465. An Antibiotic-Free Strategy for Miniplasmid Production

The segregational stability of plasmids in a recombinant bioprocess is of extreme significance. Although this is commonly achieved by the selection pressure from antibiotics, their application for the production of therapeutic DNA for gene therapy or for DNA vaccines would be undesirable. Similarly, the presence of antibiotic-resistance genes in the final product would have to be avoided. In addition to the minicircle approach, a type of miniplasmid is able to fulfil the regulatory requirements. The gene tpiA is responsible for the connection of the glycerol metabolic pathway with the essential glycolytic pathway in Escherichia coli. The knockout of genomic tpiA rendered cells completely auxotrophic in minimal medium with glycerol as sole carbon source while allowing growth at a reduced rate with glucose or in complex medium. This was advantageous for optimizing antibiotic-free cloning and selection of recombinant plasmid. Complementation of the auxotrophy by plasmid-borne tpiA led to high segregational and structural plasmid stability, resulting in stable production of a model recombinant enzyme under antibiotic-free conditions in a continuous cultivation. Thus, the complementation of tpiA represents a significant alternative to antibiotics as a selection principle and is therefore of interest for the recombinant production of biotherapeutics in the form of miniplasmids.