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

Secondary metabolites (SMs) are small molecules that are not essential for fungal growth but have promising biotechnological applications. Filamentous fungi of the genus Trichoderma sp. exhibit potential in various biotechnological applications, being capable of producing multiple SMs, such as peptaibols, gliotoxin, gliovirin, polyketides, pyrones, and terpenes, with broad applications in agriculture and medicine. Several studies have demonstrated the potential of these SMs as antitumor, antiparasitic, cytotoxic, antibiotic, antifungal, immunosuppressive, and antiviral agents.  In Trichoderma reesei, these compounds are produced in response to different stimuli, such as carbon sources, nutrient scarcity, or the presence of inducing molecules. In this context, carbohydrates like glucose and cellulose can affect SM synthesis. Fungal SM biosynthesis genes are often organized into biosynthetic gene clusters (BGCs) that contain core genes encoding multimodular enzymes responsible for SM biosynthesis. Additionally, specific biosynthetic pathway regulators and environmental factors influence SM production.  Our study aimed to understand how glucose and cellulose regulate the expression of genes involved in SM biosynthesis in T. reesei. For this purpose, the QM9414 strain was cultivated in the presence of 1% cellulose and 2% glucose, and gene expression was evaluated using RT-qPCR. Initially, in silico analyses were performed to structurally identify T. reesei gene clusters, followed by gene expression analysis of transcription factors, polyketide synthases, non-ribosomal peptide synthetases, and terpene cyclases in QM9414 cultures exposed to cellulose and glucose for 48 hours.  Our gene expression data showed that genes involved in polyketide biosynthesis were approximately 3, 10, 13, and 22 times more expressed in glucose compared to cellulose, suggesting clear carbon source-dependent regulation. Conversely, for non-ribosomal peptide synthetase expression, the highest expression levels were observed when cellulose was used as the carbon source, reaching levels ranging from 2.8- to 14-fold increases. Finally, our data also indicated that terpene biosynthesis is regulated in a carbon source-dependent manner. The expression values of these gene clusters varied from 2.8- to 14-fold, with some genes being more highly expressed in cellulose, while others showed higher expression in glucose.  Together, our results reveal differential gene expression in SM biosynthesis under the two tested carbon sources. Although these genes exhibited low expression levels, a clear differential expression pattern dependent on the carbon source was evident, suggesting that a crucial regulatory role is played in SM biosynthesis. These findings pave the way for the next steps, which will involve obtaining the metabolic profile of the culture supernatants from the QM9414 strain grown in cellulose and glucose. Thus, our study contributes to expanding knowledge on genetic regulatory mechanisms in fungi, which is essential for the sustainable exploitation of secondary metabolites in hyperproducing strains of the filamentous fungus 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).