Transcription factor engineering for the enhancement in the production of carbon storage molecules in Chlamydomonas reinhardtii

Abstract

The depletion of fossil fuels and the problems of greenhouse gas pollution has pushed the development of alternative sustainable biofuels including from microalgae. Microalgae can produce high abundance of lipids and carbohydrates that can be utilised as a biofuel feedstock. However, improvements are required throughout the production process for microalgae biofuel to become commercially viable. One of the major bottlenecks is low metabolite productivity. Microalgae cultivation conditions such as nutrient starvation that can boost the accumulation and storage of lipids and carbohydrates in microalgae also compromise growth. To overcome this problem, genetic engineering may offer solutions to improve the production of these macromolecules without compromising growth. For genetic engineering to be successful, a better knowledge of the molecular mechanism involved in the biosynthesis of carbon storage molecules is required. Transcription factors (TF) are of interest as potential targets for genetic manipulation since their engineering could provide stronger up-regulation of metabolic pathways. In an effort to contribute to the identification of key TFs involved in carbon storage metabolism, this study focused on the characterization of two TFs from Chlamydomonas reinhardtii. PSR1 is a TF that has previously been identified as a regulator of lipid and starch metabolism, however, the exact mechanisms of regulation are unknown. By using the electrophoretic mobility assay (EMSA) I tested the ability of PSR1 to bind to the promotor region of three genes involved in the carbon storage metabolism. PSR1 was able to bind specifically to the regulatory elements in the promotors of these three genes. These results showed the DNA binding activity of PSR1 for the first time and indicates a mechanism though which PSR1 is regulating starch and lipid metabolism in C. reinhardtii. A previously uncharacterised TF, MYB2 was predicted from genomic data to be upregulated under different stress conditions. MYB2 showed close phylogenetic relationship and structural similarity with MYB-type TFs from Arabidopsis thaliana that are regulators of various environmental stress responses. To determine if MYB2 is involved in the regulation of starch and lipid metabolism, MYB2 overexpression and knockdown lines were generated and characterised. Surprisingly, both knockdown and overexpression lines showed similar phenotypes with regard to carbon storage content. Both sets of these MYB2 modified lines showed an increase in the lipid accumulation under stress condition, particularly under nitrogen (N) limitation where there was a more than 2 fold increase in lipid content when compared to control lines. These results suggest that MYB2 is involved in the regulation of carbon storage metabolism. However, its role appears to be more complex than expected and potential mechanisms of genetic robustness and compensation in its function are proposed and discussed. These results expose the complex regulatory networks involved in the regulation of the carbon storage metabolism in microalgae. In conclusion, results from this investigation have provided important insights into the role of two TFs involved in the lipid and starch biosynthesis, which could potentially be targets for genetic engineering for the enhancement of lipid and starch production in microalgae in the future.

Date of Award	1 Aug 2019
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Patrick Gallois (Supervisor) & Jon Pittman (Supervisor)