Background: The filamentous fungus Trichoderma reesei is a major workhorse employed to produce cellulase, which hydrolyzes lignocellulosic biomass for the production of cellulosic ethanol and bio-based products. However, the economic efficiency of biorefineries is still low. Results: In this study, the truncation of cellulase activator ACE3 was identified and characterized in T. reesei classical mutant NG14 and its direct descendants for the first time. We demonstrated that the truncated ACE3 is the crucial cause of cellulase hyper-production in T. reesei NG14 branch. Replacing the native ACE3 with truncated ACE3 in other T. reesei strains remarkably improves cellulase production. By truncating ACE3, we engineered a T. reesei mutant, PC-3-7-A723, capable of producing more cellulase than other strains. In a 30-L fermenter, fed-batch fermentation with PC-3-7-A723 drastically increased the maximum cellulase titer (FPase) to 102.63 IU/mL at 240 h, which constitutes a 20-30% improvement to that of the parental strain PC-3-7. Conclusions: This work characterized the function of truncated ACE3 and demonstrated that analysis of classical mutants allows rational engineering of mutant strains with improved cellulase production necessary to process lignocellulosic biomass. Our rational engineering strategy might be useful for enhancing the production of other bio-based products.

Background The filamentous fungus Trichoderma reesei is one of the best producers of cellulase and has been widely studied for the production of cellulosic ethanol and bio-based products. We previously reported that Mn2+ and N,N-dimethylformamide (DMF) can stimulate cellulase overexpression via Ca2+ bursts and calcium signalling in T. reesei under cellulase-inducing conditions. To further understand the regulatory networks involved in cellulase overexpression in T. reesei, we characterised the Mn2+/DMF-induced calcium signalling pathway involved in the stimulation of cellulase overexpression. Results We found that Mn2+/DMF stimulation significantly increased the intracellular levels of cAMP in an adenylate cyclase (ACY1)-dependent manner. Deletion of acy1 confirmed that cAMP is crucial for the Mn2+/DMF-stimulated cellulase overexpression in T. reesei. We further revealed that cAMP elevation induces a cytosolic Ca2+ burst, thereby initiating the Ca2+ signal transduction pathway in T. reesei, and that cAMP signalling causes the Ca2+ signalling pathway to regulate cellulase production in T. reesei. Furthermore, using a phospholipase C encoding gene plc-e deletion strain, we showed that the plc-e gene is vital for cellulase overexpression in response to stimulation by both Mn2+ and DMF, and that cAMP induces a Ca2+ burst through PLC-E. Conclusions The findings of this study reveal the presence of a signal transduction pathway in which Mn2+/DMF stimulation produces cAMP. Increase in the levels of cAMP activates the calcium signalling pathway via phospholipase C to regulate cellulase overexpression under cellulase-inducing conditions. These findings provide insights into the molecular mechanism of the cAMP–PLC–calcium signalling pathway underlying cellulase expression in T. reesei and highlight the potential applications of signal transduction in the regulation of gene expression in fungi.

The filamentous fungus Trichoderma reesei is a model strain for cellulase production. Cellulase gene expression in T. reesei is controlled by multiple transcription factors. Here, we identified by comparative genomic screening a novel transcriptional activator ACE4 ( A ctivator of c ellulase e xpression 4) that positively regulates cellulase gene expression on cellulose in T. reesei . Disruption of the ace4 gene significantly decreased expression of four main cellulase genes, and the essential cellulase transcription factor encoding gene ace3 . Overexpression of ace4 increased cellulase production by approximately 22% compared to that in the parental strain. Further investigations using electrophoretic mobility shift assays, DNase I footprinting assays, and chromatin immunoprecipitation assays indicated that ACE4 directly binds to the promoter of cellulase genes by recognizing the two adjacent 5′-GGCC-3′ sequences. Additionally, ACE4 directly binds to the promoter of ace3 and, in turn, regulates the expression of ACE3 to facilitate cellulase production. Collectively, these results demonstrate an important role for ACE4 in regulating cellulase gene expression, which will contribute to understanding the mechanism underlying cellulase expression in T. reesei . IMPORTANCE T. reesei is commonly utilized in industry to produce cellulases, enzymes that degrade lignocellulosic biomass for the production of bioethanol and bio-based products. T. reesei is capable of rapidly initiating the biosynthesis of cellulases in the presence of cellulose, which has made it useful as a model fungus for studying gene expression in eukaryotes. Cellulase gene expression is controlled through multiple transcription factors at the transcriptional level. However, the molecular mechanisms by which transcription is controlled remain unclear. In the present study, we identified a novel transcription factor, ACE4, which regulates cellulase expression on cellulose by binding to the promoters of cellulase genes and the cellulase activator ace3 . Our study not only expands the general functional understanding of the novel transcription factor ACE4 but also provides evidence for the regulatory mechanism mediating gene expression in T. reesei .

Lignocellulosic biomass, which primarily consists of cellulose, hemicellulose, and lignin, is a renewable and abundant resource for the production of biofuels and chemicals (Peterson & Nevalainen, 2012;Ulaganathan et al., 2015). Cellulase can convert lignocellulosic biomass into fermentable sugars in an environmentally friendly manner, thus providing a promising approach for this process (Luo et al., 2020). The filamentous fungus Trichoderma reesei is a well-known industrial producer of cellulase and hemicellulase and is widely utilized in various industries, including food, fodder, paper, and textile industries. It is also particularly useful for cellulosic biofuel production (Bischof et al., 2016;Peterson & Nevalainen, 2012).The cellulase produced by T. reesei comprises primarily of two cellulobiohydrolases (CBHI and CBHII), two endoglucanases (EGI and EGII), and β-glucosidase I (BGLI), which synergistically hydrolyze lignocellulosic materials to fermentable sugars (Bischof et al., 2016;

N, N-dimethylformamide is frequently present in industrial wastewater and is environmentally detrimental. The current study aims to assess the utilization and biodegradation of N, N-dimethylformamide-containing wastewater to lessen the associated environmental load. Results show that addition of wastewater containing N, N-dimethylformamide to Trichoderma reesei fermentation media enhances cellulase production and facilitates cellulose hydrolysis. However, N, N-dimethylformamide is a cellulase enhancer that is not degraded during cellulase production in T. reesei fermentation and is retained in the N, Ndimethylformamide-enhanced cellulase solution. Indeed, the cellulosic sugar solution generated via lignocellulose hydrolysis with N, N-dimethylformamide-enhanced cellulase retains N, N-dimethylformamide. We further identified three core enzyme modules�N, N-dimethylformamidase, dimethylamine dehydrogenase, and methylamine dehydrogenase enzyme�which were inserted into Escherichia coli to develop metabolically engineered strains. These strains degraded N, N-dimethylformamide and produced succinate using N, N-dimethylformamide-enhanced cellulosic sugar as the substrate. The platform described here can be applied to effectively convert waste into valuable bioproducts.