title: Using directed evolution to improve hydrogen production in chimeric hydrogenases from Clostridia species.
abstract: Hydrogenases are enzymes that play a key role in controlling excess reducing equivalents in both photosynthetic and anaerobic organisms. This enzyme is viewed as potentially important for the industrial generation of hydrogen gas; however, insufficient hydrogen production has impeded its use in a commercial process. Here, we explore the potential to circumvent this problem by directly evolving the Fe-Fe hydrogenase genes from two species of Clostridia bacteria. In addition, a computational model based on these mutant sequences was developed and used as a predictive aid for the isolation of enzymes with even greater efficiency in hydrogen production. Two of the improved mutants have a logarithmic increase in hydrogen production in our in vitro assay. Furthermore, the model predicts hydrogenase sequences with hydrogen productions as high as 540-fold over the positive control. Taken together, these results demonstrate the potential of directed evolution to improve the native bacterial hydrogenases as a first step for improvement of hydrogenase activity, further in silico prediction, and finally, construction and demonstration of an improved algal hydrogenase in an in vivo assay of C. reinhardtii hydrogen production.
authors.affiliation: H(2)OPE Biofuels LLC, Greenwood Village, CO, USA; Dept of Chemistry, Southern Oregon University, Medford, OR, USA. Electronic address: scott@h2opebiofuels.com.
authors.forename: Scott M
authors.initials: SM
authors.lastname: Plummer
authors.affiliation: MPr&d LLC, Greenwood Village, CO, USA. Electronic address: maplummer78@gmail.com.
authors.forename: Mark A
authors.initials: MA
authors.lastname: Plummer
authors.affiliation: H(2)OPE Biofuels LLC, Greenwood Village, CO, USA.
authors.forename: Patricia A
authors.initials: PA
authors.lastname: Merkel
authors.affiliation: University of Colorado Health Sciences Center, Denver, CO, USA.
authors.forename: Moira
authors.initials: M
authors.lastname: Hagen
authors.affiliation: School of Marine Science and Policy, University of Delaware, Lewes, DE, USA.
authors.forename: Jennifer F
authors.initials: JF
authors.lastname: Biddle
authors.affiliation: H(2)OPE Biofuels LLC, Greenwood Village, CO, USA; School of Marine Science and Policy, University of Delaware, Lewes, DE, USA.
authors.forename: Lisa A
authors.initials: LA
authors.lastname: Waidner
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D003001:Cloning, Molecular; D003013:Clostridium / Q000201:enzymology* / Q000235:genetics*; D019020:Directed Molecular Evolution; D005798:Genes, Bacterial; D006859:Hydrogen / Q000378:metabolism*; D006864:Hydrogenase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D007506:Iron-Sulfur Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D007700:Kinetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D055672:Static Electricity
chemical_list: D001426:Bacterial Proteins; D007506:Iron-Sulfur Proteins; D011993:Recombinant Fusion Proteins; D006859:Hydrogen; C118830:iron hydrogenase; D006864:Hydrogenase
keywords: Bioenergy; Clostridial species; Directed evolution; Hydrogen production; Hydrogenase; Molecular modeling