Prolonged, alternating hydrogen and oxygen production cycles with the cy6Nac2 inducible chloroplast gene expression system developed for Chlamydomonas reinhardtii
Raymond Surzycki
Solarvest BioEnergy Inc., 6485 Gross Rd., Bloomington, IN 47401 USA
An inducible chloroplast gene expression system has been developed for Chlamydomonas reinhardtii by taking advantage of the properties of the copper-sensitive cytochrome c6 promoter and of the nucleus-encoded Nac2 chloroplast protein. This protein is specifically required for the stable accumulation of the chloroplast psbD RNA and acts on its 5'UTR. Our previous report demonstrated the potential of the system for hydrogen production when copper-starved cy6Nac2.49 cells grown in sealed vessel were repressed through the addition of copper. Because the Cyc6 promoter is also induced under anaerobic conditions, hydrogen production was transient, as psbD synthesis was re-initiated after a period of anaerobic growth. This property of the inducible system opened possibility for sustained cycling of hydrogen and oxygen production in a single vessel through a feedback loop mechanism that controls PSII synthesis through photosynthetic oxygen production.
We have developed an experimental platform designed to monitor the hydrogen production of the cy6Nac2 transgenic strain. The hydrogen production properties of the cy6Nac2 strain were followed in a sealed 10 L photo-bioreactor. Induction of Nac2 synthesis and hydrogen production was induced upon the onset of anaerobiosis, rather than copper starvation as we previously reported. Multiple, alternating periods of oxygen and hydrogen evolution were observed in cy6Nac2 transgenic strain. Because the method does not rely on physiological depletion of an important nutrient, these batch cultures can be sustained for weeks, rather than days. The method represents a significant technical improvement over the two-stage sulfur-starvation hydrogen production method, as the duration of hydrogen production, cell density and cell physiology are enhanced in the two-stage, single vessel hydrogen production system.
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