Final Report Summary - MISELIAS (Mechanistic Investigation of Microbial Secondary Lipid Assembly) Polyunsaturated fatty acids (PUFAs) constitute an essential class of natural products relevant to human nutrition and drug discovery. Like fatty acids, peptides and alkaloids, PUFAs are secondary metabolites produced by both prokaryotes and eukaryotes. In bacteria, they modulate numerous cellular and physiological processes including cold adaption and survival, endo- and exocytosis and activities of membrane-associated enzymes. Large amounts of PUFAs can be found in the retina and the human brain where they play an important role in optimal functioning and development. PUFAs also serve as precursors to hormones and signalling molecules, such as prostaglandins, thromboxanes and leukotrienes: indeed it has been proven that an increased uptake of long chain omega-3 PUFAs supports cardiac health and prevents cardiovascular diseases. In essence PUFAs are of vital importance to any organism’s optimal physiological function and are highly sought compounds for the development of pharmaceuticals and nutraceuticals. The current major sources of PUFAs are oceanic (e.g. fish, algae and fish oil products); however, global warming, pollution and overfishing represent major threats to the present and the future availability of these molecules. Therefore, novel and sustainable sources are constantly sought after. In this research project, we aimed to investigate microbial ‘de novo’ secondary lipid biosynthesis catalysed by polyunsaturated fatty acid synthases (Pfa synthases). A more detailed understanding of the modus operandi of this enzyme class is critical in view of best ‘reprogramming’ microbial cell factories to produce secondary lipid metabolites of pharmaceutical and industrial interest. A putative Pfa synthase from Rhodoccocus erythropolis PR4, an alkane-degrading bacterium, was chosen as a possible model for the investigation of in vitro PUFA enzymology with the aid of chemical probes: these tools have been devised to capture PfA biosynthetic intermediates making them accessible for structural characterisation.In order to heterologously express the essential Pfa enzymes in E. coli for the in vitro reconstitution of PUFA biosynthesis, Rhodococcus erythropolis PR4 was obtained from the Biological Resource Centre at the National Institute of Technology and Evaluation (Japan) and grown under recommended conditions. The genomic DNA was isolated following a standard protocol and suitable primers were designed for the amplification of a putative PfA synthase and thioesterase/dehydratase/isomerase (TE/DH/IS). After optimisation of the PCR conditions, both the Pfa and TE genes were cloned into a pET28a (+) vector to obtain histidine-tagged proteins for purification by nickel affinity chromatography. The constructs were transformed into E. coli DH5α cells and afterwards isolated, purified, preliminarily confirmed by digestion and further verified by sequence analysis. For the expression of the target proteins in an active form, the constructs were separately transformed into E. coli BAP1 cells which harbour a phosphopantetheinyl transferase for the expression of acyl carrier protein in their holo form. Both enzymes (the Pfa synthase, of approximately 220 kDa in size, and the putative TE, of approximately 58 kDa) were successfully expressed in soluble form and purified by affinity chromatography. Further protein purification by size-exclusion chromatography yielded the proteins in high purity and moderated yields. The identity of the expressed enzymes was confirmed by SDS-PAGE and Western Blot analyses. The proteins were then tested in enzyme activity assays involving PfA synthase ‘priming’ and ‘processing’ with a variety of acyl CoA derivatives. The assays showed that the enzyme can be primed with different starter units, and that its ketoreductase domain is functional, however the processing of predicted malonyl CoA as extender units is not efficient. In order to shed further light on this, and attempt the capture of Pfa intermediates in vitro, a range of novel malonyl carba(dethia) N-decanoyl cysteamine probes were prepared. These were independently and successfully tested in vivo and in vitro on iterative polyketide synthases, with which Pfa synthases share similarity of mechanism. Preliminary in vitro assays with the recombinant Rhodococcus proteins were performed in the absence and in the presence of the newly synthesised chemical probes. Advanced HR-MS analysis of the enzymatic extracts showed the off-loading of starter units from the PfA synthase but no advanced intermediate capture. This is consistent with the inefficient processing of malonyl CoA, which could be due to incorrect folding of the recombinant PfA synthase, or other factors (e.g. lack of additional proteins, cofactors, ... in the assays, or no recognition of malonyl CoA as substrate). The preparation of a DNA construct for the co-expression of the PfA and the TE proteins in E.coli (by means of a pET-DueT vector) was commenced, however this was not completed due to time constraints. In summary, within this project the genes of a putative Pfa synthase and a putative thioesterase/dehydratase/isomerase from Rhodoccocus erythropolis were successfully cloned for the very first time and the enzymes could be expressed and purified for in vitro studies. Optimisation of protein expression and purification protocols to prevent degradation led to highly 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 PKS/Pfa intermediate capture were developed. All these results lay the foundations for further work towards the ultimate reconstitution of PUFA biosynthesis in vitro and its mechanistic dissection, which may ultimately lead to novel and ‘greener’ land-based productions of PUFAs and structurally related bioactive molecules.
