| Congresso Brasileiro de Microbiologia 2023 | Resumo: 286-1 | ||||
Resumo:Trichoderma reesei is a saprophytic fungus known for its ability to secrete large amounts of cellulases, enzymes essential for the production of products from lignocellulosic biomass. The cellulolytic system of T. reesei is under the control of several transcription factors (TFs) that efficiently regulate the production of these enzymes. Analysis of RNA-Seq data from T. reesei grown in different carbon sources allowed the identification of a new potential regulator of cellulase production: a novel zinc finger TF called Nsd3, a homolog of NsdC from Aspergilli. Gene expression analysis by RT-qPCR showed that nsd3 is strongly expressed in the presence of glucose, a carbon source that represses cellulase production. At the same time, it is significantly lower in cellulose and sugarcane bagasse. A deletion mutant strain was generated using the CRISPR-Cas9 system to further investigate the function of Nsd3 in T. reesei. Deletion of nsd3 impaired vegetative growth and conidiation on a solid medium. In addition, the Δnsd3 strain is more sensitive to osmotic stress caused by NaCl (0.5 and 1 M), and the addition of CaCl2 positively affects the growth of the Δnsd3 strain to a greater extent than in the wild-type strain (WT). In addition, the Δnsd3 strain is more resistant to cell wall stress induced by Congo red (CR) and Calcofluor white (CFW) than the WT strain. Through gene expression analysis, significant upregulation of genes involved in cell wall biosynthesis and integrity, such as genes encoding chitin synthases, a β-1,3-glucan synthase (fks1), and the mitogen-activated protein kinase tmk2, was observed in the Δnsd3 strain when cells were exposed to CR 100 ug/mL for 60 min. In addition, Nsd3 acts as a repressor of cellulase expression, as its deletion increases the expression of cel7a, an essential cellulase produced by T. reesei, and the hemicellulases xyn1 and bxl1. This increase affects enzyme production, and the Δnsd3 strain showed higher xylanase and β-xylosidase activities than the WT strain. These results suggest that Nsd3 regulates multiple physiological processes and provides new insights into the regulatory system of cellulases in T. reesei. Palavras-chave: Trichoderma reesei, Cellulases, Transcription factor, Stress Response, Regulation of gene expression Agência de fomento:Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Finance Code 001 and Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), project number 2021/01580-7 | |||||