The oxygen-resistant hydrogenase of Thermotoga neapolitana: heterologous expression and perspectives for the production of bio-hydrogen from algae

One of the major challenges for our society is the development of new renewable energy sources to substitute fossil fuels, which are rapidly exhausting and whose extensive exploitation caused CO2 accumulation in the atmosphere and is probably at the origin of global climatic changes. In chapter 2 of this thesis, some of the proposed solutions to face this energetic crisis are described. Among these, today, hydrogen is considered a promising option because of its high energy content and its low environment impact, since its combustion generate electricity and produce water as the only waste. The possibility of a large scale exploitation of hydrogen as energy source, however, totally depends on the development of strategies for its large scale efficient production, which are still missing. One emerging possibility is the use of biological systems: in fact, several living organisms (bacteria or green algae) are able to develop this gas in anaerobic conditions during their metabolism. The exploitation of photosynthetic organisms, as the green alga Chlamydomonas reinhardtii, is particularly interesting because here hydrogen is produced starting from two abundant natural sources: water and light. Current knowledge about hydrogen metabolism in Chlamydomonas reinhardtii and strategies attempted to face some problems for a large scale production are described in details in chapter 1. In living organisms H2 is produced by a particular class of enzymes named hydrogenases. Chapter 1 also describes principal properties of these proteins, in particular the geometry of the catalytic site and the accessory domains that have been resolved by X-ray crystallography. These studies revealed a property of these enzymes that, in an applicative point of view, is clearly limiting: a high sensitivity to even very low oxygen concentrations. This lack of enzymatic activity in aerobic conditions is especially limiting in the case of photosynthetic organisms which evolve oxygen during photosynthesis. In nature, therefore, O2 and H2 productions in Chlamydomonas never occur at the same time. In a large scale production, however, a simultaneous photo-production of both gases would have a far higher yield. Nevertheless this ideal objective is still limited by the unavailability of an O2 insensitive hydrogenase. Until now, a H2 evolution in micro-aerobic conditions (6-8%) has been demonstrated only for the hyperthermophilic bacterium Thermotoga neapolitana. Its hydrogenase however, has never been isolated and any biochemical or structural studies is available; therefore, basis of its tolerance are still completely unknown. In chapter 3 of this thesis we describe the analysis of a T. neapolitana genome portion in which we identified the hydrogenase genes as part of an operon containing 4 additional ORFs (Open Reading Frame) and the results obtained through the heterologous expression of these proteins in E. coli. In this work we thus expressed, characterized and in some cases purified nine new proteins that are probably involved in this oxygen tolerant H2 evolving process. Finally, for the first time we could purify the T. neapolitana hydrogenase correctly folded. The long term aim of this study, conduced on isolated proteins, is the insertion of T. neapolitana hydrogenase in Chlamydomonas in order to obtain an organism able to photo-produce hydrogen also in aerobic conditions. According to several advantages, described in detail in chapter 4, the most promising strategy to express heterologous genes in Chlamydomonas is the chloroplastic transformation. This chapter also contains the description of a new vector for the tomato chloroplast transformation developed in our laboratory, as well as our experiments for the production of a cholera edible vaccine in tomato.