title: Protein engineering of hydrogenase 3 to enhance hydrogen production.
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.
authors.affiliation: Artie McFerrin Department of Chemical Engineering, Texas A&M University, College Station, TX 77843-3122, USA.
authors.forename: Toshinari
authors.initials: T
authors.lastname: Maeda
authors.forename: Viviana
authors.initials: V
authors.lastname: Sanchez-Torres
authors.forename: Thomas K
authors.initials: TK
authors.lastname: Wood
mesh_terms: D000595:Amino Acid Sequence; D032621:DNA Shuffling; D019020:Directed Molecular Evolution; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000378:metabolism*; D005561:Formates / Q000378:metabolism; D006859:Hydrogen / Q000378:metabolism*; D006864:Hydrogenase / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016133:Polymerase Chain Reaction; D015202:Protein Engineering
chemical_list: D029968:Escherichia coli Proteins; D005561:Formates; D006859:Hydrogen; C086230:hycE protein, E coli; D006864:Hydrogenase