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At least 109 records · Page 6
All sourcesNASA NTRSDOE OSTI
DOE OSTI2024

A genomic analysis reveals the diversity of cellulosome displaying bacteria

Introduction Several species of cellulolytic bacteria display cellulosomes, massive multi-cellulase containing complexes that degrade lignocellulosic plant biomass (LCB). A greater understanding of cellulosome structure and enzyme content could facilitate the development of new microbial-based methods to produce renewable chemicals and materials. Methods To identify novel cellulosome-displaying microbes we searched 305,693 sequenced bacterial genomes for genes encoding cellulosome proteins; dockerin-fused glycohydrolases (DocGHs) and cohesin domain containing scaffoldins. Results and discussion This analysis identified 33 bacterial species with the genomic capacity to produce cellulosomes, including 10 species not previously reported to produce these complexes, such asAcetivibrio mesophilus. Cellulosome-producing bacteria primarily originate from theAcetivibrio, Ruminococcus, Ruminiclostridium, andClostridiumgenera. A rigorous analysis of their enzyme, scaffoldin, dockerin, and cohesin content reveals phylogenetically conserved features. Based on the presence of a high number of genes encoding both scaffoldins and dockerin-fused GHs, the cellulosomes inAcetivibrioandRuminococcusbacteria possess complex architectures that are populated with a large number of distinct LCB degrading GH enzymes. Their complex cellulosomes are distinguishable by their mechanism of attachment to the cell wall, the structures of their primary scaffoldins, and by how they are transcriptionally regulated. In contrast, bacteria in theRuminiclostridiumandClostridiumgenera produce ‘simple’ cellulosomes that are constructed from only a few types of scaffoldins that based on their distinct complement of GH enzymes are predicted to exhibit high and low cellulolytic activity, respectively. Collectively, the results of this study reveal conserved and divergent architectural features in bacterial cellulosomes that could be useful in guiding ongoing efforts to harness their cellulolytic activities for bio-based chemical and materials production.

Microbiology↗
DOE OSTI2021

The Santorini Volcanic Complex as a Valuable Source of Enzymes for Bioenergy

Marine microbial communities are an untapped reservoir of genetic and metabolic diversity and a valuable source for the discovery of new natural products of biotechnological interest. The newly discovered hydrothermal vent field of Santorini volcanic complex located in the Aegean Sea is gaining increasing interest for potential biotechnological exploitation. The conditions in these environments, i.e., high temperatures, low pH values and high concentration of heavy metals, often resemble harsh industrial settings. Thus, these environments may serve as pools of enzymes of enhanced catalytic properties that may provide benefits to biotechnology. Here, we screened 11 metagenomic libraries previously constructed from microbial mat samples covering the seafloor and the polymetallic chimneys of Kolumbo volcano as well as mat samples from Santorini caldera, to mine, in silico, genes associated with bioenergy applications. We particularly focused on genes encoding biomass hydrolysis enzymes such as cellulases, hemicellulases and lignin-degrading enzymes. A total of 10,417 genes were found for three specific groups of enzymes—i.e., the endoglucanases, the three different beta-glucosidases BGL, bglX and bglB, and the alpha-galactosidases melA, and rafA. Overall, we concluded that the Santorini–Kolumbo volcanic ecosystems constitute a significant resource of novel genes with potential applications in bioenergy that deserve further investigation.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Characterization of the Biomass Degrading Enzyme GuxA from Acidothermus cellulolyticus

Microbial conversion of biomass relies on a complex combination of enzyme systems promoting synergy to overcome biomass recalcitrance. Some thermophilic bacteria have been shown to exhibit particularly high levels of cellulolytic activity, making them of particular interest for biomass conversion. These bacteria use varying combinations of CAZymes that vary in complexity from a single catalytic domain to large multi-modular and multi-functional architectures to deconstruct biomass. Since the discovery of CelA from Caldicellulosiruptor bescii which was identified as one of the most active cellulase so far identified, the search for efficient multi-modular and multi-functional CAZymes has intensified. One of these candidates, GuxA (previously Acel_0615), was recently shown to exhibit synergy with other CAZymes in C. bescii, leading to a dramatic increase in growth on biomass when expressed in this host. GuxA is a multi-modular and multi-functional enzyme from Acidothermus cellulolyticus whose catalytic domains include a xylanase/endoglucanase GH12 and an exoglucanase GH6, representing a unique combination of these two glycoside hydrolase families in a single CAZyme. These attributes make GuxA of particular interest as a potential candidate for thermophilic industrial enzyme preparations. Here, we present a more complete characterization of GuxA to understand the mechanism of its activity and substrate specificity. In addition, we demonstrate that GuxA exhibits high levels of synergism with E1, a companion endoglucanase from A. cellulolyticus. We also present a crystal structure of one of the GuxA domains and dissect the structural features that might contribute to its thermotolerance.

09 BIOMASS FUELS↗
DOE OSTI2022

Insight into CAZymes of Alicyclobacillus mali FL18: Characterization of a New Multifunctional GH9 Enzyme

In the bio-based era, cellulolytic and hemicellulolytic enzymes are biocatalysts used in many industrial processes, playing a key role in the conversion of recalcitrant lignocellulosic waste biomasses. In this context, many thermophilic microorganisms are considered as convenient sources of carbohydrate-active enzymes (CAZymes). In this work, a functional genomic annotation of Alicyclobacillus mali FL18, a recently discovered thermo-acidophilic microorganism, showed a wide reservoir of putative CAZymes. Among them, a novel enzyme belonging to the family 9 of glycosyl hydrolases (GHs), named AmCel9, was identified; in-depth in silico analyses highlighted that AmCel9 shares general features with other GH9 members. The synthetic gene was expressed in Escherichia coli and the recombinant protein was purified and characterized. The monomeric enzyme has an optimal catalytic activity at pH 6.0 and has comparable activity at temperatures ranging from 40 °C to 70 °C. It also has a broad substrate specificity, a typical behavior of multifunctional cellulases; the best activity is displayed on β-1,4 linked glucans. Very interestingly, AmCel9 also hydrolyses filter paper and microcrystalline cellulose. This work gives new insights into the properties of a new thermophilic multifunctional GH9 enzyme, that looks a promising biocatalyst for the deconstruction of lignocellulose.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Comparative Genomics and Transcriptomics Analyses Reveal Divergent Plant Biomass-Degrading Strategies in Fungi

Plant biomass is one of the most abundant renewable carbon sources, which holds great potential for replacing current fossil-based production of fuels and chemicals. In nature, fungi can efficiently degrade plant polysaccharides by secreting a broad range of carbohydrate-active enzymes (CAZymes), such as cellulases, hemicellulases, and pectinases. Due to the crucial role of plant biomass-degrading (PBD) CAZymes in fungal growth and related biotechnology applications, investigation of their genomic diversity and transcriptional dynamics has attracted increasing attention. In this project, we systematically compared the genome content of PBD CAZymes in six taxonomically distant species, Aspergillus niger, Aspergillus nidulans, Penicillium subrubescens, Trichoderma reesei, Phanerochaete chrysosporium, and Dichomitus squalens, as well as their transcriptome profiles during growth on nine monosaccharides. Considerable genomic variation and remarkable transcriptomic diversity of CAZymes were identified, implying the preferred carbon source of these fungi and their different methods of transcription regulation. In addition, the specific carbon utilization ability inferred from genomics and transcriptomics was compared with fungal growth profiles on corresponding sugars, to improve our understanding of the conversion process. This study enhances our understanding of genomic and transcriptomic diversity of fungal plant polysaccharide-degrading enzymes and provides new insights into designing enzyme mixtures and metabolic engineering of fungi for related industrial applications.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Protective effects of non–catalytic proteins on endoglucanase activity at air and lignin interfaces

The manner in which added non-catalytic proteins during enzymatic hydrolysis of lignocellulosic substrates enhances hydrolysis mechanisms is not completely understood. Prior research has indicated that a reduction in the non-specific adsorption of enzymes on lignin, and deactivation of enzymes exposed to air–liquid interface provide rationale. This work investigated root causes including effects of the air–liquid interface on non-catalytic proteins, and effects of lignin on endoglucanase. Three different experimental designs and three variables (air–liquid interfacial area, the types of lignin (acid or enzymatic lignin), and the presence of non-enzymatic protein (bovine serum albumin [BSA] or soy proteins ) were used. The results showed that acid isolated lignin adsorbed almost all endoglucanase activity initially present in supernatant, independent of air interface conditions (25 or 250 ml flasks) with the presence of BSA preventing this effect. Endoglucanase lost 30%–50% of its activity due to an air–liquid interface in the presence of lignin while addition of non-enzymatic protein helped to preserve this enzyme's activity. Langmuir and Freundlich models applied to experimental data indicated that the adsorption increases with increasing temperature for both endoglucanase and BSA. Adsorption of the enzyme and protein were endothermic with an increase in entropy. Finally, these results, combined, show that hydrophobicity plays a strong role in the adsorption of both endoglucanase and BSA on lignin.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Effect of pelleting on the enzymatic digestibility of corn stover

Pelleting of lignocellulosic biomass to improve its transportation, storage and handling impacts subsequent processing and conversion. Here, this work reports the role of high moisture pelleting in the enzymatic digestibility of corn stover prior to pretreatment, together with associated substrate characteristics. Pelleting increases the digestibility of unpretreated corn stover, from 8.2 to 15.5% glucan conversion, at 5% solid loading using 1 FPU Cellic® CTec2 per g solids. Compositional analysis indicates that loose and pelleted corn stover have similar non-dissolvable compositions, although their extractives are different. Enzymatic hydrolysis of corn stover after size reduction to normalize particle sizes and removal of extractives confirms that pelleting improves corn stover digestibility. Such differences may be explained by the decreased particle size, improved substrate accessibility, and hydrolysis of cross-linking structures induced by pelleting. These findings are useful for the development of processing schemes for sustainable and efficient use of lignocellulose.

09 BIOMASS FUELS↗
DOE OSTI2020

The Role of Evolving Interfacial Substrate Properties on Heterogeneous Cellulose Hydrolysis Kinetics

Interfacial enzyme reactions require formation of an enzyme–substrate complex at the surface of a heterogeneous substrate, but often multiple modes of enzyme binding and types of binding sites complicate analysis of their kinetics. Excess heterogeneous substrate is often used as a justification to model the substrate as unchanging, but using the study of the enzymatic hydrolysis of insoluble cellulose as an example, we argue that reaction rates are dependent on evolving substrate interfacial properties. We hypothesize that the relative abundance of binding sites on cellulose where hydrolysis can occur (productive binding sites) and binding sites where hydrolysis cannot be initiated or is inhibited (nonproductive binding sites) contribute to rate limitations. We show that the initial total number of productive binding sites (the productive binding capacity) determines the magnitude of the initial burst phase of cellulose hydrolysis, while productive binding site depletion explains overall hydrolysis kinetics. Furthermore, we show that irreversibly bound surface enzymes contribute to the depletion of productive binding sites. Our model shows that increasing the ratio of productive to nonproductive binding sites promotes hydrolysis, while maintaining an elevated productive binding capacity throughout conversion is key to preventing hydrolysis slowdown.

09 BIOMASS FUELS↗
DOE OSTI2022

Enzyme kinetics by GH7 cellobiohydrolases on chromogenic substrates is dictated by non‐productive binding: insights from crystal structures and MD simulation

Cellobiohydrolases (CBHs) in the glycoside hydrolase family 7 (GH7) ( EC3.2.1.176 ) are the major cellulose degrading enzymes both in industrial settings and in the context of carbon cycling in nature. Small carbohydrate conjugates such as p‐ nitrophenyl‐β‐ d ‐cellobioside (pNPC), p‐ nitrophenyl‐β‐ d ‐lactoside (pNPL) and methylumbelliferyl‐β‐ d ‐cellobioside have commonly been used in colorimetric and fluorometric assays for analysing activity of these enzymes. Despite the similar nature of these compounds the kinetics of their enzymatic hydrolysis vary greatly between the different compounds as well as among different enzymes within the GH7 family. Through enzyme kinetics, crystallographic structure determination, molecular dynamics simulations, and fluorometric binding studies using the closely related compound o‐nitrophenyl‐β‐ d ‐cellobioside (oNPC), in this work we examine the different hydrolysis characteristics of these compounds on two model enzymes of this class, TrCel7A from Trichoderma reesei and PcCel7D from Phanerochaete chrysosporium . Protein crystal structures of the E212Q mutant of TrCel7A with pNPC and pNPL, and the wildtype TrCel7A with oNPC, reveal that non‐productive binding at the product site is the dominating binding mode for these compounds. Enzyme kinetics results suggest the strength of non‐productive binding is a key determinant for the activity characteristics on these substrates, with PcCel7D consistently showing higher turnover rates ( k cat ) than TrCel7A, but higher Michaelis–Menten ( K M ) constants as well. Furthermore, oNPC turned out to be useful as an active‐site probe for fluorometric determination of the dissociation constant for cellobiose on TrCel7A but could not be utilized for the same purpose on PcCel7D, likely due to strong binding to an unknown site outside the active site.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Cellulosome Localization Patterns Vary across Life Stages of Anaerobic Fungi

Anaerobic fungi ( Neocallimastigomycota ) isolated from the guts of herbivores excel at degrading ingested plant matter, making them attractive potential platform organisms for converting waste biomass into valuable products, such as chemicals and fuels. Major contributors to their biomass-hydrolyzing power are the multienzyme cellulosome complexes that anaerobic fungi produce, but knowledge gaps in how cellulosome production is controlled by the cellular life cycle and how cells spatially deploy cellulosomes complicate the use of anaerobic fungi and their cellulosomes in industrial bioprocesses.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Biodiesel and higher value products from stillage fiber

Global climate change - due largely to the emission of anthropic CO2 - has been recognized by the scientific community for many decades and is becoming increasingly obvious to the public due to extremely hot summers, drought, and frequent fires. Destruction of tropical forests especially contributes to climate change and loss of species diversity.

09 BIOMASS FUELS↗
DOE OSTI2025

Methodology for Determination of Cellulosic Glucans and Hemicellulose Content in a Fuel Ethanol Production Facility (CRADA Final Report)

Develop a single methodology for determination of the total cellulosic glucan, including both cellulose and beta-glucans, along with hemicellulose content in corn kernel fiber which could be utilized by the dry grind corn ethanol production industry to determine the cellulosic converted fraction of the corn kernel fiber. The work will build upon a methodology already described in literature but will specifically focus on a more complete determination of the structural polysaccharides in corn kernel fiber to make it more applicable to the corn ethanol industry.

09 BIOMASS FUELS↗
DOE OSTI2022

Revisiting Theoretical Tools and Approaches for the Valorization of Recalcitrant Lignocellulosic Biomass to Value-Added Chemicals

Biorefinery processes for converting lignocellulosic biomass to fuels and chemicals proceed via an integrated series of steps. Biomass is first pretreated and deconstructed using chemical catalysts and/or enzymes to liberate sugar monomers and lignin fragments. Deconstruction is followed by a conversion step in which engineered host organisms assimilate the released sugar monomers and lignin fragments, and produce value-added fuels and chemicals. Over the past couple of decades, a significant amount of work has been done to develop innovative biomass deconstruction and conversion processes that efficiently solubilize biomass, separate lignin from the biomass, maximize yields of bioavailable sugars and lignin fragments and convert the majority of these carbon sources into fuels, commodity chemicals, and materials. Herein, we advocate that advanced in silico approaches provide a theoretical framework for developing efficient processes for lignocellulosic biomass valorization and maximizing yields of sugars and lignin fragments during deconstruction and fuel and chemical titers during conversion. This manuscript surveys the latest developments in lignocellulosic biomass valorization with special attention given to highlighting computational approaches used in process optimization for lignocellulose pretreatment; enzyme engineering for enhanced saccharification and delignification; and prediction of the genome modification necessary for desired pathway fine-tuning to upgrade products from biomass deconstruction into value-added products. Physics-based modeling approaches such as density functional theory calculations and molecular dynamics simulations have been most impactful in studies aimed at exploring the molecular level details of solvent-biomass interactions, reaction mechanisms occurring in biomass-solvent systems, and the catalytic mechanisms and engineering of enzymes involved in biomass degradation. More recently, with ever increasing amounts of data from, for example, advanced mutli-omics experiments, machine learning approaches have begun to make important contributions in synthetic biology and optimization of metabolic pathways for production of biofuels and chemicals.

09 BIOMASS FUELS↗