Abstract
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 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 byproduct that is synthesized aerobically when the glucose rate exceeds the limits.
In recent years, several 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. So, the main goal of this chapter is to show how the development of an experimental design directed to aromatic amino acids pathway can improve the yield of a therapeutic plasmid DNA by culture of a new strain of Escherichia coli VH33.
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Acknowledgments
L.A. Passarinha acknowledges a sabattical fellowship (SFRH/BSAB/150376/2019) from the Portuguese Foundation for Science and Technology (FCT) within the scope of POCH—Advanced Formation programs co-funded by European Social Fund and MCTES. This work was also supported by the Applied Molecular Biosciences Unit—UCIBIO, which is financed by national funds from FCT/MCTES (UID/Multi/04378/2019). A special thanks to Professor Guillermo Gosset (Instituto de Biotecnología from Universidad Nacional Autónoma de México) for providing us the E.coli VH33 strain and to Dr. Thomas Roberts for providing the pcDNA3-FLAG construct through Addgene (reference 10838).
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Passarinha, L.A. (2021). Enhanced Biosynthesis of Plasmid DNA from Escherichia coli Applying Experimental Design. In: Sousa, Â. (eds) DNA Vaccines. Methods in Molecular Biology, vol 2197. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0872-2_7
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DOI: https://doi.org/10.1007/978-1-0716-0872-2_7
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