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

Abstract

BackgroundPenicillium 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.ResultsGenes 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.ConclusionsThe 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.

Heterologous protein production in filamentous fungi

“…Subsequently, the BGL and lytic polysaccharide monooxygenases (LPMOs) from T. reesei were produced by P . verruculosum B1-537 strain (Bulakhov et al 2017 ). P. verruculosum was also used to produce heterologous endo-xanthanase (Denisenko et al 2021 ).…”

Tailoring in fungi for next generation cellulase production with special reference to CRISPR/CAS system

“…The resultant recombinant yeast strain can be utilized cellobiose as a carbon source [ 40 ]. Simultaneous expression of bgl1 encoding BGL from A. niger (AnBGL) and egl4 encoding LPMO (previously endoglucanase IV) from T. reesei (TrLPMO) have been cloned in Penicillium verruculosum utilizing the inducible gla1 promoter, led to the far better hydrolysis of lignocellulosic biomass in contrast with the wild type strain [ 41 ]. The gpdA promoter (isolated from A. nidulans ) mediated overexpression of clrB in P. oxalicum , resulting in higher cellulase levels [ 42 ].…”

Developments and opportunities in fungal strain engineering for the production of novel enzymes and enzyme cocktails for plant biomass degradation

“…Combining the introduction of several genes can further enhance the effectivity of an enzyme mixture. Simultaneous expression of bglI, encoding a β-glucosidase from A. niger (AnBGL), and eglIV, encoding a lytic polysaccharide monooxygenase (LPMO) from T. reesei (TrLPMO), in Penicillium verruculosum under the control of the inducible gla1 promoter resulted in more efficient hydrolysis of a lignocellulosic substrate than the control enzyme preparations (Bulakhov et al, 2017). Similarly, modification of the expression of a major regulator can also affect the enzyme mixture as a whole, such as the overexpression of clrB in Penicillium oxalicum using the gpdA promoter from Aspergillus nidulans that resulted in higher cellulase levels (Yao et al, 2015).…”