Abstract
Filamentous fungal strains of Trichoderma reesei have been widely used for cellulase production, and great effort has been devoted to enhancing their cellulase titers for the economic biorefinery of lignocellulosic biomass. In our previous studies, artificial zinc finger proteins (AZFPs) with the Gal4 effector domain were used to enhance cellulase biosynthesis in T. reesei, and it is of great interest to modify the AZFPs to further improve cellulase production. In this study, the endogenous activation domain from the transcription activator Xyr1 was used to replace the activation domain of Gal4 of the AZFP to explore impact on cellulase production. The cellulase producer T. reesei TU-6 was used as a host strain, and the engineered strains containing the Xyr1 and the Gal4 activation domains were named as T. reesei QS2 and T. reesei QS1, respectively. Compared to T. reesei QS1, activities of filter paper and endoglucanases in crude cellulase produced by T. reesei QS2 increased 24.6 and 50.4%, respectively. Real-time qPCR analysis also revealed significant up-regulation of major genes encoding cellulase in T. reesei QS2. Furthermore, the biomass hydrolytic performance of the cellulase was evaluated, and 83.8 and 97.9% more glucose was released during the hydrolysis of pretreated corn stover using crude enzyme produced by T. reesei QS2, when compared to the hydrolysis with cellulase produced by T. reesei QS1 and the parent strain T. reesei TU-6. As a result, we proved that the effector domain in the AZFPs can be optimized to construct more effective artificial transcription factors for engineering T. reesei to improve its cellulase production.
Highlights
Lignocellulosic biomass is abundantly available as a renewable resource, which is mainly composed of cellulose, hemicelluloses, and lignin
Transformants containing artificial zinc finger proteins (AZFPs) coding sequences driven by the constitutive promoter pki were screened (Zhang et al, 2016), and one hyper-cellulolytic mutant T. reesei M2 was selected by detecting FPase activity during liquid fermentation for cellulase production in flasks (Meng et al, 2020)
The expression of AZFPs in the cell lysate of T. reesei QS1 and QS2 mutants was confirmed by Western blot analysis (Figure 1D), indicating the observed phenotypic changes of transformants in cellulase production were resulted from the expression of the integrated AZFPs
Summary
Lignocellulosic biomass is abundantly available as a renewable resource, which is mainly composed of cellulose, hemicelluloses, and lignin. The cellulase enzymatic complex of T. reesei has been shown to consist of at least two cellobiohydrolases (CBHs), eight endo-β-1,4-glucanases (EGs), and seven β-glucosidases (BGLs) that act synergistically upon insoluble cellulose substrate (Druzhinina and Kubicek, 2017). The synthesis of these cellulase components is strictly controlled by various regulators, including at least six positive transcriptional activators (Xyr, Ace, Ace, Vib, BglR, and the Hap2/3/5 complex) as well as three repressors (Ace, Rce, and the carbon catabolite repressor Cre1) (Aro et al, 2001, 2003; Cao et al, 2017; Zhang F. et al, 2018). It is of great interest to investigate the effect of endogenous effector domains in the AZFPs on regulation of cellulase production in T. reesei
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