Engineering Trichoderma reesei for Hyperproduction of Cellulases on Glucose to Efficiently Saccharify Pretreated Corncobs

Abstract

The filamentous fungus Trichoderma reesei (teleomorph Hypocrea jecorina) is widely used as a cellulase producer in the industry. Herein, we describe the rational engineering of the publicly available T. reesei QM9414 strain to achieve a remarkable high-level production of cellulase on glucose. Overexpression of the key cellulase regulator XYR1 by the copper-repressible promoter Ptcu1 was first implemented to achieve a full cellulase production in the context of catabolite repression (CCR) while eliminating the requirement of inducing sugars for enzyme production. The T. reesei bgl1 gene was further overexpressed to compensate for its low β-glucosidase activity on glucose. This overexpression resulted in a 102% increase in FPase activity compared with the CCR-released RUT-C30 strain cultured on Avicel. Moreover, the saccharification efficiency toward pretreated corncob residues by crude enzymes from the engineered strain on glucose increased by 85% compared with that treated by enzymes from RUT-C30 cultivated on Avicel. The engineered T. reesei strain thus shows great potential as a viable alternative to deliver commercial cellulases after further optimization for efficient saccharification of agricultural waste.

Inducer-Free Cellulase Production System Based on the Constitutive Expression of Mutated Xyr1 and Ace3 in the Industrial Fungus Trichoderma Reesei

“…That is, the expression of the cellulase/hemicellulase genes was not regulated only by the transcription of Xyr1, suggesting that there is a regulation that occurs only in the presence of the inducer. In contrast, only the overexpression of Xyr1 by the copperrepressible promoter Ptcu1 in a T. reesei QM9414 strain resulted in the high-level production of cellulase on glucose [45]. Further research is required because the degree of the mutation of CCR-related factors and activator/repressors is different in the ancestral lines.…”

Metabolic Engineering for the Comprehensive Utilization of N,N-Dimethylformamide-Containing Wastewater

“…26 Lignocellulose is a sustainable and renewable resource that is inexpensive and abundantly available throughout the year. 22,24 Separate enzymatic hydrolysis and fermentation (SHF) processes have been widely used and are a promising approach for the production of high-value-added and environmentfriendly products from low cost and inexhaustible sources of lignocellulose wastes. 22 However, the cost of cellulase enzymes remains a major hurdle in lignocellulose conversion.…”

“…22,24 Separate enzymatic hydrolysis and fermentation (SHF) processes have been widely used and are a promising approach for the production of high-value-added and environmentfriendly products from low cost and inexhaustible sources of lignocellulose wastes. 22 However, the cost of cellulase enzymes remains a major hurdle in lignocellulose conversion. 25,35 In the present study, the production of cellulase and cellulosic sugar was significantly increased with the addition of DMFcontaining wastewater (Figure 1 and Table 1), demonstrating the potential to reduce the cost associated with conversion of lignocellulose.…”

Carbon catabolite regulation of secondary metabolite formation, an old but not well‐established regulatory system

“…Some recent reports on CCR have focused on understanding the use of hardly assimilable substrates such as lignocellulose (Shiwa et al ., 2020 ; Zheng et al ., 2020 ; Wu et al ., 2021 ). However, the mechanism of CCR has effects beyond the use of carbon sources in phenomena such as virulence or the production of secondary metabolites.…”

“…In contrast, less stringent CCR has a fitness cost under stable conditions, but allows for a robust and more uniform adaptation, leading to better fitness in a changing environment (New et al, 2014). Some recent reports on CCR have focused on understanding the use of hardly assimilable substrates such as lignocellulose (Shiwa et al, 2020;Zheng et al, 2020;Wu et al, 2021). However, the mechanism of CCR has effects beyond the use of carbon sources in phenomena such as virulence or the production of secondary metabolites.…”