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Designing an optimal microbial cell factory often requires overexpression, knock-down, and knock-out of multiple gene targets. Unfortunately, such rewiring of cellular metabolism is often carried out sequentially and with low throughput. Here, we report a combinatorial metabolic engineering strategy based on an orthogonal tri-functional CRISPR system that combines transcriptional activation, transcriptional interference, and gene deletion (CRISPR-AID) in the yeast Saccharomyces cerevisiae . This strategy enables perturbation of the metabolic and regulatory networks in a modular, parallel, and high-throughput manner. We demonstrate the application of CRISPR-AID not only to increase the production of β-carotene by 3-fold in a single step, but also to achieve 2.5-fold improvement in the display of an endoglucanase on the yeast surface by optimizing multiple metabolic engineering targets in a combinatorial manner. Metaboli engineering through gene overexpression, knock-down and knock-out is often carried out sequentially in a high labor, low-throughput manner. Here, the authors use CRISPR-mediated gene activation, interference and deletion to rapidly rewire S. cerevisiae metabolism in a single step.

Cellulases, as environmentally appropriate catalysts specifically acting on cellulosic substrates, are important for the industrial conversion of lignocellulose and modification of cellulose products. After decades of research, a fundamental understanding of cellulase-mediated hydrolysis of cellulose is that its ability to processively act as a key for the complete enzymatic hydrolysis of natural crystalline cellulose. Two types of processive cellulases are known: exoglucanases and processive endoglucanases. Exoglucanases are typical processive enzymes, and they have been studied in detail so that their modes of action and mechanisms are reasonably well characterized. Conversely, endoglucanases are less well characterized because of the non-universality and structural diversity. However, processive endoglucanases have certain characteristics that exoglucanases lack such as hydrolysis product diversity and independent hydrolyze natural crystalline cellulose. Therefore, besides the conversion of cellulose to monosaccharide, they might be useful for modification of fibrous substrates and preparation of cellulose oligosaccharides. Herein, we review in detail the sources, hydrolysis products, application, and possible hydrolysis mechanisms of processive endoglucanases.

The advent of user-friendly instruments for measuring force/deflection curves of plant surfaces at high spatial resolution has resulted in a recent outpouring of reports of the 'Young's modulus' of plant cell walls. The stimulus for these mechanical measurements comes from biomechanical models of morphogenesis of meristems and other tissues, as well as single cells, in which cell wall stress feeds back to regulate microtubule organization, auxin transport, cellulose deposition, and future growth directionality. In this article I review the differences between elastic modulus and wall extensibility in the context of cell growth. Some of the inherent complexities, assumptions, and potential pitfalls in the interpretation of indentation force/deflection curves are discussed. Reported values of elastic moduli from surface indentation measurements appear to be 10- to >1000-fold smaller than realistic tensile elastic moduli in the plane of plant cell walls. Potential reasons for this disparity are discussed, but further work is needed to make sense of the huge range in reported values. The significance of wall stress relaxation for growth is reviewed and connected to recent advances and remaining enigmas in our concepts of how cellulose, hemicellulose, and pectins are assembled to make an extensible cell wall. A comparison of the loosening action of α-expansin and Cel12A endoglucanase is used to illustrate two different ways in which cell walls may be made more extensible and the divergent effects on wall mechanics.

Anaerobic fungi found in the guts of large herbivores are prolific biomass degraders whose genomes harbor a wealth of carbohydrate-active enzymes (CAZymes), of which only a handful are structurally or biochemically characterized. Here, we report the structure and kinetic rate parameters for a glycoside hydrolase (GH) family 5 subfamily 4 enzyme (CelD) from Piromyces finnis, a modular, cellulosome-incorporated endoglucanase that possesses three GH5 domains followed by two C-terminal fungal dockerin domains (double dockerin). We present the crystal structures of an apo wild-type CelD GH5 catalytic domain and its inactive E154A mutant in complex with cellotriose at 2.5 and 1.8 Å resolution, respectively, finding the CelD GH5 catalytic domain adopts the (β/α)8-barrel fold common to many GH5 enzymes. Structural superimposition of the apo wild-type structure with the E154A mutant-cellotriose complex supports a catalytic mechanism in which the E154 carboxylate side chain acts as an acid/base and E278 acts as a complementary nucleophile. Further analysis of the cellotriose binding pocket highlights a binding groove lined with conserved aromatic amino acids that when docked with larger cellulose oligomers is capable of binding seven glucose units and accommodating branched glucan substrates. Activity analyses confirm P. finnis CelD can hydrolyze mixed linkage glucan and xyloglucan, as well as carboxymethylcellulose (CMC). Measured kinetic parameters show the P. finnis CelD GH5 catalytic domain has CMC endoglucanase activity comparable to other fungal endoglucanases with kcat = 6.0 ± 0.6 s−1 and Km = 7.6 ± 2.1 g/L CMC. Enzyme kinetics were unperturbed by the addition or removal of the native C-terminal dockerin domains as well as the addition of a non-native N-terminal dockerin, suggesting strict modularity among the domains of CelD.Key points• Anaerobic fungi host a wealth of industrially useful enzymes but are understudied.• P. finnis CelD has endoglucanase activity and structure common to GH5_4 enzymes.• CelD’s kinetics do not change with domain fusion, exhibiting high modularity.

A novel endoglucanase gene was cloned from Thermobifida halotolerans YIM 90462ᵀ, designated as thcel6A for being a member of glycoside hydrolase family 6. The gene was 1332 bp long and encoded a 443-amino-acid protein with a molecular mass of 45.9 kDa. The purified recombinant endoglucanase had optimal activity at 55 °C and pH 8.5. Thcel6A showed high hydrolytic activities at 25–55 °C and retained 58 % of initial activity after incubation at 90 °C for 1 h. It retained more than 80 % of activity after incubation for 12 h at pH values from 4 to 12. Thcel6A displayed higher hydrolytic activities in 5–15 % NaCl (w/v) than at 0 % NaCl. Activity increased 2.5-fold after incubation with 20 % (w/v) NaCl at 37 °C for 10 min. These properties suggest that this novel endoglucanase has potential for specific industrial application.

[This corrects the article DOI: 10.1371/journal.pone.0151017.].

The enzyme endoglucanase is responsible for the depolymerization of cellulose. This study focuses on characterization and purification of endoglucanase from Rhizopus oryzae MTCC 9642 through a simple size exclusion method and its effective application as an antibiofilm agent. Extracellular ß-1,4-endoglucanase, an enzyme that catalyzes the hydrolysis of carboxymethyl cellulose, was found to be synthesized by Rhizopus oryzae MTCC 9642. The enzyme was purified up to homogeneity simply by size exclusion process through ultrafiltration and gel chromatography. The molecular weight of purified enzyme protein was estimated to be 39.8 kDa and it showed the highest substrate affinity towards carboxymethyl-cellulose with Km and Vmax values of 0.833 mg ml−1 and of 0.33 mmol glucose min−1 mg−1protein, respectively. The purified enzyme exhibited optimal activity at pH 6 with a broad stability range of pH 3–8. The most preferred temperature was 35 °C and 50% of activity could be retained after the thermal exposure at 40 °C for 25 min. The purified enzyme protein was inactivated by Cu2+, while the activity could be enhanced by the addition of exogenous thiols. Since biofilm is a challenge for health sector, with the aim of eradicating the biofilm, the purified endoglucanase was used to remove biofilm produced by two nosocomial bacteria. As predicted by in silico molecular docking interaction, the purified enzyme could effectively degrade biofilm architecture of bacterial strains S. aureus and P. aeruginosa by 76.52 ± 6.52% and 61.67 ± 8.76%, respectively. The properties of purified enzyme protein, as elucidated by in vitro and in silico characterization, may be favourable for its commercial applications as a potent antibiofilm agent.

Volvariella volvacea endoglucanase EG1 was used to treat bleached softwood kraft pulp (BSKP) and hardwood pulp (BHKP) to improve the refinability and physical strength, as well as to reduce vessel picking in Eucalyptus pulp. The results indicated that BSKP was treated with an enzyme dosage of 3 U/g for 2 h at 12,000 refining revolutions, which increased the tensile index from 71.4 N·m/g to 86.7 N·m/g. For BHKP, treatment with 10 U/g of EG1 for 2 h at 15,000 refining revolutions improved the tensile index from the control of 47.7 N·m/g to 56.9 N·m/g. Vessel-removed and vessel-enriched fractions of Eucalyptus pulp were obtained by screening and treated with EG1, respectively. It was found that EG1-assisted refining increased the physical strength and surface strength of both pulp fractions, and the latter improved even more, with increases of 22.4% and 160%, respectively.

In this study, fermentable sugars and cellulose nanocrystals (CNCs) were co-produced from endoglucanase treatment of wood pulp, followed by acid hydrolysis. Enzymatic hydrolysis was performed using two endoglucanases differentiated by the presence or absence of a cellulose-binding domain (CBD). The enzyme with an intact CBD gave the higher glucan conversion (up to 14.1 ± 1.2 wt %) and improved the degree of crystallinity of the recovered wood pulp fiber (up to 83.0 ± 1.0%). Thus, this endoglucanase-assisted treatment successfully removed amorphous content from the original cellulosic feedstock. CNC recovery (16.9 ± 0.7 wt %) from the feedstock going into the acid hydrolysis was improved relative to untreated pulp (13.2 ± 0.6 wt %). The mass loss from enzymatic treatment did not cause a decrease in the CNC yield from the starting material. The characteristics of CNCs obtained through acid hydrolysis (with or without enzyme treatment of pulp) were analyzed using X-ray diffraction, transmission electron microscopy, dynamic light scattering, Fourier transform infrared spectroscopy, and differential scanning calorimetry as characterization techniques. The CNCs generated through acid hydrolysis of endoglucanase-treated wood pulp displayed comparable properties relative to those generated using untreated pulp. Thus, endoglucanase treatment can enable co-production of CNCs and sugars for biofuel fermentation.

The extracellular space (apoplast) of plant tissue represents a critical battleground between plants and attacking microbes. Here we show that a pathogen-secreted apoplastic xyloglucan-specific endoglucanase, PsXEG1, is a focus of this struggle in the Phytophthora sojae–soybean interaction. We show that soybean produces an apoplastic glucanase inhibitor protein, GmGIP1, that binds to PsXEG1 to block its contribution to virulence. P. sojae, however, secretes a paralogous PsXEG1-like protein, PsXLP1, that has lost enzyme activity but binds to GmGIP1 more tightly than does PsXEG1, thus freeing PsXEG1 to support P. sojae infection. The gene pair encoding PsXEG1 and PsXLP1 is conserved in many Phytophthora species, and the P. parasitica orthologs PpXEG1 and PpXLP1 have similar functions. Thus, this apoplastic decoy strategy may be widely used in Phytophthora pathosystems.