Engineering robust cellulases for tailored lignocellulosic degradation cocktails

Lignocellulosic biomass is a most promising feedstock in the production of second-generation biofuels. Efficient degradation of lignocellulosic biomass requires a synergistic action of several cellulases and hemicellulases. Cellulases depolymerize cellulose, the main polymer of the lignocellulosic biomass, to its building blocks. The production of cellulase cocktails has been widely explored, however, there are still some main challenges that enzymes need to overcome in order to develop a sustainable production of bioethanol. The main challenges include low activity, product inhibition, and the need to perform fine-tuning of a cellulase cocktail for each type of biomass. Protein engineering and directed evolution are powerful technologies to improve enzyme properties such as increased activity, decreased product inhibition, increased thermal stability, improved performance in non-conventional media, and pH stability, which will lead to a production of more efficient cocktails. In this review, we focus on recent advances in cellulase cocktail production, its current challenges, protein engineering as an efficient strategy to engineer cellulases, and our view on future prospects in the generation of tailored cellulases for biofuel production. © 2020 by the authors. Licensee MDPI, Basel, Switzerland

Engineering Robust Cellulases for Tailored Lignocellulosic Degradation Cocktails

Lignocellulosic biomass is a most promising feedstock in the production of second-generation biofuels. Efficient degradation of lignocellulosic biomass requires a synergistic action of several cellulases and hemicellulases. Cellulases depolymerize cellulose, the main polymer of the lignocellulosic biomass, to its building blocks. The production of cellulase cocktails has been widely explored, however, there are still some main challenges that enzymes need to overcome in order to develop a sustainable production of bioethanol. The main challenges include low activity, product inhibition, and the need to perform fine-tuning of a cellulase cocktail for each type of biomass. Protein engineering and directed evolution are powerful technologies to improve enzyme properties such as increased activity, decreased product inhibition, increased thermal stability, improved performance in non-conventional media, and pH stability, which will lead to a production of more efficient cocktails. In this review, we focus on recent advances in cellulase cocktail production, its current challenges, protein engineering as an efficient strategy to engineer cellulases, and our view on future prospects in the generation of tailored cellulases for biofuel production. © 2020 by the authors. Licensee MDPI, Basel, Switzerland

Using an Inducible Promoter of a Gene Encoding <i>Penicillium verruculosum</i> Glucoamylase for Production of Enzyme Preparations with Enhanced Cellulase Performance

<div><p>Background</p><p><i>Penicillium verruculosum</i> is an efficient producer of highly active cellulase multienzyme system. One of the approaches for enhancing cellulase performance in hydrolysis of cellulosic substrates is to enrich the reaction system with β -glucosidase and/or accessory enzymes, such as lytic polysaccharide monooxygenases (LPMO) displaying a synergism with cellulases.</p><p>Results</p><p>Genes <i>bglI</i>, encoding β-glucosidase from <i>Aspergillus niger</i> (AnBGL), and <i>eglIV</i>, encoding LPMO (formerly endoglucanase IV) from <i>Trichoderma reesei</i> (TrLPMO), were cloned and expressed by <i>P</i>. <i>verruculosum</i> B1-537 strain under the control of the inducible <i>gla1</i> gene promoter. Content of the heterologous AnBGL in the secreted multienzyme cocktails (hBGL1, hBGL2 and hBGL3) varied from 4 to 10% of the total protein, while the content of TrLPMO in the hLPMO sample was ~3%. The glucose yields in 48-h hydrolysis of Avicel and milled aspen wood by the hBGL1, hBGL2 and hBGL3 preparations increased by up to 99 and 80%, respectively, relative to control enzyme preparations without the heterologous AnBGL (at protein loading 5 mg/g substrate for all enzyme samples). The heterologous TrLPMO in the hLPMO preparation boosted the conversion of the lignocellulosic substrate by 10–43%; however, in hydrolysis of Avicel the hLPMO sample was less effective than the control preparations. The highest product yield in hydrolysis of aspen wood was obtained when the hBGL2 and hLPMO preparations were used at the ratio 1:1.</p><p>Conclusions</p><p>The enzyme preparations produced by recombinant <i>P</i>. <i>verruculosum</i> strains, expressing the heterologous AnBGL or TrLPMO under the control of the <i>gla1</i> gene promoter in a starch-containing medium, proved to be more effective in hydrolysis of a lignocellulosic substrate than control enzyme preparations without the heterologous enzymes. The enzyme composition containing both AnBGL and TrLPMO demonstrated the highest performance in lignocellulose hydrolysis, providing a background for developing a fungal strain capable to express both heterologous enzymes simultaneously.</p></div

Progress kinetics of Avicel hydrolysis by different <i>P. verruculosum</i> preparations.

<p>Conditions: substrate concentration 100 mg/mL; protein loading 5 mg/g substrate; CDH loading (when applied) 0.1 mg/g substrate; 50°C; pH 5.0.</p

Production of Biomodified Bleached Kraft Pulp by Catalytic Conversion Using <i>Penicillium verruculosum</i> Enzymes: Composition, Properties, Structure, and Application

The global development of the bioeconomy is impossible without technologies for comprehensive processing of plant renewable resources. The use of proven pretreatment technologies raises the possibility of the industrial implementation of the enzymatic conversion of polysaccharides from lignocellulose considering the process’s complexity. For instance, a well-tuned kraft pulping produces a substrate easily degraded by cellulases and hemicelulases. Enzymatic hydrolysis of bleached hardwood kraft pulp was carried out using an enzyme complex of endoglucanases, cellobiohydrolases, β-glucosidases, and xylanases produced by recombinant strains of Penicillium verruculosum at a 10 FPU/g mixture rate and a 10% substrate concentration. As a result of biocatalysis, the following products were obtained: sugar solution, mainly glucose, xylobiose, xylose, as well as other minor reducing sugars; a modified complex based on cellulose and xylan. The composition of the biomodified kraft pulp was determined by HPLC. The method for determining the crystallinity on an X-ray diffractometer was used to characterize the properties. The article shows the possibility of producing biomodified cellulose cryogels by amorphization with concentrated 85% H3PO4 followed by precipitation with water and supercritical drying. The analysis of the enzymatic hydrolysate composition revealed the predominance of glucose (55–67%) among the reducing sugars with a maximum content in the solution up to 6% after 72 h. The properties and structure of the modified kraft pulp were shown to change during biocatalysis; in particular, the crystallinity increased by 5% after 3 h of enzymatic hydrolysis. We obtained cryogels based on the initial and biomodified kraft pulp with conversion rates of 35, 50, and 70%. The properties of these cryogels are not inferior to those of cryogels based on industrial microcrystalline cellulose, as confirmed by the specific surface area, degree of swelling, porosity, and SEM images. Thus, kraft pulp enzymatic hydrolysis offers prospects not only for producing sugar-rich hydrolysates for microbiological synthesis, but also cellulose powders and cryogels with specified properties

SDS-PAGE of <i>P. verruculosum</i> preparations.

<p><i>M</i>, molecular markers (in kDa); <i>1</i>, hBGL1; <i>2</i>, hBGL2; <i>3</i>, hBGL3; <i>4</i>, PvC1; <i>5</i>, PvC2; <i>6</i>, hLPMO.</p

Progress kinetics of hydrolysis of pretreated aspen wood by different <i>P. verruculosum</i> preparations.

<p>Conditions: substrate concentration 100 mg/mL; protein loading 5 mg/g substrate; 50°C; pH 5.0.</p

The scheme of cloning pGA-BGL and pGA-EGIV plasmids.

<p>The scheme of cloning pGA-BGL and pGA-EGIV plasmids.</p

Specific activities (U/mg protein) of enzyme preparations.

<p>Specific activities (U/mg protein) of enzyme preparations.</p