making microbiology easy

Disarming Pseudomonas project

Construction of an alternative host derived from Pseudomonas aeruginosa


The construction of plasmids for cloning and protein expression was one of the key factors in the development of biotechnology in the 1980s. Advances in the field of molecular biology have, at the same time, made genetic manipulations more and more easy. Thus, steps of amplification, recombination, transfer and selection of specific nucleotide sequences have been optimized, and are now accessible to many laboratories, thanks to commercial kits. However, these tools and the protocols for implementing them are often adapted to work with Escherichia coli strains. This bacterial species has many advantages such as a rapid growth, sensitivity to a wide range of antibiotics allowing the selection and maintenance  of plasmid in vitro, the ability to integrate recombinant DNA by transformation and its relative safety. For all of these reasons and because its genetic potential is one of the best known in the bacterial world, E. coli has established itself as the microbial reference host in molecular biology for the expression of heterologous genes. The applications resulting from this model are numerous, both in fundamental and applied research. On the other hand, its use for biotechnological purposes is limited by several factors, including the high cost of the culture media required for its growth. Although not very demanding for its development, this enterobacterium requires complex nutrients to produce sufficient biomasses. The need to use antibiotics for the maintenance of plasmid  is another obstacle to its widespread use in biotechnology as their cost is relatively high. Moireover, they are also at the center of a major ecological problem because of the impact of industrial waste on the resistance of environmental bacteria to antibiotics. It is now well established that antibiotic resistance genes, particularly those carried by plasmids, circulate between environmental, animal and human bacteria. These genetic exchanges are at the origin of a progressive loss of sensitivity of pathogenic bacteria to anti-infective agents and of more and more failures in the treatment of nosocomial infections and human community infections. It should also be emphasized that laboratory strains of E. coli remain able to exchange genes with intestinal bacteria of the same species (commensal E. coli) or other enterobacteria (eg, Klebsiella pneumoniae, Enterobacter sp. , Proteus sp.), Hence the potential risk of these strains contributing to the overall evolution of antibiotic resistance. Finally, among the weaknesses of the E. coli model is its limited number of secretion systems (Sec, TAT).


In this context, species of the genus Pseudomonas and in particular Pseudomonas aeruginosa have genetic characteristics making them an extraordinary tool especially for the production of recombinant proteins. However, a current brake on the use of P. aeruginosa is its pathogenicity (BLS2: biosafety level 2) which limits its use in BSL1 laboratories. Since 2014, SMALTIS has started the construction of a platform strain derived from strain PAO1. For this, our R&D team has undertaken to remove from the genome of this strain all the genetic elements coding for mechanisms conferring to this bacterium with antibiotic resistance and production of several virulence factors. Thus, nearly 1% of the genome has been removed from the chromosome. In parallel, plasmid tools compatible with P. aeruginosa are also under construction.


In the long term, this project aims to bring to the biotechnology market a new model and new technical solutions to improve the production of biologicals such as recombinant proteins or chemical molecules from plant resources.


The Disarming Pseudomonas project received the support of the Ministry of Higher Education and Research in partnership with BPI France Financement as part of the 2013 and 2014 innovative business creation competitions. This project also received support of the National Agency for Technological Research (ANRT) through the financing of PhD program.