chemical heterogeneity and generating molecules which can be valorized into performance-advantaged materials. Pseudomonas putida KT2440 (P. putida) catabolizes lignin-derived aromatic monomers rapidly and efficiently via the b-ketoadipate (bKA) pathway and our data suggest P. putida may depolymerize HMW lignin as well [1]. The mechanism by which HMW lignin is depolymerized by bacteria remains unknown. Intriguingly, we found that P. putida secretes outer membrane vesicles (OMVs) of two distinct sizes in the presence of lignin. Here, we describe the temporal proteome of OMVs and the vesicle-free secretome (VFS) in the presence or absence of lignin. Enzymes involved in aromatic catabolism via the bKA pathway are temporally enriched into OMVs in the presence of lignin. Isolated OMVs enhance growth of P. putida mutants unable to grow on model lignin-derived aromatics, and turnover protocatechuic acid in vitro. Together, this work leads us to hypothesize mechanisms by which OMVs contribute to extracellular aromatic catabolism by P. putida. Ongoing and future work seeks to further understand this phenomena in the context of both fundamental an applied lignin conversion research areas.
09 BIOMASS FUELS↗
Leveraging Super High Optical Resolution Microscopy to Probe the Interaction Zone Between Clostridium thermocellum and Biomass
This poster, part of the Pacific Northwest National Laboratory-sponsored Integration 2020: Visualizing the Proteome virtual conference, discusses super high optical resolution microscopy to probe the interaction zone between Clostritium thermocellum and biomass. Clostridium thermocellum is one of the most efficient microorganisms for the deconstruction of biomass. To achieve this high level of cellulolytic activity, C. thermocellum uses large multienzyme complexes known as cellulosomes to sugars in 4-5 break down polysaccharides found in plant cell walls. The attachment of bacterial switchgrass 70% cells to the nearby substrate via the cellulosome has been hypothesized to be the reason for this high efficiency. The region lying between the cell and the substrate has shown great variation and dynamics that are affected by the growth stage of cells and the substrate used for growth.
bacteria↗
Aromatic Monomer Production from Lignin Through Catalytic Carbon-Carbon Bond Cleavage
The conversion of lignin, the heterogeneous aromatic polymer found in terrestrial plant cell walls, into renewable bio-derived chemicals is critical to enable a viable lignocellulose-based bioeconomy. While lignin finds some limited industrial uses today, cost-effective and robust processes for the depolymerization of lignin to aromatic monomers would substantially expand lignin applications to larger markets, such as sustainable polymers and commodity chemicals. Historically, catalytic lignin depolymerization has focused on aryl-ether bond cleavage. A large fraction of aromatics in lignin, however, is linked by various recalcitrant carbon-carbon (C-C) bonds, which present a challenging substrate for depolymerization to aromatic monomers at high yields. To increase monomer yields above those accessible through aryl-ether bond cleavage alone, here we report that autoxidation catalysis cleaves C-C bonds in various lignin substrates with high atom economy. The catalyzed autoxidations of phenol-protected reductive catalytic fractionation (RCF) oil oligomers and hydrodeoxygenation (HDO) oil are described. The stream oxygenated monomers produced from the various lignin substrates are bioavailable as demonstrated by their bioconversion to cis,cis-muconate by engineered strains of Pseudonomas putida.
autoxidation↗
Investigating the Effect of Water on the Mechanical Properties of Cellulose from Multiscale Molecular Dynamics Simulations
Classical molecular dynamics (MD) simulations provide insight into the structure and physicochemical properties of materials with atomic resolution. However, the length and time scales accessible to atomistic MD are orders of magnitude smaller than many relevant processes such as the response of a bulk material to experimentally accessible strain rates, which presents challenges when comparing models to experimental measurements. Bottom-up coarse-graining provides a means for systematically mapping atomistic information to lower resolution models to increase the length and time scales achievable by simulation. Cellulose is an abundant carbohydrate biopolymer with applications to many fields of research, such as materials science and renewable energy, due to its desirable mechanical properties and viability for conversion into biofuel. The effect of moisture content on the Young's modulus of cellulose is of special interest due to its native environment often being in the hydrated secondary plant cell wall and the grinding energy requirements for biomass feedstock preprocessing. The current work investigates the effects of water solvent on the Young's modulus of cellulose calculated from coarse-grained MD mechanical stress simulations. The coarse-grained model was parametrized from atomistic MD calculations of cellulose-cellulose potentials of mean force using umbrella sampling techniques under vacuum and solvated conditions. The Young's moduli of the coarse-grained cellulose assemblies parametrized from cellulose in vacuum or solvated in water were computed via mechanical stress simulations to highlight the importance of capturing solvent interactions for modeling the mechanical behavior of cellulose.
BASIC BIOLOGICAL SCIENCES,RADIATION PROTECTION AND↗
The regulatory and transcriptional landscape associated with carbon utilization in a filamentous fungus
Filamentous fungi, such as Neurospora crassa , are very efficient in deconstructing plant biomass by the secretion of an arsenal of plant cell wall-degrading enzymes, by remodeling metabolism to accommodate production of secreted enzymes, and by enabling transport and intracellular utilization of plant biomass components. Although a number of enzymes and transcriptional regulators involved in plant biomass utilization have been identified, how filamentous fungi sense and integrate nutritional information encoded in the plant cell wall into a regulatory hierarchy for optimal utilization of complex carbon sources is not understood. Here, we performed transcriptional profiling of N. crassa on 40 different carbon sources, including plant biomass, to provide data on how fungi sense simple to complex carbohydrates. From these data, we identified regulatory factors in N. crassa and characterized one (PDR-2) associated with pectin utilization and one with pectin/hemicellulose utilization (ARA-1). Using in vitro DNA affinity purification sequencing (DAP-seq), we identified direct targets of transcription factors involved in regulating genes encoding plant cell wall-degrading enzymes. In particular, our data clarified the role of the transcription factor VIB-1 in the regulation of genes encoding plant cell wall-degrading enzymes and nutrient scavenging and revealed a major role of the carbon catabolite repressor CRE-1 in regulating the expression of major facilitator transporter genes. These data contribute to a more complete understanding of cross talk between transcription factors and their target genes, which are involved in regulating nutrient sensing and plant biomass utilization on a global level.
59 BASIC BIOLOGICAL SCIENCES↗
A core of cell wall proteins functions in wall integrity responses in Arabidopsis thaliana
Abstract Cell walls surround all plant cells, and their composition and structure are tightly regulated to maintain cellular and organismal homeostasis. In response to wall damage, the cell wall integrity (CWI) system is engaged to ameliorate effects on plant growth. Despite the central role CWI plays in plant development, our current understanding of how this system functions at the molecular level is limited. Here, we investigated the transcriptomes of etiolated seedlings of mutants of Arabidopsis thaliana with defects in three major wall polysaccharides, pectin ( quasimodo2 ), cellulose ( cellulose synthase3 je5 ), and xyloglucan ( xyloglucan xylosyltransferase1 and 2 ), to probe whether changes in the expression of cell wall‐related genes occur and are similar or different when specific wall components are reduced or missing. Many changes occurred in the transcriptomes of pectin‐ and cellulose‐deficient plants, but fewer changes occurred in the transcriptomes of xyloglucan‐deficient plants. We hypothesize that this might be because pectins interact with other wall components and/or integrity sensors, whereas cellulose forms a major load‐bearing component of the wall; defects in either appear to trigger the expression of structural proteins to maintain wall cohesion in the absence of a major polysaccharide. This core set of genes functioning in CWI in plants represents an attractive target for future genetic engineering of robust and resilient cell walls.
59 BASIC BIOLOGICAL SCIENCES↗
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↗
Flexible Pectin Nanopatterning Drives Cell Wall Organization in Plants
Not Available
59 BASIC BIOLOGICAL SCIENCES↗
A Glycan Array-Based Assay for the Identification and Characterization of Plant Glycosyltransferases
Growing plants with modified cell wall compositions is a promising strategy to improve resistance to pathogens, increase biomass digestibility, and tune other important properties. In order to alter biomass architecture, a detailed knowledge of cell wall structure and biosynthesis is a prerequisite. We report here a glycan array-based assay for the high-throughput identification and characterization of plant cell wall biosynthetic glycosyltransferases (GTs). We demonstrate that different heterologously expressed galactosyl-, fucosyl-, and xylosyltransferases can transfer azido-functionalized sugar nucleotide donors to selected synthetic plant cell wall oligosaccharides on the array and that the transferred monosaccharides can be visualized “on chip” by a 1,3-dipolar cycloaddition reaction with an alkynyl-modified dye. The opportunity to simultaneously screen thousands of combinations of putative GTs, nucleotide sugar donors, and oligosaccharide acceptors will dramatically accelerate plant cell wall biosynthesis research.
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
Plant Cell Growth and Cell Wall Enlargement
Irreversible cell enlargement begins with cell wall loosening which induces wall stress relaxation, leading to cell water uptake and cell enlargement. Growing cell walls consist of a cohesive network of cellulose microfibrils embedded in hydrophilic pectins and cellulose-binding hemicelluloses. Models of the growing cell wall are provisional hypotheses about how these wall polymers interact to make a strong yet extensible wall. Recent results clarify how wall strength, plasticity and elasticity depend mostly on the stretching, straightening and sliding of cellulose microfibril networks in the wall. Passive sliding of cellulose microfibrils is facilitated by nonenzymic proteins named expansins while various enzymes modify pectins and hemicelluloses, altering their interactions with each other and with cellulose. Growth cessation is correlated with reduced expression of genes that promote wall loosening as well as changes in the direction of cellulose deposition and the structure of matrix polysaccharides, leading to a less extensible cell wall.
59 BASIC BIOLOGICAL SCIENCES↗
Mutations in the Pectin Methyltransferase QUASIMODO2 Influence Cellulose Biosynthesis and Wall Integrity in Arabidopsis
Abstract Pectins are abundant in the cell walls of dicotyledonous plants, but how they interact with other wall polymers and influence wall integrity and cell growth has remained mysterious. Here, we verified that QUASIMODO2 (QUA2) is a pectin methyltransferase and determined that QUA2 is required for normal pectin biosynthesis. To gain further insight into how pectin affects wall assembly and integrity maintenance, we investigated cellulose biosynthesis, cellulose organization, cortical microtubules, and wall integrity signaling in two mutant alleles of Arabidopsis (Arabidopsis thaliana) QUA2, qua2 and tsd2. In both mutants, crystalline cellulose content is reduced, cellulose synthase particles move more slowly, and cellulose organization is aberrant. NMR analysis shows higher mobility of cellulose and matrix polysaccharides in the mutants. Microtubules in mutant hypocotyls have aberrant organization and depolymerize more readily upon treatment with oryzalin or external force. The expression of genes related to wall integrity, wall biosynthesis, and microtubule stability is dysregulated in both mutants. These data provide insights into how homogalacturonan is methylesterified upon its synthesis, the mechanisms by which pectin functionally interacts with cellulose, and how these interactions are translated into intracellular regulation to maintain the structural integrity of the cell wall during plant growth and development.
Biochemistry & Molecular Biology↗
Overexpression of REDUCED WALL ACETYLATION C increases xylan acetylation and biomass recalcitrance in Populus
Abstract Plant lignocellulosic biomass, i.e. secondary cell walls of plants, is a vital alternative source for bioenergy. However, the acetylation of xylan in secondary cell walls impedes the conversion of biomass to biofuels. Previous studies have shown that REDUCED WALL ACETYLATION (RWA) proteins are directly involved in the acetylation of xylan but the regulatory mechanism of RWAs is not fully understood. In this study, we demonstrate that overexpression of a Populus trichocarpa PtRWA-C gene increases the level of xylan acetylation and increases the lignin content and S/G ratio, ultimately yielding poplar woody biomass with reduced saccharification efficiency. Furthermore, through gene coexpression network and expression quantitative trait loci (eQTL) analysis, we found that PtRWA-C was regulated not only by the secondary cell wall hierarchical regulatory network but also by an AP2 family transcription factor HARDY (HRD). Specifically, HRD activates PtRWA-C expression by directly binding to the PtRWA-C promoter, which is also the cis-eQTL for PtRWA-C. Taken together, our findings provide insights into the functional roles of PtRWA-C in xylan acetylation and consequently saccharification and shed light on synthetic biology approaches to manipulate this gene and alter cell wall properties. These findings have substantial implications for genetic engineering of woody species, which could be used as a sustainable source of biofuels, valuable biochemicals, and biomaterials.
59 BASIC BIOLOGICAL SCIENCES↗
Metatranscriptomics sheds light on the links between the functional traits of fungal guilds and ecological processes in forest soil ecosystems
Soil fungi belonging to different functional guilds, such as saprotrophs, pathogens, and mycorrhizal symbionts, play key roles in forest ecosystems. To date, no study has compared the actual gene expression of these guilds in different forest soils. We used metatranscriptomics to study the competition for organic resources by these fungal groups in boreal, temperate, and Mediterranean forest soils. Using a dedicated mRNA annotation pipeline combined with the JGI MycoCosm database, we compared the transcripts of these three fungal guilds, targeting enzymes involved in C- and N mobilization from plant and microbial cell walls. Genes encoding enzymes involved in the degradation of plant cell walls were expressed at a higher level in saprotrophic fungi than in ectomycorrhizal and pathogenic fungi. However, ectomycorrhizal and saprotrophic fungi showed similarly high expression levels of genes encoding enzymes involved in fungal cell wall degradation. Transcripts for N-related transporters were more highly expressed in ectomycorrhizal fungi than in other groups. Here, we showed that ectomycorrhizal and saprotrophic fungi compete for N in soil organic matter, suggesting that their interactions could decelerate C cycling. Metatranscriptomics provides a unique tool to test controversial ecological hypotheses and to better understand the underlying ecological processes involved in soil functioning and carbon stabilization.
59 BASIC BIOLOGICAL SCIENCES↗