Dhara Batista Leles Azevedo¹, Thiago de Andrade Simon¹, Renan Alves Lucio da Silva¹, Cleberson dos Santos Loureiro da Victoria¹, Leonardo Martins Santana², Juliana Barbosa Coitinho¹, Renato Graciano de Paula^1,3

Filamentous fungi are highly diverse organisms that have attracted significant attention due to their ability to produce secondary metabolites (SMs) with a wide range of chemical structures, many of which are crucial for human health. The biosynthesis of these SMs is regulated by signaling pathways triggered by extracellular signals such as light and carbon sources. However, while most studies focus on gene expression data, few have validated these findings through metabolomic analysis. One of the key regulatory pathways in fungi is the calcineurin-CRZ1 pathway, where Ca²⁺ serves as a second messenger. When intracellular calcium levels rise, the calcineurin pathway is activated, leading to the activation of the transcription factor CRZ1. This factor, in turn, modulates the expression of various genes. In this study, we aimed to explore how the Ca²⁺ pathway, mediated by CRZ1, influences the regulation of genes involved in SM biosynthesis in T. reesei. To investigate this, we cultivated both the parental strain and the CRZ1 knockout strain of T. reesei in media containing 1% glycerol and 1% cellulose. We then analyzed fungal mycelia for gene expression and culture supernatants for metabolomic profiling. Our gene expression data revealed that the expression of gene clusters associated with the biosynthesis of polyketides (PKSs), non-ribosomal peptides (NRPSs), and terpenes (TCs) varied significantly between the strains, depending on the carbon source. For PKSs, the genes analyzed showed expression levels 2.5 to 1000-fold higher in the Δcrz1 knockout strain when grown in glycerol. This suggests that CRZ1 acts as a repressor of these genes in response to glycerol. In contrast, when cellulose was the carbon source, CRZ1 exhibited either positive or negative regulatory effects, depending on the specific gene. For NRPSs, three genes were more highly expressed in the knockout strain under glycerol conditions, one gene showed increased expression in response to cellulose, and two genes were downregulated in the presence of cellulose. This indicates that CRZ1 can either enhance or suppress NRPS gene expression, depending on the gene and carbon source. As for TCs, glycerol had a more varied impact on gene expression regulation, with two genes showing either increased or decreased expression in the knockout strain, depending on the gene cluster. In cellulose, however, all analyzed genes exhibited lower expression levels in the knockout strain compared to the parental strain, suggesting that CRZ1 plays a key positive regulatory role in response to cellulose. To further elucidate the regulatory role of CRZ1 in SM biosynthesis in T. reesei, we examined the promoter regions of target genes for predicted CRZ1 binding sites. Our in silico analysis revealed that most of the analyzed genes contain predicted CRZ1 binding sites, supporting the idea that this transcription factor regulates the expression of PKS, NRPS, and TC biosynthesis genes through diverse molecular mechanisms, depending on the carbon source. These findings lay the groundwork 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 calcium signaling pathway regulates SM biosynthesis in T. reesei.

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).