Directed evolution for optimization of industrially-relevant protists

Thraustochytrids are being recognized as important producers of omega-3 polyunsaturated fatty acids (PUFAs), particularly docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), which have proven to have various beneficial effects on animal and human health [1]. The consumption of DHA especially is shown to improve brain and heart functions, as well as exhibiting positive effects on the development of both retinal and immune systems and helping in the prevention of cardiovascular diseases, by functioning as a key factor in membrane fluidity, cell interactions and cell signalling. Therefore, thraustochytrids are emerging as a sustainable alternative in human and animal feeds, especially in the aquaculture industry. Their vast biotechnological potential lies, as well, in their ability to produce other bioactive compounds such as carotenoid pigments, squalene, exopolysaccharides and extracellular enzymes.
This project would aim to exploit this biotechnological potential to produce industrially relevant compounds by enhancing the phenotype of these protists through the process of directed evolution. Adaptive laboratory evolution (ALE) has so far proven to be effective in strain optimization of bacteria, yeast and microalgae under chosen evolutionary pressures [2]. Some of the potential biotechnological applications for this method include improving biomass production, enhancing tolerance of strains to stresses that generally occur in industrial processing, inducing activation of latent pathways to improve product tolerance and production of non-native compounds, as well as identifying essential genetic bases of strain adaptation [3]. In contrast to genetic engineering, directed evolution strategies allow multiple beneficial mutations to occur in various genes and regulatory gene networks at a time. Additionally, depending on the selection pressure, ALE can mediate many different evolutionary trajectories [3].
The project itself is expected to have numerous outcomes depending on the chosen stress factors and the environment of experiments. Molecular biology tools will be used to characterize chosen strains biochemically and to shed light on various metabolic pathways and how they interconnect to produce a desired compound. Next-generation sequencing technology and transcriptome analysis will be used to analyse the expression of key genes involved in fatty acid production. The project will examine the diversity of polyunsaturated fatty acid metabolism pathways and aim to develop a mutant strain with increased production of omega-3 PUFAs. To facilitate the ALE experiments, screening will have to be optimized to identify variants with desired function. Additionally, growth characteristics, quantification of fatty acids and chemical composition analysis of the evolved thraustochytrid strains will be studied throughout by using analytical chemical methods including gas chromatography and Nile red lipid visualization combined with light microscopy.
The project is in collaboration with MiAlgae Ltd., a start-up company that cultivates Omega-3 rich microalgae by recycling industrial co-products, in specially-designed fermenting vessels tailored for optimal microalgal growth. By producing algal oil with proven human and animal health benefits, MiAlgae contribute to decreasing global over-reliance on fish as a source of Omega-3 PUFAs for human and animal consumption. As the project is in collaboration with an industrial partner, selected mutant strains could be industrially relevant.