As the global population grows, there is increasing concern over the future costs and availability of essential resources such as food, drinking water and energy. Fuel is a particularly vital resource, accounting for 80% of the global energy usage, and is used for the transport of people, the production and distribution of food and broadly throughout industry. Oil and gas are the predominant transport fuels, however, the discovery of new reserves is dropping, while demand is increasing. More critically, fossil fuels are major contributors to greenhouse gas emissions. Scalable, renewable, CO2-neutral fuels are therefore urgently required to protect against climate change and increase energy security. Microalgae provide a solution, as they can tap into the vast supply of solar energy (1300 ZJ yr-1 of photosynthetically active radiation) and use it to drive photosynthesis to produce fuel, food and renewable chemical feedstocks.
Green microalgae are attractive biofuel feedstocks. They can be used to produce a wide range of biofuels, including bio-hydrogen, bio-ethanol, biodiesel, biomass-to-liquid and bio-methane. Importantly, microalgae assimilate and convert CO2, and can contribute to CO2 emission reduction strategies. The energy returned on energy invested of microalgae is projected to be sufficient to justify their scale-up to a significant percentage of global fuel use; the main limitation is cost competitiveness with existing fossil fuels. Specifically, low photon capture efficiency relative to infrastructure costs hinders the economic feasibility of current algal biofuels. Biotechnological improvements are needed to improve the economic viability of these systems before they can become feasible on a large scale.
Light capture is the first step of biofuel production and its optimisation is central to high efficiency process development. Genetic engineering of the light capturing machinery has already proven effective in the case of the green microalga and model organism, Chlamydomonas reinhardtii, where down-regulation of the light harvesting antenna complex (LHC) led to improved photosynthetic efficiencies and biomass production. To be most effective, however, genetic modification should be preceded by a detailed understanding of the pathways underlying photosynthesis and its two main regulatory goals: efficient light harvesting versus protection of the photosynthetic apparatus from light stress. Improving our understanding of the regulation of the LHC proteins is therefore essential.
This thesis explores the regulation of LHC gene expression in C. reinhardtii at both the transcriptional and translational level using two systems of current interest to the study of photosynthetic regulation. The transcriptional regulation of the PSII-associated LHCBM9 protein was examined through analysis of its promoter. LHCBM9 is of great interest because it is regulated differently from the other LHCII proteins and thus provides a window to a unique suite of signal transduction events. Its induction is also potentially linked to hydrogen production, which is theoretically the most efficient biofuel production method. Second, the structure of the N-terminal cold shock domain (CSD) of the LHC translational regulator, NAB1, was probed using nuclear magnetic resonance (NMR) spectroscopy. NAB1 is the only translational regulator of the photosynthetic apparatus described so far in C. reinhardtii, making its mechanism of action a key starting point for the study of photosynthetic regulation at this level.
1. LHCBM9 transcriptional regulation
In contrast to the other LHCII proteins and the majority of photosynthesis proteins, LHCBM9 is induced under sulfur deprivation conditions. Promoter deletion analysis was used to identify a 264 bp region between +54 and -209 of the transcription start site that was essential for S‑responsiveness. Electrophoretic mobility shift assays (EMSA) were then used to screen the +54 to -117 region for the exact binding motif. Differences were seen in the banding patterns of the +S and –S EMSA, confirming that this promoter region is responsive to changing S levels, although specific cis-elements remain to be identified. The LHCBM9 promoter was also tested for its responsiveness to anaerobiosis, light, heat, reactive oxygen species, nitrogen-, phosphorus‑, copper- and calcium-deprivation. Light and anaerobiosis were found to induce LHCBM9 gene expression, but only in combination with S deprivation, indicating that S is the main transcriptional regulator of LHCBM9 but that the promoter is also responsive to other stimuli.
2. LHCBM4 and LHCBM6 translational regulation
The RNA-binding protein, NAB1, is a translational repressor of LHCBM4 and LHCBM6. Loss of NAB1 function results in increased levels of LHC subunits and a 30‑40% increase in LHCII antenna size. NAB1 contains two putative RNA binding domains - an N-terminal CSD, which binds LHCBM4 and LHCBM6 mRNA in a sequence-specific manner, and a C‑terminal RNA recognition motif (RRM), thought to bind RNA non-specifically – separated by a 96 residue linker region that is predicted to be largely unstructured. Due to problems encountered previously with obtaining soluble, folded full-length protein, here, the CSD (residues 1 to 70) and RRM (residues 147 to 244) were expressed and purified separately. A CSD construct (CSD1) was purified with relatively high yields, allowing NMR experiments to be performed. RNA titration experiments employing the 15N-labelled protein confirmed the binding specificity of the CSD, while experiments with 15N/13C-labelled CSD1 were used to interrogate protein structure. More than 90% of the backbone resonances in the heteronuclear single-quantum correlation (HSQC) spectrum and 60% of the side chain resonances were sequence specifically assigned, which allowed the determination of a preliminary tertiary structure. The overall fold was a five-stranded β‑barrel, which was consistent with the consensus CSD fold.