Applied Microbiology and Biotechnology

, Volume 79, Issue 1, pp 77–86 | Cite as

Protein engineering of hydrogenase 3 to enhance hydrogen production

  • Toshinari Maeda
  • Viviana Sanchez-Torres
  • Thomas K. WoodEmail author
Biotechnologically Relevant Enzymes and Proteins


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.


Protein engineering Hydrogenase 3 Error-prone PCR DNA shuffling Saturation mutagenesis 



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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Copyright information

© Springer-Verlag 2008

Authors and Affiliations

  • Toshinari Maeda
    • 1
  • Viviana Sanchez-Torres
    • 1
  • Thomas K. Wood
    • 1
    • 2
    • 3
    Email author
  1. 1.Artie McFerrin Department of Chemical EngineeringTexas A & M UniversityCollege StationUSA
  2. 2.Department of BiologyTexas A & M UniversityCollege StationUSA
  3. 3.Zachry Department of Civil and Environmental EngineeringTexas A & M UniversityCollege StationUSA

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