Working with engineered bacteria involves important safety issues that we considered in the design of our project.
The Polylactic acid (PLA) produced by Pseudomonas putida that we want to manufacture is designed to be use in everybody's daily life, and also in different application such as drug delivery, so it is key that we detail its biosafety to reassure this usability.
Choosing Pseudomonas putida as chassis has multiple advantages; not only for its ability to produce lactate and making good polymerization but also because it is a versatile saprophytic soil bacterium that has been certified as a biosafety host for the cloning of foreign genes.
Moreover, our strain, KT2440, is the first Gram-negative soil bacterium to be certified as a safe strain by the Recombinant DNA Advisory Committee and is the preferred host for cloning and gene expression in Gram-negative soil bacteria. Pseudomonas putida KT2440 belongs to the risk group 1 of the non-pathogenic organisms.[1, 2, 3]
- Clostridium propionicum is a risk group 1 organism . We used the gene coding the propionyl-CoA transferase (Pct) that has no virulence factor. This part catalyzes the transformation of lactate into lactyl-CoA (necessary for the synthesis of PLA).
- Pseudomonas sp. MBEL 6-19 NA (the risk group is not indicated in ). We used coding for Polyhydroxyalkanoate synthase, this part is necessary for the polymerization of the lactyl-CoA into PLA. It has any virulent factor
- Lactobacillus delbrueckii 11842 is a risk group 1 organism . We used the part coding for D-lactate dehydrogenase (LDH) that will allow to increase the amount of D-lactate in Pseudomonas putida.
- Pseudomonas resinovorans is risk group 2 organism . We used the gene coding for the Polyhydroxyalkanoate synthase. This enzyme catalyzes the polymerization of the lactyl-CoA into PLA and it has no virulence factor, which makes it safe.
If released in the environment, our engineered bacteria won't be able to produce PLA due to the fact that the PLA producing operon is under control of an IPTG inducible promoter.
Other risks linked to our project context, would be the dissemination of the PLA product waste into the environment. Because of being announced as biodegradable compound, people may think that throwing away their PLA items into the environment would be harmless. Providing contrasted information on PLA degradability should be done, as well as stating the fact that the degradation of PLA into lactate could result in an acidification of the ground. Thus, it would be important to avoid the accumulation of PLA waste in the environment.
Another main risk would be the "contamination" of the recycling trashes with PLA waste. Indeed, in France, recycling waste has a great importance but people are often not enough aware of the different type of waste and could easily throw away the PLA articles with the other plastics. The PLA can be reused if it undergoes a special treatment, different from the one performed for common plastics.
In order to reduce these two main risks, a sensitivization campaign on the correct way to throw away PLA waste should be prepared and spread with the items produced with our PLA and during events involving public audience.
- Federal Register (1982) Certified host–vector systems, p. 17197
- Ramos, J.L., Wasserfallen, A., Rose, K., and Timmis, K.N. Redesigning metabolic routes: manipulation of TOL plasmid pathway for catabolism of alkylbenzoates. Science 235: 593–596. (1987)
- American Biological Safety Association. Risk Groups: Bacteria (1998). Retrieved from: http://www.fbcb.unl.edu.ar/media/Investigacion/RG_Bacteria.pdf