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Title: Molecular tools and approaches for increasing complexity of transplastomic engineering in Chlamydomonas reinhardtii
Author: Larrea-Alvarez, Marco
ISNI:       0000 0004 7232 1241
Awarding Body: UCL (University College London)
Current Institution: University College London (University of London)
Date of Award: 2018
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Microalgae have been grown commercially for many years as a feedstock for aquaculture, and as a source of natural products such as pigments and omega-3 oils. More recently, the focus has turned to the development of microalgae as industrial biotechnological platforms, and several species are being explored as possible cell factories. One promising species is the green alga Chlamydmonas reinhardtii, which has been used extensively as a lab- oratory model for basic genetic and biochemistry studies. The repertoire of molecular and ’omics tools available for its genetic manipulation has in- creased significantly in recent years, and there have been numerous reports of recombinant proteins produced successfully in this alga. However, examples of metabolic engineering are limited, and this reflects the more challeng- ing problem of introducing multiple transgenes, and also regulating their expression so that biomass production and redirecting metabolic flux to- wards a desired product can be temporally separated. The algal chloroplast is a unique sub-cellular compartment with its own genetic system, which houses core metabolic pathways for carbohydrates, fatty acids, terpenoids and tetrapyrroles. It is therefore an attractive site for metabolic engineering. Modification of the C. reinhardtii chloroplast genome is routine and many single proteins have been synthesised in the organelle. Thus, the challenge is to increase the complexity of chloroplast engineering to allow sophisticated metabolic engineering. To contribute to this goal, my study has focused on several aspects: i) the simultaneous expression of multiple transgenes; ii) possible novel metabolism that could be introduced into the organelle: iii) the development of a novel fluorescence reporter for assessing transgene regulation. For the first project, a combinatorial/serial approach where var- ious expression cassettes were combined into two transformation plasmids, was demonstrated to be efficient for expressing simultaneously three recombinant proteins. In the second project, nitrogen fixation was investigated as a potential metabolic pathway. The advantages of the organelle to meet the nitrogenase enzymes requirements are discussed; furthermore, the expression of nifV from a suggested minimal set of nif genes, is demonstrated. Finally, the expression of a cyan fluoresecent reporter (mTurq2cp) was achieved. Emission at the expected wavelength was detected, and the fold increase in fluorescence above the background was determined. The presented results help to enlarge the repertoire of molecular tools and know-how available for chloroplast engineering in C. reinhardtii.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available