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J. Biol. Chem., Vol. 280, Issue 40, 34170-34177, October 7, 2005

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Improved Photobiological H₂ Production in Engineered Green Algal Cells^*

Olaf Kruse1, Jens Rupprecht, Klaus-Peter Bader, Skye Thomas-Hall¶, Peer Martin Schenk¶, Giovanni Finazzi||, and Ben Hankamer

From the Department of Biology VIII, Molecular Cell Physiology, University Bielefeld, 33501 Bielefeld, Germany, ^||UPR 1261-CNRS Institut de Biologie Physico-Chimique, 75005 Paris, France, Institute for Molecular Bioscience, University of Queensland, St. Lucia Campus, Queensland 4072, Australia, and ^¶Faculty of Biological and Chemical Sciences, University of Queensland, Queensland 4072, Australia

Oxygenic photosynthetic organisms use solar energy to split water (H₂O) into protons (H⁺), electrons (e^-), and oxygen. A select group of photosynthetic microorganisms, including the green alga Chlamydomonas reinhardtii, has evolved the additional ability to redirect the derived H⁺ and e^- to drive hydrogen (H₂) production via the chloroplast hydrogenases HydA1 and A2 (H₂ ase). This process occurs under anaerobic conditions and provides a biological basis for solar-driven H₂ production. However, its relatively poor yield is a major limitation for the economic viability of this process. To improve H₂ production in Chlamydomonas, we have developed a new approach to increase H⁺ and e^- supply to the hydrogenases. In a first step, mutants blocked in the state 1 transition were selected. These mutants are inhibited in cyclic e^- transfer around photosystem I, eliminating possible competition for e^- with H₂ase. Selected strains were further screened for increased H₂ production rates, leading to the isolation of Stm6. This strain has a modified respiratory metabolism, providing it with two additional important properties as follows: large starch reserves (i.e. enhanced substrate availability), and a low dissolved O₂ concentration (40% of the wild type (WT)), resulting in reduced inhibition of H₂ase activation. The H₂ production rates of Stm6 were 5-13 times that of the control WT strain over a range of conditions (light intensity, culture time, ± uncoupler). Typically, 540 ml of H₂ liter^-1 culture (up to 98% pure) were produced over a 10-14-day period at a maximal rate of 4 ml h^-1 (efficiency = 5 times the WT). Stm6 therefore represents an important step toward the development of future solar-powered H₂ production systems.

Received for publication, April 8, 2005 , and in revised form, August 12, 2005.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EBIData Bank with accession number(s) AF531421.

^* This work was supported by Deutsche Forschungsgemeinschaft Grant DFG-FOR387, the University of Bielefeld (to O. K), and the University of Queensland (to B. H. and J. R.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed. Tel.: 49-521-1065611; Fax: 49-521-1066410; E-mail: olaf.kruse{at}uni-bielefeld.de.

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