Secondary supervisor: Professor Dylan Owen
PhD project title: Metabolic engineering of yeast species for medium-chain fatty acid production
University of Registration: University of Birmingham
Schizosaccharomyces japonicus (S. japonicus) is a relative of well-studied cell biology yeast model organism Schizosaccharomyces pombe (S. pombe). These two organisms belong to a small monophyletic clade at the base of the Ascomycete phylogenetic tree, with S. japonicus forming an early diverging branch with respect to the other Schizosaccharomyces species. In spite of their kinship these species have evolved markedly distinct strategies of managing several cell biological processes linked to membrane function and the two sister species exhibit profound differences in the composition of phospholipid fatty acyl (FA) chains. The S. pombe membrane lipidome is similar to that of budding yeast and to many other eukaryotes, where most of the phospholipid FA chains contain either sixteen (C16) or eighteen (C18) carbon atoms with various number of double bonds. Remarkably, a large fraction of S. japonicus phospholipids (both structural and storage triacyl glycerides) contain medium-length C10 and C12 chains along with the C16 and C18 species. Medium chain fatty acids (MCFAs) are commercially valuable for various industries including food production, detergents and cosmetics. Since the main source for commercially available MCFAs and MCFA-containing triacyl glycerides are coconut and palm oil, it’s important to search for sustainable alternatives. In this light, developing S. japonicus as a new model organism with a potential for commercial usage might be of a great interest.
Aims and methods:
Firstly, we will investigate the molecular machinery responsible for the incorporation of MCFAs into phospholipids. We hypothesise that enzymes catalysing acylation in S. japonicus have evolved different specificities to incorporate two distinct fatty acid lengths. We will knockout candidate genes encoding a range of acyltransferases and assess the generated strains by lipid mass spectrometry. This will allow us to probe the rules for enhanced MCFA incorporation into phospholipids and storage lipids and facilitate the generation of tailored acyl chain compositions.
Secondly, we will elucidate mechanisms of fatty acid degradation in both fission yeast species. We will generate knockout mutants along with retro-engineered interspecies swaps to address the role of divergence in processing distinct length fatty acids. Generated mutants will be assessed by lipid mass spectrometry approaches. To further elucidate mechanisms of lipid degradation, we will generate strains where enzymes are tagged with fluorescent proteins in order to visualise their sub-cellular localisation and co-localisation with cellular organelles.
Finally, we will investigate the main pathways of phospholipid biosynthesis in S. japonicus to generate strains with extreme oil accumulation capacities. Genetic modifications of phospholipid biosynthesis and development of genetically encoded medium chain fatty acid biosensors will allow us to test multiple backgrounds and conditions for tailored fatty acid production.
This complex mapping of the enzymatic pathways involved in phospholipid production and degradation will allow us to engineer optimal genetic background of S. japonicus cells for the further optimizations in various growth conditions utilising different energy sources. Ultimately, we will aim to find a combination of modifications to the culture conditions and the regulated expression of key enzymes (native or exogenous) to produce specific lipid compounds with added value and of potential interest in the oleochemical and petrochemical field.
BBSRC Strategic Research Priority: Renewable Resources and Clean Growth: Industrial Biotechnology
Techniques that will be undertaken during the project:
- Molecular biology, cloning, generating genetically modified yeast strains
- Various methods for lipid mass spectrometry
- Advanced fluorescence microscopy
Contact: Dr Maria Makarova, University of Birmingham