Research Article: Using an Inducible Promoter of a Gene Encoding Penicillium verruculosum Glucoamylase for Production of Enzyme Preparations with Enhanced Cellulase Performance

Date Published: January 20, 2017

Publisher: Public Library of Science

Author(s): Alexander G. Bulakhov, Pavel V. Volkov, Aleksandra M. Rozhkova, Alexander V. Gusakov, Vitaly A. Nemashkalov, Aidar D. Satrutdinov, Arkady P. Sinitsyn, Shihui Yang.

http://doi.org/10.1371/journal.pone.0170404

Abstract

Penicillium verruculosum 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.

Genes bglI, encoding β-glucosidase from Aspergillus niger (AnBGL), and eglIV, encoding LPMO (formerly endoglucanase IV) from Trichoderma reesei (TrLPMO), were cloned and expressed by P. verruculosum B1-537 strain under the control of the inducible gla1 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.

The enzyme preparations produced by recombinant P. verruculosum strains, expressing the heterologous AnBGL or TrLPMO under the control of the gla1 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.

Partial Text

Filamentous fungi from the Ascomycota phylum proved to be efficient producers of highly active extracellular cellulase systems [1]. They include various species belonging to the genera Trichoderma (T. reesei, T. longibrachiatum, T. harzianum, T. koningii, etc.), Penicillium (P. funiculosum, P. verruculosum, P. oxalicum, P. decumbens, etc.), Myceliophthora (M. thermophila), Chaetomium (C. thermophilum) and others [2–5]. However, quite many fungal species produce multienzyme cocktails having a low level of the β-glucosidase (BGL) activity that is not enough for a fast conversion of cellobiose and other oligosaccharides, formed in cellulose hydrolysis under the action of cellulolytic enzymes (endoglucanases and cellobiohydrolases), to the final product, glucose [2, 6, 7]. Cellobiose released during the enzymatic hydrolysis of cellulose rather strongly inhibits cellobiohydrolases, thus reducing the cellulase system performance [8].

The enzyme preparations produced by recombinant P. verruculosum strains, expressing the heterologous AnBGL or TrLPMO under the control of the gla1 gene promoter in a starch-containing medium (pretreated wheat meal), proved to be more effective in hydrolysis of a lignocellulosic substrate than control enzyme preparations without the heterologous enzymes, including that grown in a standard medium optimized for cellulase production. At the same time, the enzyme sample containing TrLPMO (hLPMO) could not compete with the control preparations in hydrolysis of Avicel. 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.

 

Source:

http://doi.org/10.1371/journal.pone.0170404

 

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