The primary producers of essential omega-3 polyunsaturated fatty acids are marine microalgae, which form the base of the aquatic food web. One alternative source of our ever diminishing stocks of fish and fish oil is via the cultivation of these microorganisms. Unfortunately, these microalgae, of which diatoms are the dominant class, only accumulate oil during specific stages of their life-cycle and/or under nutritional states which are incompatible with the required high density of growth and target fatty acid profile. Additionally, the endogenous levels of desirable fatty acids such as eicospantaenoic acid (EPA; 20:5, n-3) and docosahexaenoic acid (DHA; 22:6, n-3) are usually relatively modest (in the range of 10-35% of total fatty acids) and therefore present an opportunity for enhancement. A database search carried out on the genomes of omega-3-producing unicellular photoautotrophic green alga Ostreococcus sp. RCC809 and cold-water diatom Fragilariopsis cylindrus led to the identification of four novel genes involved in omega-3 biosynthesis. These genes encoded an omega-3-specific Δ6-desaturase, a Δ4-desaturase, a Δ6-elongase and a Δ5- elongase. Overexpression of genes encoding Δ6-desaturase and Δ6-elongase activities in Thalassiosira pseudonana impacted the fatty acid and acyl-CoA profiles of this model centric diatom. Changes to chloroplast and lipid droplet phenotype were also observed. Targeted knock-down of native genes involved in the omega-3 biosynthetic pathway was carried out in T. pseudonana to further understand endogenous omega-3 fatty acid production. Cells targeted for the knock-down of Δ9-desaturase exhibited a drastically altered growth phenotype, but maintained a wild type-like fatty acid profile. This phenotype was attributed to the possibility of another, functionally redundant, protein that escaped sequence-based silencing, masking the knock-down of Δ9-desaturase. The results and observations provided in this thesis contribute new valuable information to the field of lipidomic research in microalgae, breaking new ground in metabolic engineering of lipid metabolism in diatoms.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:656120 |
| Date | January 2015 |
| Creators | Vaezi, Royah |
| Publisher | University of East Anglia |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | https://ueaeprints.uea.ac.uk/53379/ |
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