Development of a factorial design for a therapeutic plasmid DNA biosynthesis

• Autor(a) principal: Martins, Luís Miguel Candeias
• Data de Publicação: 2013
• Tipo de documento: Dissertação
• Idioma: eng
• Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
• Texto Completo: http://hdl.handle.net/10400.6/3206
• topic: Biomoléculas terapêuticas - DNA plasmídico; Escherichia coli VH33 - DNA plasmídico

Therapeutic applications of plasmid DNA (pDNA) have significantly advanced during the last years. Currently, several pDNA-based drugs are already in the market, whereas several others have entered to phases 2 and 3 of clinical trials. The present and future demand for pDNA requires the development of efficient bioprocesses to produce it. Commonly, pDNA is produced by cultures of Escherichia coli. It has been previously demonstrated that specific strains of E. coli with a modified substrate transport system can be able to attain high cell densities in batch mode, due to the very low overflow metabolism displayed. However, the large amounts of oxygen demanded can lead to microaerobic conditions after some hours of cultivation, even at small-scale. Typically, the inherent problems for these cultures are the high oxygen demand and the accumulation of acetate, a metabolic by-product that is synthesized aerobically when the glucose rate exceeds limits. In recent years, various researches have been focused on the study of induction of plasmid DNA as well as strategies for fermentation using semi-defined mediums. These studies conceived relevant results that allow us to design a production platform for enhanced plasmid DNA. The main goal of this study is to optimize the yield of therapeutic plasmid DNA by culture of recent developed strain of Escherichia coli. The strategy is based on the variation of composition of the fermentation media in terms of nutrients and by a development of an experimental design directed to aromatic amino acids pathway. Monitoring through analytical methods is an advantage both in control and optimization of the intervenients in fermentation process. The change of composition and concentration of the substrates affect the growth of Escherichia coli VH33 and simultaneously, the quantity and quality of the plasmid in study. The experimental model confirms that the disturbing on the aromatic amino acid pathway influences the production plasmid DNA.