Analysis of Structural and Functional Characterisation of TetR/AcrR Regulators in Streptomyces fildesensis So13.3: An in silico CRISPR-Based Strategy to Influence the Suppression of Actinomycin D Production

The growing threat of antimicrobial resistance has driven the search for new bioactive compounds in extreme environments such as Antarctica. Streptomyces fildesensis So13.3, isolated from Antarctic soil, has been shown to contain a biosynthetic gene cluster associated with producing actinomycin D, an antibiotic with therapeutic potential. In this study, we analysed the regulatory role of TetR/AcrR family transcription factors present within this BGC, focusing on their activation under different nutritional conditions and their structural scharacterisation using bioinformatics tools and molecular dynamics simulations. The results showed that TetR/AcrR expression increased significantly in ISP4 and IMA media, suggesting their involvement in nutrient-dependent regulation of the cluster. At the structural level, two TetR proteins (TetR-206 and TetR-279) were modelled, with the latter standing out due to a C-terminal tetracycline repressor-like domain. A 200 ns molecular dynamics simulation was performed in GROMACS to evaluate the stability and flexibility of TetR-279, including analysis of point mutations (S166P, V167A, V167I). The S166P mutation significantly impacted structural flexibility, while V167A and V167I caused only minor alterations. This work demonstrates the value of integrating omics approaches, structural modelling, and gene editing with CRISPR to study and potentially activate silent BGCs in non-model bacteria such as Antarctic Streptomyces. In particular, the targeted inhibition of TetR-279 may trigger metabolic rewiring and facilitate the expression of novel antibiotics encoded in cryptic biosynthetic gene clusters.