The availability of a Chlamydomonas Reinhardtii strain without a nuclear hydrogenase gene has opened the door for exploration of the Hyd gene in the chloroplast. Biolistics and homologous recombination provide a well understood tool for integrating recombinant genetic material into the C. Reinhardtii chloroplast. The effects of different 5’- and 3’-untranslated regions on hydrogen production catalyzed by a eukaryotic hydrogenase will be measured.
With the reduced stability of fossil fuel supplies and prices, interest in transportation fuels based on biological sources has been renewed. While terrestrial sources (e.g. corn ethanol and soybean biodiesel) currently replace a small fraction of fossil fuels, these fuel sources compete with agriculture for food. Aquatic phototrophs (algae and seaweed) are able to flourish in conditions unsuitable for agriculture, including saltwater, and may be easier to process into a usable fuel product.[12, 3, 6] So far, algae have been investigated for the production of lipids for biodiesel, starches for ethanol production, and direct production of hydrogen gas.[9, 2]
The natural propensity of certain algae to produce hydrogen gas under anaerobic condi- tions has lead to interest in optimizing the metabolic processes involved, such that algae could be made to efficiently convert water into hydrogen gas[9, 2] (in contrast to photovoltaic sys- tems, which involve expensive solar panels and inefficient electrolytic processes). The purpose of this project is to study the effects of untranslated DNA regions related to the hydrogenase enzyme on hydrogen production in Chlamydomonas Reinhardtii.
2 Background 2.1 Chlamydomonas Reinhardtii
Chlamydomonas Reinhardtii has become a model cell for the study of photosynthetic and metabolic processes in monocellular phototrophs. The abundance of research, availability of a
published genome, and established culture and transformation protocols provide a foundation for exploration in metabolism, and simplify the development of novel genetic methods. C. Reinhardtii has been extensively studied in the area of biofuels, as well as the production of pharmaceutical products for human consumption, as complex proteins may be generated and assembled in the chloroplast, which shares many characteristics with eukaryotic cells in terms of transcription and protein assembly. Thus, a preponderance of research exists regarding the transformation of both the Chlamydomonas nucleus and chloroplast.
2.2 Hydrogen Production
In the chloroplast’s photosynthetic process, high-energy electrons are transferred via the pro- tein ferredoxin (FD). Ferredoxin fuels many of the chloroplast’s metabolic processes via the protein FNR (ferredoxin NADP+ oxidoreductase), which reduces NADP+ to NADPH. Under anaerobic conditions, FD may also transfer electrons to hydrogenase (HYD), which uses protons as an alternative electron sink, and produces H2 as a product.[9, 12, 2]