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Rubisco mutants of Chlamydomonas reinhardtii enhance photosynthetic hydrogen production

  • Bioenergy and biofuels
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Abstract

Molecular hydrogen (H2) is an ideal fuel characterized by high enthalpy change and lack of greenhouse effects. This biofuel can be released by microalgae via reduction of protons to molecular hydrogen catalyzed by hydrogenases. The main competitor for the reducing power required by the hydrogenases is the Calvin cycle, and rubisco plays a key role therein. Engineered Chlamydomonas with reduced rubisco levels, activity and stability was used as the basis of this research effort aimed at increasing hydrogen production. Biochemical monitoring in such metabolically engineered mutant cells proceeded in Tris/acetate/phosphate culture medium with S-depletion or repletion, both under hypoxia. Photosynthetic activity, maximum photochemical efficiency, chlorophyll and protein levels were all measured. In addition, expression of rubisco, hydrogenase, D1 and Lhcb were investigated, and H2 was quantified. At the beginning of the experiments, rubisco increased followed by intense degradation. Lhcb proteins exhibited monomeric isoforms during the first 24 to 48 h, and D1 displayed sensitivity under S-depletion. Rubisco mutants exhibited a significant decrease in O2 evolution compared with the control. Although the S-depleted medium was much more suitable than its complete counterpart for H2 production, hydrogen release was observed also in sealed S-repleted cultures of rubisco mutated cells under low-moderate light conditions. In particular, the rubisco mutant Y67A accounted for 10–15-fold higher hydrogen production than the wild type under the same conditions and also displayed divergent metabolic parameters. These results indicate that rubisco is a promising target for improving hydrogen production rates in engineered microalgae.

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Acknowledgments

We thank Sónia R. Vieira for the technical assistance on the last chlorophyll fluorescence measurements. This work was supported by project MICROPHYTE (PTDC/EBB-EBI/102728/2008), funded by FCT (Fundação para a Ciência e Tecnologia, Portugal) and supervised by F. X. Malcata.

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Authors and Affiliations

  1. Instituto Superior de Agronomia (ISA), Centro de Botânica Aplicada à Agricultura (CBAA), Calçada da Tapada, 1349-017, Lisbon, Portugal

    T. S. Pinto & M. G. Esquível

  2. Department of Chemical Engineering, University of Porto, Rua Dr. Roberto Frias, 4200-465, Porto, Portugal

    F. X. Malcata

  3. Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Avenida da República, 2780-157, Oeiras, Portugal

    F. X. Malcata

  4. BioFIG, Centro de Biodiversidade, Genómica Integrativa e Funcional and Departamento de Biologia Vegetal, Faculdade de Ciências da Universidade de Lisboa, 1749-016, Lisbon, Portugal

    J. D. Arrabaça & J. M. Silva

  5. Department of Biochemistry, University of Nebraska, Lincoln, NE, 68588, USA

    R. J. Spreitzer

Authors
  1. T. S. Pinto
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  2. F. X. Malcata
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  3. J. D. Arrabaça
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  4. J. M. Silva
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  5. R. J. Spreitzer
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  6. M. G. Esquível
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Corresponding author

Correspondence to M. G. Esquível.

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Pinto, T.S., Malcata, F.X., Arrabaça, J.D. et al. Rubisco mutants of Chlamydomonas reinhardtii enhance photosynthetic hydrogen production. Appl Microbiol Biotechnol 97, 5635–5643 (2013). https://doi.org/10.1007/s00253-013-4920-z

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  • DOI: https://doi.org/10.1007/s00253-013-4920-z

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