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Engineering Escherichia coli for Fermentative Dihydrogen Production: Potential Role of NADH-Ferredoxin Oxidoreductase from the Hydrogenosome of Anaerobic Protozoa

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Abstract

Trichomonas vaginalis generates reduced ferredoxin within a unique subcellular organelle, hydrogenosome that is used as a reductant for H2 production. Pyruvate ferredoxin oxidoreductase and NADH dehydrogenase (NADH-DH) are the two enzymes catalyzing the production of reduced ferredoxin. The genes encoding the two subunits of NADH-DH were cloned and expressed in Escherichia coli. Kinetic properties of the recombinant heterodimer were similar to that of the native enzyme from the hydrogenosome. The recombinant holoenzyme contained 2.15 non-heme iron and 1.95 acid-labile sulfur atoms per heterodimer. The EPR spectrum of the dithionite-reduced protein revealed a [2Fe–2S] cluster with a rhombic symmetry of gxyz = 1.917, 1.951, and 2.009 corresponding to cluster N1a of the respiratory complex I. Based on the Fe content, absorption spectrum, and the EPR spectrum of the purified small subunit, the [2Fe–2S] cluster was located in the small subunit of the holoenzyme. This recombinant NADH-DH oxidized NADH and reduced low redox potential electron carriers, such as viologen dyes as well as Clostridium ferredoxin that can couple to hydrogenase for H2 production from NADH. These results show that this unique hydrogenosome NADH dehydrogenase with a critical role in H2 evolution in the hydrogenosome can be produced with near-native properties in E. coli for metabolic engineering of the bacterium towards developing a dark fermentation process for conversion of biomass-derived sugars to H2 as an energy source.

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Acknowledgements

We thank Dr. A. Rooney, USDA-ARS, for providing C. acetobutylicum strain. This work was supported in part by US Department of Energy grant DE-FG36-04GO14019 and funds from the Florida Agricultural Experiment Station. This work was also supported in part by the In-House Research Program of the National High Magnetic Field Laboratory (AA) and by a grant by the Grant Agency of the Czech Republic no. 204/06/0944 (IH). L.B. was supported by grant MSM0021620858.

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Authors and Affiliations

  1. Department of Microbiology and Cell Science, University of Florida, P.O. Box 110700, Gainesville, FL, 32611, USA

    Phi Minh Do, Lonnie O. Ingram & K. T. Shanmugam

  2. Department of Chemistry, University of Florida, Gainesville, FL, 32611, USA

    Alexander Angerhofer

  3. Department of Parasitology, Faculty of Science, Charles University, Prague, Czech Republic

    Ivan Hrdy & Lucie Bardonova

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  1. Phi Minh Do
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Correspondence to K. T. Shanmugam.

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Do, P.M., Angerhofer, A., Hrdy, I. et al. Engineering Escherichia coli for Fermentative Dihydrogen Production: Potential Role of NADH-Ferredoxin Oxidoreductase from the Hydrogenosome of Anaerobic Protozoa. Appl Biochem Biotechnol 153, 21–33 (2009). https://doi.org/10.1007/s12010-008-8508-5

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