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Protein engineering of hydrogenase 3 to enhance hydrogen production

  • Biotechnologically Relevant Enzymes and Proteins
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Abstract

The large subunit (HycE, 569 amino acids) of Escherichia coli hydrogenase 3 produces hydrogen from formate via its Ni–Fe-binding site. In this paper, we engineered HycE for enhanced hydrogen production by an error-prone polymerase chain reaction (epPCR) using a host that lacked hydrogenase activity via the hyaB hybC hycE mutations. Seven enhanced HycE variants were obtained with a novel chemochromic membrane screen that directly detected hydrogen from individual colonies. The best epPCR variant contained eight mutations (S2T, Y50F, I171T, A291V, T366S, V433L, M444I, and L523Q) and had 17-fold higher hydrogen-producing activity than wild-type HycE. In addition, this variant had eightfold higher hydrogen yield from formate compared to wild-type HycE. Deoxyribonucleic acid shuffling using the three most-active HycE variants created a variant that has 23-fold higher hydrogen production and ninefold higher yield on formate due to a 74-amino acid carboxy-terminal truncation. Saturation mutagenesis at T366 of HycE also led to increased hydrogen production via a truncation at this position; hence, 204 amino acids at the carboxy terminus may be deleted to increase hydrogen production by 30-fold. This is the first random protein engineering of a hydrogenase.

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Acknowledgments

The authors are grateful for the Keio clones sent by the National of Institute of Genetics (Japan), for the chemochromic membrane sensor provided by Dr. Hilton G. Pryce Lewis of GVD, and for protein modeling provided by Dr. Thammajun Leungsakul. This research was supported by DARPA (HR0011-06-1-0001).

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Authors and Affiliations

  1. Artie McFerrin Department of Chemical Engineering, Texas A & M University, 200 Jack E. Brown Building, College Station, TX, 77843-3122, USA

    Toshinari Maeda, Viviana Sanchez-Torres & Thomas K. Wood

  2. Department of Biology, Texas A & M University, College Station, TX, 77843-3258, USA

    Thomas K. Wood

  3. Zachry Department of Civil and Environmental Engineering, Texas A & M University, College Station, TX, 77843-3136, USA

    Thomas K. Wood

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  1. Toshinari Maeda
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  2. Viviana Sanchez-Torres
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Correspondence to Thomas K. Wood.

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Maeda, T., Sanchez-Torres, V. & Wood, T.K. Protein engineering of hydrogenase 3 to enhance hydrogen production. Appl Microbiol Biotechnol 79, 77–86 (2008). https://doi.org/10.1007/s00253-008-1416-3

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  • DOI: https://doi.org/10.1007/s00253-008-1416-3

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