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Title: Metabolic engineering of the algal chloroplast for terpenoid production
Author: Al Hoqani, U. H. A.
ISNI:       0000 0004 7225 3058
Awarding Body: UCL (University College London)
Current Institution: University College London (University of London)
Date of Award: 2017
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Microalgal biotechnology has attracted considerable interest owing to it is potential to provide renewable energy and its capacity to produce molecules such as pigments, fatty acids and other high value compounds, which can be used in the biomaterials, cosmetics and pharmaceutical industries. One class of compounds are the terpenoids: a diverse group of molecules derived from C5 isoprene units that are exploited for their aromatic and bioactive properties. Terpenoid production in microalgae offers an alternative to extraction from plant species or chemical synthesis. However, metabolic engineering technology for microalgae is still in its infancy and far from economic viability. Thus, the aim of this study was to develop engineering tools for the industrial algal species Nannochloropsis gaditana, with the goal of manipulating the main terpenoid pathway located in the chloroplast. In parallel, the effects of such manipulation was studied using the laboratory species Chlamydomonas reinhardtii, for which chloroplast genetic engineering is already established. N. gaditana is a robust marine species well suited to industrial scale cultivation. The availability of a draft genomic sequence, nuclear transformation methodology and a high lipid productivity have positioned N. gaditana as a promising oleaginous alga for metabolic engineering. However, to develop it as an industrially relevant platform, further molecular tools are needed; in particular a reliable chloroplast transformation method. Thus, the aim of the first project was to develop chloroplast transformation for the alga. This involved optimizing the cultivation conditions for N. gaditana, evaluating its sensitivity to herbicides and chloroplast specific compounds in order to identify suitable selectable markers, and to construct chloroplast transformation vectors. In addition, the temporary increase in cell size by inhibition of cytokinesis was investigated in order to facilitate the delivery of DNA into the small chloroplast. C. reinhardtii is the most developed algal model with well-established tools for genetic manipulation, and can be used to study the effect of chloroplast metabolic engineering in other species such as Nannochloropsis. Thus, the second project focused on the manipulation of the terpenoid biosynthetic pathway: specifically, the chloroplast-localized methylerythritol phosphate pathway by over-expressing the rate limiting enzyme; 1-deoxy-D-xylulose-5-phosphate synthase (DXS). An additional dxs gene from the cyanobacterium Synechocystis 6803 was introduced into the chloroplast genome in the hope of improving the productivity of downstream terpenoid metabolites. A number of transgenic lines were obtained and the successful integration was confirmed by molecular analysis. The effects of up-regulating DXS enzyme activity on overall algal growth and terpenoid profile are studied.
Supervisor: Purton, S. Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available