Inducer-free cellulase production system based on the constitutive expression of mutated XYR1 and ACE3 in the industrial fungus Trichoderma reesei

Abstract

Trichodermareesei is a widely used host for producing cellulase and hemicellulase cocktails for lignocellulosic biomass degradation. Here, we report a genetic modification strategy for industrial T.reesei that enables enzyme production using simple glucose without inducers, such as cellulose, lactose and sophorose. Previously, the mutated XYR1V821F or XYR1A824V was known to induce xylanase and cellulase using only glucose as a carbon source, but its enzyme composition was biased toward xylanases, and its performance was insufficient to degrade lignocellulose efficiently. Therefore, we examined combinations of mutated XYR1V821F and constitutively expressed CRT1, BGLR, VIB1, ACE2, or ACE3, known as cellulase regulators and essential factors for cellulase expression to the T.reesei E1AB1 strain that has been highly mutagenized for improving enzyme productivity and expressing a ß-glucosidase for high enzyme performance. The results showed that expression of ACE3 to the mutated XYR1V821F expressing strain promoted cellulase expression. Furthermore, co-expression of these two transcription factors also resulted in increased productivity, with enzyme productivity 1.5-fold higher than with the conventional single expression of mutated XYR1V821F. Additionally, that productivity was 5.5-fold higher compared to productivity with an enhanced single expression of ACE3. Moreover, although the DNA-binding domain of ACE3 had been considered essential for inducer-free cellulase production, we found that ACE3 with a partially truncated DNA-binding domain was more effective in cellulase production when co-expressed with a mutated XYR1V821F. This study demonstrates that co-expression of the two transcription factors, the mutated XYR1V821F or XYR1A824V and ACE3, resulted in optimized enzyme composition and increased productivity.

Single Mutation in Transcriptional Activator Xyr1 Enhances Cellulase and Xylanase Production in Trichoderma reesei on Glucose

“…Previous studies revealed that the expression of Xyr1 with the constitutive promoter tcu1 or pdc1 led to glucose-blind cellulase hyperproduction but not that of β-glucosidase and xylanase. , Unlike WT Xyr1, Xyr1 A824V and Xyr1 V821F mutants resulted in significantly improved xylanase production but weak activation of cellulase expression . We report here that the constitutive expression of Xyr1 R434L improves the production of both cellulolytic enzymes, including cellobiohydrolases, endoglucanases, β-glucosidases, and xylanolytic enzymes.…”

“…12,25 Unlike WT Xyr1, Xyr1 A824V and Xyr1 V821F mutants resulted in significantly improved xylanase production but weak activation of cellulase expression. 39 We report here that the constitutive expression of Xyr1 R434L improves the production of both cellulolytic enzymes, including cellobiohydrolases, endoglucanases, β-glucosidases, and xylanolytic enzymes. Consistent with the master regulatory role of Xyr1, 40,41 Xyr1 R434L with improved transcriptional activity enhances the expression of a suite of cellulolytic and xylanolytic enzymes in T. reesei.…”

Optimum Fermentation Conditions for Bovine Lactoferricin-Lactoferrampin-Encoding LimosiLactobacillus reuteri and Regulation of Intestinal Inflammation

“…The expression system of lactic acid bacteria has gained attention due to its stable expression level and efficient expression of target proteins [33]. Constitutive expression systems are particularly advantageous as they exhibit consistent expression levels under varying environmental conditions, thereby eliminating the need for inducers [34]. The present study reports the construction of a recombinant strain, namely pPG-LFCA-E/LR-CO21 (LR-LFCA), utilizing LimosiLactobacillus reuteri as a carrier for expressing LFCA, a compound that possesses both probiotic and antimicrobial peptide properties [11].…”

Engineering cellulases for conversion of lignocellulosic biomass

“…Perhaps the most remarkable (well-documented) example of strain engineering is the development of the RUT-C30 cellulase hyperproducer prototype strain, which is derived from strain QM6a, and shows a 20-fold increase in extracellular protein levels, reaching about 30 g/L on a lactose-containing growth medium ( Bischof et al , 2016 ). Recent tools such as RNAi-mediated gene silencing ( Sun et al , 2022 ), inducer-free expression systems ( Arai et al , 2022 ), CRISPR/Cas9-based methods ( Rantasalo et al , 2019 , Fonseca et al , 2020 ) and synthetic expression systems with engineered transcription factors and promoters ( Rantasalo et al , 2018 ) provide new tools for engineering cellulase production strains ( Mojzita et al , 2019 ). For example, Arai et al (2022) reported an inducer-free expression system for T. reesei that enables protein overproduction in glucose– containing media without inducers like cellulose, lactose and sophorose.…”

“…Recent tools such as RNAi-mediated gene silencing ( Sun et al , 2022 ), inducer-free expression systems ( Arai et al , 2022 ), CRISPR/Cas9-based methods ( Rantasalo et al , 2019 , Fonseca et al , 2020 ) and synthetic expression systems with engineered transcription factors and promoters ( Rantasalo et al , 2018 ) provide new tools for engineering cellulase production strains ( Mojzita et al , 2019 ). For example, Arai et al (2022) reported an inducer-free expression system for T. reesei that enables protein overproduction in glucose– containing media without inducers like cellulose, lactose and sophorose. In another study, Fonseca et al (2020) used CRISPR/Cas9-based rational engineering of the RUT-C30 strain to increase protein secretion and β-glucosidase (72-fold) and xylanase (42-fold) activities in the secretome.…”