title: A selection assay for haloalkane dehalogenase activity based on toxic substrates.
abstract: Based on natural selection and the survival of the fittest by evolutionary adaption, a smart high-throughput system was developed to select active haloalkane dehalogenase variants from a large mutant library. Only active enzyme variants can hydrolyse toxic halogenated alkanes to promote growth, whereas inactive mutants starve or die due to the toxic compound. With this powerful tool, huge enzyme mutant libraries can be screened within a few days. The selection is done without any artificial substrates that are hard to synthesize and they also resemble typical ones for haloalkane dehalogenases. Three saturation libraries, with a size of more than 10(6) cells, based on inactive variants of the haloalkane dehalogenases DhaA or DhlA were successfully screened to retrieve active enzymes. The enrichment of the active wild-type enzyme in contrast to the inactive variants was about 340-fold. In addition, this selection approach can be applied for continuous directed evolution experiments for the enrichment of cells expressing adapted haloalkane dehalogenases.
authors.affiliation: Department of Biotechnology and Enzyme Catalysis, Institute of Biochemistry, University of Greifswald, Felix-Hausdorff-Straße 4, D-17491, Greifswald, Germany.
authors.forename: Michael P C
authors.initials: MP
authors.lastname: Fibinger
authors.affiliation: Davids Biotechnologie GmbH, Röntgenstraße 3, D-93055, Regensburg, Germany.
authors.forename: Timo
authors.initials: T
authors.lastname: Davids
authors.affiliation: Department of Biotechnology and Enzyme Catalysis, Institute of Biochemistry, University of Greifswald, Felix-Hausdorff-Straße 4, D-17491, Greifswald, Germany.
authors.forename: Dominique
authors.initials: D
authors.lastname: Böttcher
authors.affiliation: Department of Biotechnology and Enzyme Catalysis, Institute of Biochemistry, University of Greifswald, Felix-Hausdorff-Straße 4, D-17491, Greifswald, Germany. uwe.bornscheuer@uni-greifswald.de.
authors.forename: Uwe T
authors.initials: UT
authors.lastname: Bornscheuer
mesh_terms: D000475:Alkenes / Q000378:metabolism* / Q000633:toxicity; D001681:Biological Assay / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development*; D005820:Genetic Testing / Q000379:methods*; D006846:Hydrocarbons, Halogenated / Q000378:metabolism* / Q000633:toxicity; D006867:Hydrolases / Q000032:analysis* / Q000235:genetics*; D050505:Mutant Proteins / Q000032:analysis / Q000235:genetics
chemical_list: D000475:Alkenes; D006846:Hydrocarbons, Halogenated; D050505:Mutant Proteins; D006867:Hydrolases; C046137:haloalkane dehalogenase
keywords: Continuous selection process; Growth assay; Haloalkane dehalogenase; Toxicity