Since the beginning of DNA engineering, plasmid instability has been a significant concern, especially in recombinant protein production. Typically, protein-production processes in prokaryotes require the use of bacterial plasmids as vectors to carry the gene of interest encoding the protein to be overexpressed.
It is well known that the growth of plasmid-bearing cells is significantly reduced relative to plasmid-free hosts simply because the protein production (corresponding to the gene of interest overexpression) and the vector represent a burden on host metabolism.
Antibiotic-resistant genes are the selectable markers typically used in fermentation processes to avoid plasmid-free cells overgrowing the culture. Antibiotic and antibiotic markers represent a problem from a regulatory standpoint, and today, only the Kanamycin resistance gene is still tolerated by regulatory authorities. The Kanamycin resistance gene, however, represents a metabolic burden that limits the production yield of the bioproduct of interest.
The only way to avoid antibiotic-resistance spread in the environment and transfer to pathogenic strains is the complete absence of antibiotic-resistance genes in recombinant constructions. Thus, it is expected that, in the near future, there may be zero tolerance toward antibiotic-based selection and production systems at least in the biopharmaceutical field.
Several alternatives to antibiotics have been proposed but none of them has been widely adopted. This can be explained by the complexity of the proposed systems, which requires complex or specific media, or nonconventional strains.
An alternative to antibiotic markers that is less energy consuming for the host metabolism is required for more efficient recombinant bioproduction processes.
With these challenges in mind, Delphi Genetics developed a stabilization system called StabyExpress™ based on the use of natural bacterial antidote/poison genes and more precisely the ccd operon. This operon was isolated from the F plasmid naturally present in E. coli and encodes two genes (ccdA and ccdB) that are very well characterized.
The ccdB poison gene codes for a small stable protein, whereas the ccdA antidote gene codes for a small unstable protein that neutralizes the poison protein both transcriptionally and via protein-protein interactions. These genes were chosen because they are not toxic to humans.
StabyExpress technology takes full advantage of the natural properties of these genetic elements. Indeed, the functions of these genes have been polished by evolution in order to interact very efficiently with E. coli physiology so as to trigger bacterial death in very defined conditions.
The observed efficiency of the StabyExpress technology comes from this natural evolution. The ccd operon belongs to the poison/antidote bacterial gene family. These loci share common features and are widespread in plasmids and bacterial chromosomes. They represent a natural toolbox for applications in bacterial species presenting an industrial interest.