title: Activity screening of environmental metagenomic libraries reveals novel carboxylesterase families.
abstract: Metagenomics has made accessible an enormous reserve of global biochemical diversity. To tap into this vast resource of novel enzymes, we have screened over one million clones from metagenome DNA libraries derived from sixteen different environments for carboxylesterase activity and identified 714 positive hits. We have validated the esterase activity of 80 selected genes, which belong to 17 different protein families including unknown and cyclase-like proteins. Three metagenomic enzymes exhibited lipase activity, and seven proteins showed polyester depolymerization activity against polylactic acid and polycaprolactone. Detailed biochemical characterization of four new enzymes revealed their substrate preference, whereas their catalytic residues were identified using site-directed mutagenesis. The crystal structure of the metal-ion dependent esterase MGS0169 from the amidohydrolase superfamily revealed a novel active site with a bound unknown ligand. Thus, activity-centered metagenomics has revealed diverse enzymes and novel families of microbial carboxylesterases, whose activity could not have been predicted using bioinformatics tools.
authors.affiliation: Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, ON, M5S 3E5, Canada.
authors.forename: Ana
authors.initials: A
authors.lastname: Popovic
authors.affiliation: School of Biological Sciences, Bangor University, Gwynedd LL57 2UW, UK.
authors.forename: Tran
authors.initials: T
authors.lastname: Hai
authors.affiliation: Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, ON, M5S 3E5, Canada.
authors.forename: Anatoly
authors.initials: A
authors.lastname: Tchigvintsev
authors.affiliation: Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, ON, M5S 3E5, Canada.
authors.forename: Mahbod
authors.initials: M
authors.lastname: Hajighasemi
authors.affiliation: Midwest Center for Structural Genomics and Structural Biology Center, Biosciences Division, Argonne National Laboratory, Argonne, Illinois 60439, USA.
authors.forename: Boguslaw
authors.initials: B
authors.lastname: Nocek
authors.affiliation: Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, ON, M5S 3E5, Canada.
authors.forename: Anna N
authors.initials: AN
authors.lastname: Khusnutdinova
authors.affiliation: Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, ON, M5S 3E5, Canada.
authors.forename: Greg
authors.initials: G
authors.lastname: Brown
authors.affiliation: Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, ON, M5S 3E5, Canada.
authors.forename: Julia
authors.initials: J
authors.lastname: Glinos
authors.affiliation: Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, ON, M5S 3E5, Canada.
authors.forename: Robert
authors.initials: R
authors.lastname: Flick
authors.affiliation: Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, ON, M5S 3E5, Canada.
authors.forename: Tatiana
authors.initials: T
authors.lastname: Skarina
authors.affiliation: School of Biological Sciences, Bangor University, Gwynedd LL57 2UW, UK.
authors.forename: Tatyana N
authors.initials: TN
authors.lastname: Chernikova
authors.affiliation: Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, ON, M5S 3E5, Canada.
authors.forename: Veronica
authors.initials: V
authors.lastname: Yim
authors.affiliation: Commissariat à l'Energie Atomique et aux Energies Alternatives (CEA), Direction de la Recherche Fondamentale, Institut de Génomique, Université de d'Evry Val d'Essonne (UEVE), Centre National de la Recherche Scientifique (CNRS), UMR8030, Génomique métabolique, Evry, France.
authors.forename: Thomas
authors.initials: T
authors.lastname: Brüls
authors.affiliation: Université de d'Evry Val d'Essonne (UEVE), Centre National de la Recherche, Scientifique (CNRS), UMR8030, Génomique métabolique, Commissariat à l'Energie, Atomique et aux Energies Alternatives (CEA), Direction de la Recherche, Fondamentale, Institut de Génomique, Evry, France.
authors.forename: Denis Le
authors.initials: DL
authors.lastname: Paslier
authors.affiliation: Institute for Coastal Marine Environment, CNR, 98122 Messina, Italy.
authors.forename: Michail M
authors.initials: MM
authors.lastname: Yakimov
authors.affiliation: Midwest Center for Structural Genomics and Structural Biology Center, Biosciences Division, Argonne National Laboratory, Argonne, Illinois 60439, USA.
authors.forename: Andrzej
authors.initials: A
authors.lastname: Joachimiak
authors.affiliation: Institute of Catalysis, CSIC, Madrid 28049, Spain.
authors.forename: Manuel
authors.initials: M
authors.lastname: Ferrer
authors.affiliation: School of Biological Sciences, Bangor University, Gwynedd LL57 2UW, UK.
authors.forename: Olga V
authors.initials: OV
authors.lastname: Golyshina
authors.affiliation: Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, ON, M5S 3E5, Canada.
authors.forename: Alexei
authors.initials: A
authors.lastname: Savchenko
authors.affiliation: School of Biological Sciences, Bangor University, Gwynedd LL57 2UW, UK.
authors.forename: Peter N
authors.initials: PN
authors.lastname: Golyshin
authors.affiliation: Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, ON, M5S 3E5, Canada.
authors.forename: Alexander F
authors.initials: AF
authors.lastname: Yakunin
mesh_terms: D002265:Carboxylic Ester Hydrolases / Q000737:chemistry / Q000235:genetics*; D004783:Environmental Microbiology; D004926:Escherichia coli / Q000235:genetics; D015723:Gene Library; D054892:Metagenome; D056186:Metagenomics
chemical_list: D002265:Carboxylic Ester Hydrolases