Exploring Vibrio natriegens potential as a new microbial chassis for plasmid DNA production

Modern, state-of-the-art therapies rely at some point, in their manufacture or mode of action, in plasmid DNA (pDNA). Plasmids play a supporting role in the production of recombinant proteins and mRNA, and function as direct applications, in context of gene therapies and DNA vaccination. Large-scale manufacture of pDNA depends exclusively on one host platform – Escherichia coli. However, the growing demand for pDNA justifies the search for a novel host, more suitable for its manufacture . One bacterium that has emerged as an attractive chassis is Vibrio natriegens. This bacterium is the fastest-growing organism known to date. Additionally, it is non-pathogenic and metabolically proficient. The aim of this project was to evaluate whether the remarkable characteristics of V. natriegens could be tapped to develop a novel, preferable host for pDNA production.

V. natriegens cells were transformed with a pDNA containing an eGFP gene under the control of a eukaryotic promotor. Plasmids replicated in V. natriegens were isolated and analysed by agarose gel electrophoresis. Supercoiled pDNA obtained from V. natriegens were purified by multimodal chromatography and used to transfect mammalian cells (HEK293T). Plasmids replicated in E. coli were used as control. Our results showed that the plasmid can be replicated in V. natriegens and show a similar topology to those replicated in E. coli. Supercoiled pDNA obtained from V. natriegenswere able to drive GFP expression in HEK293T cells cultured in vitro, showing a level of expression comparable to that obtained with pDNA from E. coli. Although productivity was lower in V. natriegens than in E. coli, we concluded that this bacterium is a promising alternative as a host for pDNA manufacturing.