About the Project
Secondary lipid metabolites are specialised lipids produced in both prokaryotes and eukaryotes through biosynthetic pathways distinct from (or complementary to) that of primary fatty acids. These ‘accessory’ lipids are of vital importance for any organism’s optimal physiological functions.1 The most well-known products of this kind are polyunsaturated fatty acids (PUFAs) such as omega-3 and -6 fatty acids, which are essential membrane components and the precursors of many lipid-derived signalling molecules. Their benefit as nutraceuticals is well known and drives the high demand for these compounds. However their current major sources are still oceanic and threatened by pollution and climate change. Bacterial fermentation has recently emerged as a potentially novel sustainable source of secondary lipids,2-4 yet much remains unknown about the mechanistic details of their formation due to the lack of direct information on the nature and the processing of the biosynthetic intermediates. This lack of knowledge hampers the full utilisation of bacterial fermentation as a sustainable source of fatty acids.
The main aims of this project are 1) to dissect the mechanism of polyunsaturated fatty acid biosynthesis with the aid of chemical probes 5-8 and 2) to reconstitute and manipulate the activity of candidate polyunsaturated fatty acid synthases from Rhodococcus, Streptomyces and Frankia bacteria in tractable in vitro and in vivo systems. Specific research objectives for the project student will include: the cloning and the heterologous expression of candidate polyunsaturated fatty acid synthases in E.coli and Actinobacteria hosts; enzyme purification and activity reconstitution in vitro; where possible, enzyme structural characterisation by X-ray crystallography in collaboration with other research groups within and outside Warwick; in vivo genetic manipulation of bacteria; synthesis of chemical probes; the analytical characterisation of the lipid intermediates first by mass spectrometry and, if possible, by NMR.
The results from these experiments will provide a comprehensive picture of bacterial secondary lipid assembly, with important implications for the engineering of secondary lipid- producing bacteria.
BBSRC Strategic Research Priority: Renewable Resources and Clean Growth: Bio-energy & Industrial Biotechnology. Understanding the Rules of Life: Microbiology. Integrated Understanding of Health: Diet and Health & Pharmaceuticals
Techniques that will be undertaken during the project:
• Organic chemistry: synthesis, purification and characterisation of organic compounds;
• Microbiology: growing and handling of coli and Actinobacteria strains;
• Molecular biology: gene cloning and gene editing in Actinobacteria ;
• Protein chemistry and enzymology: protein expression in heterologous hosts, enzyme activity reconstitution and characterisation;
• Analytical chemistry: identification, isolation and characterisation of natural products and biosynthetic intermediates by LC-MS, HPLC, HR-MS, GC, NMR.
2. Jiang, H.; Rajski, S. R.; Shen, B. 2009, Method Enzymol., 459, 79-96.
3. Shulse, C. N.; Allen, E. E. 2011, PLoS ONE, 6, e20146.
4. Metz, J. G.; Roessler, P.; Facciotti, D.; Levering, C.; Dittrich, F.; Lassner, M.; Valentine, R.; Lardizabal, K.; Domergue, F.; Yamada, A.; Yazawa, K.; Knauf, V.; Browse, J. 2001, Science, 293, 290- 293.
5. Riva, E.; Wilkening, I.; Gazzola, S.; Li, A.; Smith, L.; Leadlay, P. F. L.; Tosin, M., Chem. Int. Ed. 2014, 53, 11944-9 (VIP article).
6. Parascandolo, J. S.; Havemann; Potter, H. K. P.; Huang, F.; Riva, E.; Connolly, J.; Wilkening, I.; Song, L.; Leadlay, P. F. L.; Tosin, M., Chem. Int. Ed. 2016, 55, 3463-3467.
7. Wilkening, I.; Gazzola, S.; Riva, E.; Parascandolo, J. S.; Song, L.; Tosin, M., Commun. 2016, 52, 10392-10395.
8. Kilgour, S. L.; Kilgour, D. P. A.; Prasongpholchai, P; O’ Connor, P. B. O.; Tosin, M. Eur. J. 2019 (VIP article), 25, 16515-16518.
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