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KATAYAMA, K.Y. Evaluation of plasmidial stabilization system toxin-antitoxin for recombinant proteins production in Escherichia coli. 2016. 66 p. Master thesis (Biotechnology) – Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo, 2016. The search for renewable energy sources has driven research into more efficient and economically viable technologies for second generation ethanol production. A promising approach to obtain efficient enzyme cocktails for the hydrolysis step is the enrichment of cocktails in terms of activities that are lacking, but can be obtained by heterologous systems. The objective of this study was to evaluate the toxin-antitoxin (TA) system for plasmid stabilization in E. coli, to reduce costs by eliminating the antibiotic in the production of recombinant proteins. The results obtained in kinetic and expression assays indicated that the expression system in the strain with TA stabilization is as effective as the use of antibiotic-dependent strain for plasmid stabilization, widely used at industrial scale. Besides effective, TA stabilization system was more efficient as it does not allow the survival of cells without the plasmid. It has been found that there is a leak of up to 10% of cells without the plasmid in antibioticdependent strain for plasmid maintenance, even in the presence of the antibiotic, and up to 34% without antibiotics during cultivation, which does not occur when using the TA system. Studies have indicated the minimum amount of 0.05 mM of IPTG for expression of proteins in this strain, about 20 times less than the concentration usually applied, but it is important to consider the cell density at time of induction. Thus, the TA plasmid stabilization system proved to be a great tool for the development of an alternative platform for producing recombinant proteins.

Avaliação do sistema de estabilização plasmidial toxina-antitoxina para a produção de proteínas recombinantes em <i>Escherichia coli</i>.