Bacteria factories to produce bioactive molecules
PUFAs are consumed as nutraceuticals for their numerous health benefits, including anti-inflammatory and immunoregulatory actions. It has been shown an increased uptake of long chain omega-3 PUFAs can help prevent cardiovascular disease. Currently, the major sources of PUFAs are fish, algae and fish oil products. However, global warming, overfishing and pollution threaten the availability of these molecules. Hence, the MISELIAS (Mechanistic Investigation of Microbial Secondary Lipid Assembly) project was established to address this problem. Researchers investigated ′de novo′ secondary biosynthesis catalyzed by polyunsaturated fatty acid synthases (Pfa synthases) to gain a better understanding of how this class of enzyme operates. This information will enable scientists to ′reprogramme′ microbe cell factories to produce secondary lipid metabolites of pharmaceutical and industrial interest. A putative Pfa synthase from the alkane degrading bacterium Rhodococcus erythropolis PR4 was chosen as a model for investigating enzymes and captured and characterised using a chemical probe. The genes of a putative Pfa synthase and a putative thioesterase/dehydratase/isomerase from Rhodococcus erythropolis were successfully cloned for the very first time and the enzymes expressed and purified for in vitro studies. Optimisation of protein expression and purification protocols to prevent degradation led to extremely pure enzymes in acceptable yields that allowed first attempts for the in vitro reconstitution of PUFA biosynthesis. Moreover, novel chemicals probes of improved efficiency for polyketide synthases (PKS)/Pfa intermediate capture were developed. Results from the MISELIAS project have laid the foundation for further work on the ultimate reconstitution of PUFA biosynthesis in vitro and its mechanistic dissection. This will ultimately lead to new and sustainable land-based productions of PUFAs and structurally related bioactive molecules.
Keywords
Secondary lipid metabolites, polyunsaturated fatty acids, MISELIAS, polyunsaturated fatty acid synthases, Rhodoccocus erythropolis PR4, polyketide synthases