Thiago de Andrade Simon¹, Dhara Batista Leles Azevedo¹, Renan Alves Lucio da Silva¹, Cleberson dos Santos Loureiro da Victoria¹, David Batista Maues², Roberto Nascimento Silva^2,3, Juliana Barbosa Coitinho¹, Renato Graciano de Paula^1,3

The filamentous fungus Trichoderma reesei is a highly relevant organism in the industrial and biotechnological fields due to its production of secondary metabolites (SMs) with diverse properties, such as antitumor, antibiotic, antiviral, and antiparasitic activities. Given the growing issue of antibiotic resistance, the search for new bioactive molecules is intensifying, and T. reesei stands out prominently in the field of secondary metabolism. It is one of the most significant organisms in the agrobiotechnology industry and the most important producer of cellulases for second-generation biofuels. In filamentous fungi, the synthesis of SMs is controlled by complex regulatory networks, including transcription factors and intracellular signaling pathways. Despite its ability to synthesize a wide range of SMs, a complete understanding of the genes, transcription factors, and regulatory pathways involved in controlling SM biosynthesis in this fungus remains limited. Interestingly, the MAPK pathway influences SM production in Trichoderma. Therefore, this study aims to understand how the MAPK pathway regulates the expression of gene clusters involved in SM biosynthesis in T. reesei in response to cellulose. To achieve this, the knockout strains Δtmk1, Δtmk2, and Δtmk3, as well as the parental strain QM6a, were cultivated in the presence of cellulose, and gene expression was analyzed using RT-qPCR. Our results showed that MAPK signaling variably influences the expression of genes and transcription factors associated with SM biosynthesis in T. reesei, with positive or negative effects depending on the specific gene or transcription factor analyzed. Additionally, in silico analyses for phosphorylation site prediction revealed that while the MAPK pathway regulates the expression of genes within SM gene clusters, the biosynthesis of these molecules may also be regulated at the post-translational level through phosphorylation, primarily by other protein kinases beyond MAPKs. Many of these kinases may be directly regulated by the MAPK pathway and, consequently, modulate secondary metabolism in this fungus. Our results pave the way for future research, which will involve metabolomic profiling of the fungal culture supernatants to quantify the production of these SMs and provide a more comprehensive understanding of how the MAPK signaling pathway regulates SM biosynthesis in T. reesei. Moreover, these findings highlight its potential as a target for future genetic and metabolic engineering strategies aimed at enhancing the production of these highly valuable biotechnological molecules.

Agradecimentos: Funding agencies: FAPES (process number 2021-RZN24 (TO 438/2021), Capes, CNPq (grant number 405934/2022-0 - The National Institute of Science and Technology INCT Funvir, Brazil); Laboratório Multiusuário de Análises Biomoleculares (Labiom/UFES), Laboratório de Química de Proteínas (LQP/UFES), Laboratório de Bioquímica e Biofísica Molecular de Proteínas (LB2MP/UFES), Laboratório Neuroquímica e Comportamento (LabNec), Laboratório de Caracterização Física, Química e Microbiológica (LACAR) no Centro de Pesquisa, Inovação e Desenvolvimento (CPID).