title: The dual effect of a ferredoxin-hydrogenase fusion protein in vivo: successful divergence of the photosynthetic electron flux towards hydrogen production and elevated oxygen tolerance.
abstract: BACKGROUND
  Hydrogen photo-production in green algae, catalyzed by the enzyme [FeFe]-hydrogenase (HydA), is considered a promising source of renewable clean energy. Yet, a significant increase in hydrogen production efficiency is necessary for industrial scale-up. We have previously shown that a major challenge to be resolved is the inferior competitiveness of HydA with NADPH production, catalyzed by ferredoxin-NADP(+)-reductase (FNR). In this work, we explored the in vivo hydrogen production efficiency of Fd-HydA, where the electron donor ferredoxin (Fd) is fused to HydA and expressed in the model organism Chlamydomonas reinhardtii.
  
  
  RESULTS
  We show that once the Fd-HydA fusion gene is expressed in micro-algal cells of C. reinhardtii, the fusion enzyme is able to intercept photosynthetic electrons and use them for efficient hydrogen production, thus supporting the previous observations made in vitro. We found that Fd-HydA has a ~4.5-fold greater photosynthetic hydrogen production rate standardized for hydrogenase amount (PHPRH) than that of the native HydA in vivo. Furthermore, we provide evidence suggesting that the fusion protein is more resistant to oxygen than the native HydA.
  
  
  CONCLUSIONS
  The in vivo photosynthetic activity of the Fd-HydA enzyme surpasses that of the native HydA and shows higher oxygen tolerance. Therefore, our results provide a solid platform for further engineering efforts towards efficient hydrogen production in microalgae through the expression of synthetic enzymes.
authors: Department of Molecular Biology and Ecology of Plants, The George S. Wise Faculty of Life Science, Tel Aviv University, Ramat Aviv, 69978 Tel Aviv, Israel.
authors: Haviva
authors: H
authors: Eilenberg
authors: Department of Molecular Biology and Ecology of Plants, The George S. Wise Faculty of Life Science, Tel Aviv University, Ramat Aviv, 69978 Tel Aviv, Israel.
authors: Iddo
authors: I
authors: Weiner
authors: Department of Molecular Biology and Ecology of Plants, The George S. Wise Faculty of Life Science, Tel Aviv University, Ramat Aviv, 69978 Tel Aviv, Israel.
authors: Oren
authors: O
authors: Ben-Zvi
authors: Department of Molecular Biology and Ecology of Plants, The George S. Wise Faculty of Life Science, Tel Aviv University, Ramat Aviv, 69978 Tel Aviv, Israel.
authors: Carmel
authors: C
authors: Pundak
authors: Department of Molecular Biology and Ecology of Plants, The George S. Wise Faculty of Life Science, Tel Aviv University, Ramat Aviv, 69978 Tel Aviv, Israel.
authors: Abigail
authors: A
authors: Marmari
authors: Department of Molecular Biology and Ecology of Plants, The George S. Wise Faculty of Life Science, Tel Aviv University, Ramat Aviv, 69978 Tel Aviv, Israel.
authors: Oded
authors: O
authors: Liran
authors: GeneBiologics, LLC, Boulder, CO USA.|National Renewable Energy Laboratory, Golden, CO USA.
authors: Matt S
authors: MS
authors: Wecker
authors: Department of Molecular Biology and Ecology of Plants, The George S. Wise Faculty of Life Science, Tel Aviv University, Ramat Aviv, 69978 Tel Aviv, Israel.
authors: Yuval
authors: Y
authors: Milrad
authors: Department of Molecular Biology and Ecology of Plants, The George S. Wise Faculty of Life Science, Tel Aviv University, Ramat Aviv, 69978 Tel Aviv, Israel.
authors: Iftach
authors: 0000-0003-0177-0624
authors: I
authors: Yacoby
keywords: Chlamydomonas reinhardtii; Ferredoxin; Fusion enzyme; H2 production; Hydrogenase; Oxygen sensitivity