Enhancement of cellulosome-mediated deconstruction of cellulose by improving enzyme thermostability

Background: The concerted action of three complementary cellulases from Clostridium thermocellum, engineered to be stable at elevated temperatures, was examined on a cellulosic substrate and compared to that of the wild-type enzymes. Exoglucanase Cel48S and endoglucanase Cel8A, both key elements of the natural cellulosome from this bacterium, were engineered previously for increased thermostability, either by SCHEMA, a structure-guided, site-directed protein recombination method, or by consensus-guided mutagenesis combined with random mutagenesis using error-prone PCR, respectively. A thermostable β-glucosidase BglA mutant was also selected from a library generated by error-prone PCR that will assist the two cellulases in their methodic deconstruction of crystalline cellulose. The effects of a thermostable scaffoldin versus those of a largely mesophilic scaffoldin were also examined. By improving the stability of the enzyme subunits and the structural component, we aimed to improve cellulosome-mediated deconstruction of cellulosic substrates. Results: The results demonstrate that the combination of thermostable enzymes as free enzymes and a thermostable scaffoldin was more active on the cellulosic substrate than the wild-type enzymes. Significantly, “thermostable” designer cellulosomes exhibited a 1.7-fold enhancement in cellulose degradation compared to the action of conventional designer cellulosomes that contain the respective wild-type enzymes. For designer cellulosome formats, the use of the thermostabilized scaffoldin proved critical for enhanced enzymatic performance under conditions of high temperatures. Conclusions: Simple improvement in the activity of a given enzyme does not guarantee its suitability for use in an enzyme cocktail or as a designer cellulosome component. The true merit of improvement resides in its ultimate contribution to synergistic action, which can only be determined experimentally. The relevance of the mutated thermostable enzymes employed in this study as components in multienzyme systems has thus been confirmed using designer cellulosome technology. Enzyme integration via a thermostable scaffoldin is critical to the ultimate stability of the complex at higher temperatures. Engineering of thermostable cellulases and additional lignocellulosic enzymes may prove a determinant parameter for development of state-of-the-art designer cellulosomes for their employment in the conversion of cellulosic biomass to soluble sugars

Enhancement of cellulosome-mediated deconstruction of cellulose by improving enzyme thermostability

Background: The concerted action of three complementary cellulases from Clostridium thermocellum, engineered to be stable at elevated temperatures, was examined on a cellulosic substrate and compared to that of the wild-type enzymes. Exoglucanase Cel48S and endoglucanase Cel8A, both key elements of the natural cellulosome from this bacterium, were engineered previously for increased thermostability, either by SCHEMA, a structure-guided, site-directed protein recombination method, or by consensus-guided mutagenesis combined with random mutagenesis using error-prone PCR, respectively. A thermostable β-glucosidase BglA mutant was also selected from a library generated by error-prone PCR that will assist the two cellulases in their methodic deconstruction of crystalline cellulose. The effects of a thermostable scaffoldin versus those of a largely mesophilic scaffoldin were also examined. By improving the stability of the enzyme subunits and the structural component, we aimed to improve cellulosome-mediated deconstruction of cellulosic substrates. Results: The results demonstrate that the combination of thermostable enzymes as free enzymes and a thermostable scaffoldin was more active on the cellulosic substrate than the wild-type enzymes. Significantly, “thermostable” designer cellulosomes exhibited a 1.7-fold enhancement in cellulose degradation compared to the action of conventional designer cellulosomes that contain the respective wild-type enzymes. For designer cellulosome formats, the use of the thermostabilized scaffoldin proved critical for enhanced enzymatic performance under conditions of high temperatures. Conclusions: Simple improvement in the activity of a given enzyme does not guarantee its suitability for use in an enzyme cocktail or as a designer cellulosome component. The true merit of improvement resides in its ultimate contribution to synergistic action, which can only be determined experimentally. The relevance of the mutated thermostable enzymes employed in this study as components in multienzyme systems has thus been confirmed using designer cellulosome technology. Enzyme integration via a thermostable scaffoldin is critical to the ultimate stability of the complex at higher temperatures. Engineering of thermostable cellulases and additional lignocellulosic enzymes may prove a determinant parameter for development of state-of-the-art designer cellulosomes for their employment in the conversion of cellulosic biomass to soluble sugars

DataSheet_1_2-NBDG Uptake in Gossypium hirsutum in vitro ovules: exploring tissue-specific accumulation and its impact on hexokinase-mediated glycolysis regulation.pdf

Fluorescent glucose derivatives are valuable tools as glucose analogs in plant research to explore metabolic pathways, study enzyme activity, and investigate cellular processes related to glucose metabolism and sugar transport. They allow visualization and tracking of glucose uptake, its utilization, and distribution within plant cells and tissues. This study investigates the phenotypic and metabolic impact of the exogenously fed glucose derivative, 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose) (2-NBDG) on the fibers of Gossypium hirsutum (Upland cotton) ovule in vitro cultures. The presence of 2-NBDG in the culture medium did not lead to macroscopic morphological alterations in ovule and fiber development or to the acquisition of fluorescence or yellow coloration. Confocal laser scanning microscope imaging and chromatographic analysis of cotton ovules’ outer rim cross-sections showed that the 2-NBDG is transported from the extracellular space and accumulated inside some outer integument cells, epidermal cells, and fertilized epidermal cells (fibers), but is not incorporated into the cell walls. Untargeted metabolic profiling of the fibers revealed significant changes in the relative levels of metabolites involved in glycolysis and upregulation of alternative energy-related pathways. To provide biochemical and structural evidence for the observed downregulation of glycolysis pathways in the fibers containing 2-NBDG, kinetics analysis and docking simulations were performed on hexokinase from G. hirsutum (GhHxk). Notably, the catalytic activity of heterologously expressed recombinant active GhHxk exhibited a five-fold decrease in reaction rates compared to D-glucose. Furthermore, GhHxk exhibited a linear kinetic behavior in the presence of 2-NBDG instead of the Michaelis-Menten kinetics found for D-glucose. Docking simulations suggested that 2-NBDG interacts with a distinct binding site of GhHxk9, possibly inducing a conformational change. These results highlight the importance of considering fluorescent glucose derivatives as ready-to-use analogs for tracking glucose-related biological processes. However, a direct comparison between their mode of action and its extrapolation into biochemical considerations should go beyond microscopic inspection and include complementary analytical techniques.</p

Significance of Relative Position of Cellulases in Designer Cellulosomes for Optimized Cellulolysis

<div><p>Degradation of cellulose is of major interest in the quest for alternative sources of renewable energy, for its positive effects on environment and ecology, and for use in advanced biotechnological applications. Due to its microcrystalline organization, celluose is extremely difficult to degrade, although numerous microbes have evolved that produce the appropriate enzymes. The most efficient known natural cellulolytic system is produced by anaerobic bacteria, such as <i>C</i>. <i>thermocellum</i>, that possess a multi-enzymatic complex termed the cellulosome. Our laboratory has devised and developed the designer cellulosome concept, which consists of chimaeric scaffoldins for controlled incorporation of recombinant polysaccharide-degrading enzymes. Recently, we reported the creation of a combinatorial library of four cellulosomal modules comprising a basic chimaeric scaffoldin, i.e., a CBM and 3 divergent cohesin modules. Here, we employed selected members of this library to determine whether the position of defined cellulolytic enzymes is important for optimized degradation of a microcrystalline cellulosic substrate. For this purpose, 10 chimaeric scaffoldins were used for incorporation of three recombinant <i>Thermobifida fusca</i> enzymes: the processive endoglucanase Cel9A, endoglucanase Cel5A and exoglucanase Cel48A. In addition, we examined whether the characteristic properties of the <i>T</i>. <i>fusca</i> enzymes as designer cellulosome components are unique to this bacterium by replacing them with parallel enzymes from <i>Clostridium thermocellum</i>. The results support the contention that for a given set of cellulosomal enzymes, their relative position within a scaffoldin can be critical for optimal degradation of microcrystaline cellulosic substrates.</p></div

Analysis of complex formation.

<p>All chimaeric enzymes and scaffoldins were first assayed individually for binding to a cellulosic substrate. Relevant enzymes and scaffoldin were then mixed together at equimolar ratios and subsequently introduced to a cellulosic substrate (Avicel). The cellulose-binding ability of both individual proteins and the resultant complex was determined by examining the cellulose-bound (lanes 7–11) and unbound (lanes 2–6) fractions by SDS-PAGE. Samples include: lane 1 and 12, molecular weight markers. Lane 2 to lane 6, unbound fractions with the following details: lane 2, <i>a</i>-9A; lane 3, <i>b</i>-48A; lane 4, 5A-<i>t</i>; lane 5, Scaf20; lane 6, Complex of <i>a</i>-9A, <i>b</i>-48A, 5A-<i>t</i> and Scaf20. Lane 7 to lane 11 are bound fractions: lane 7, <i>a</i>-9A; lane 8, <i>b</i>-48A; lane 9, 5A-<i>t</i>; lane 10, Scaf 20; lane 11, Complex of <i>a</i>-9A, <i>b</i>-48A, 5A-<i>t</i> and Scaf20. In the presence of the chimaeric scaffoldin, the enzymatic components were associated with the cellulose-bound fraction; whereas in its absence, they remained in the unbound fraction.</p

Comparative Avicel degradation by recombinant enzymes from <i>C</i>. <i>thermocellum</i>, either (i) in the free state, (ii) bound to respective monovalent scaffoldins (CBM-restored) or (iii) bound to different chimaeric scaffoldins.

<p>Enzymatic activity is defined as mM total reducing sugars following a 24-h reaction period at 50°C with each enzyme and scaffoldin concentration fixed at 0.4 μM. Stars indicate a statistically significant difference (lower than) with the two most efficient scaffoldins Scaf 20 and Scaf 22.</p

MOESM1 of Enhancement of cellulosome-mediated deconstruction of cellulose by improving enzyme thermostability

Additional file 1. Non-denaturing PAGE analysis of wild-type and mutant forms of Cel48S complexed with the monovalent scaffoldin Scaf¡T. Complexes were produced at different molar ratios and applied to non-denaturing polyacrylamide gels. For each gel, lanes 1 to 7 correspond to the respective enzyme/scaffoldin complexes at the following ratios: 0.4, 0.6, 0.8, 1.0, 1.2, 1.4 and 1.6. (Lane 4 signifies the calculated 1:1 ratio). Lane 8 corresponds to the specified enzyme alone at the presumed molar ratio. Red rectangle indicated the determined exact equimolar ratio