A cell-free microtiter plate screen for improved [FeFe] hydrogenases

• Published in: PloS one Vol. 5; no. 5; p. e10554
• Main Authors: Stapleton, James A, Swartz, James R
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 10-05-2010; Public Library of Science (PLoS)
• Subjects: Adaptation, Physiological - drug effects; Adenosine; Bacteriology; Binding sites; Biocatalysts; Bioengineering; Biotechnology; Biotechnology/Biocatalysis; Biotechnology/Bioengineering; Catalysis; Catalysts; Cell-Free System; Chemical Biology/Directed Molecular Evolution; Chemical engineering; Chlamydomonas reinhardtii; Chlamydomonas reinhardtii - drug effects; Chlamydomonas reinhardtii - enzymology; Clostridium; Complexity; Deoxyribonucleic acid; Directed evolution; Dissociation; DNA; DNA methylation; E coli; Electronic Data Processing; Enzyme Assays; Enzymes; Evolution; Feasibility studies; Fuel cells; Gene expression; Gene Library; Hydrogen; Hydrogen fuels; Hydrogenase; Hydrogenase - genetics; Hydrogenase - isolation & purification; Hydrogenase - metabolism; Laboratories; Metabolism; Molecular Biology/Molecular Evolution; Mutagenesis; Mutant Proteins - isolation & purification; Mutant Proteins - metabolism; Mutants; Mutation; Mutation - genetics; Organisms; Oxidation; Oxygen; Oxygen - pharmacology; Poisson Distribution; Polymerase Chain Reaction; Protein Biosynthesis - drug effects; Protein Engineering - instrumentation; Protein Engineering - methods; Protein synthesis; Proteins; Reproducibility; Screening; Synthetic biology; Technology application; United States > US; California
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0010554

[FeFe] hydrogenase enzymes catalyze the production and dissociation of H(2), a potential renewable fuel. Attempts to exploit these catalysts in engineered systems have been hindered by the biotechnologically inconvenient properties of the natural enzymes, including their extreme oxygen sensitivity. Directed evolution has been used to improve the characteristics of a range of natural catalysts, but has been largely unsuccessful for [FeFe] hydrogenases because of a lack of convenient screening platforms.
Here we describe an in vitro screening technology for oxygen-tolerant and highly active [FeFe] hydrogenases. Despite the complexity of the protocol, we demonstrate a level of reproducibility that allows moderately improved mutants to be isolated. We have used the platform to identify a mutant of the Chlamydomonas reinhardtii [FeFe] hydrogenase HydA1 with a specific activity approximately 4 times that of the wild-type enzyme.
Our results demonstrate the feasibility of using the screen presented here for large-scale efforts to identify improved biocatalysts for energy applications. The system is based on our ability to activate these complex enzymes in E. coli cell extracts, which allows unhindered access to the protein maturation and assay environment.