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

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

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

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 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

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

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

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