Spencer-Milnes, Xenia; (2019) Towards engineering the microalga Chlorella sorokiniana for the production of tailored high-value oils. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

This project explores the production of LC-PUFAs, such as the nutritionally relevant omega-3 fatty acids EPA and DHA, in the freshwater microalga Chlorella sorokiniana (UTEX 1230). C. sorokiniana is of interest due to its rapid growth rate, tolerance to high light and temperature, and ability to accumulate a large proportion of cell weight as lipids. Since wild-type C. sorokiniana terminates fatty acid synthesis at ALA (C18:3n3), a genetic engineering approach is required to produce LC-PUFAs. Changes in FAME and lipid content in different growth conditions including carbon source, nitrogen source, and trophic state were assessed in this microalga. These investigations confirmed the common finding that nitrogen stress is a robust way to induce neutral lipid accumulation, but also highlighted the potential of pH change as an alternate stressor for TAG production. To facilitate the heterologous gene expression needed to increase the range of fatty-acids produced by C. sorokiniana, this work develops and begins to characterise a toolbox of genetic parts for nuclear transformation. This combinatorial parts library, compatible with a standard cloning syntax to enable sharing between groups, comprises five coding sequences, four promoter/5’UTRs and four 3’UTR/terminators. The coding sequences include two antibiotic reporters, two fluorescent reporters and one lipid gene. Among the regulatory sequences, two were novel putative promoters from chlorovirus, as identified from detailed bioinformatics analysis of published transcriptomic and genomic data. An attempt to improve an existing Agrobacterium-mediated transformation strategy was utilised to validate some of the parts within the library. Preliminary evidence suggests the putative chlorovirus promoters may be active within both C. sorokiniana and Chlamydomonas reinhardtii. Challenges with appropriate selectable markers and transformation efficiency highlight the difficulty in genetic engineering of microalgal strains, especially where these tools are not well established, but also showcase the pressing need for such foundational research to be conducted.

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