MiRNAs: expressão heteróloga e sua aplicação como componente de novas tecnologias na aquicultura

Abstract

Aquaculture faces critical challenges, such as environmental stressors and diseases, which impact productivity. This thesis proposes an innovative solution based on the heterologous production of microRNAs (miRNAs) in bacteria and their use as a dietary supplement for teleosts, combining a systematic review of the state of the art with the development of a patent. Initially, a comprehensive analysis of bacterial platforms for miRNA expression was conducted, identifying 832 studies across three databases, of which 15 were selected. The results demonstrated that Escherichia coli, Salmonella typhimurium, and Rhodovulum sulfidophilum are the main species used, with 78 miRNAs produced heterologously—75 in E. coli (e.g., miR-21 and miR-155), 1 in R. sulfidophilum (miR-29b), and 2 in S. typhimurium (mi-INHA and miRNA CCL22). R. sulfidophilum stood out for its ability to secrete nucleic acids, absence of RNases, and cultivation in saltwater, while E. coli remained the preferred model for large-scale production. Plasmids with the lpp promoter and multiple cloning sites proved to be the most efficient for miRNA expression, highlighting the importance of vector design in production optimization. Based on these findings, a patent was developed for a dietary supplement containing synthetic miRNAs and antagomiRs, aimed at enhancing fish tolerance to low temperatures. The method involves: 1) Designing recombinant DNA constructs with specific miRNA sequences; 2) Transformation into E. coli for production in bioreactors; 3) Thermal inactivation of bacterial biomass; 4) Encapsulation in feed with a fatty adhesive, ensuring stability and intestinal absorption. The formulation works by modulating antioxidant and metabolic pathways (e.g., the glucose pathway), reducing the effects of thermal stress. It differs from conventional approaches by being non-transgenic, orally administered, and seasonal, facilitating commercial adoption. The results indicate that combining bacterial production and nutraceutical application of miRNAs offers a viable solution to aquaculture challenges. In conclusion, the developed platform not only advances knowledge about heterologous miRNA expression in prokaryotes but also establishes a new paradigm for RNA-based interventions in animal production, with potential adaptation to other environmental stressors or economically relevant species.