Activity-Based Screening of Metagenomic Libraries for Hydrogenase Enzymes

Nicole Adam; Mirjam Perner

doi:10.1007/978-1-4939-6691-2_17

Activity-Based Screening of Metagenomic Libraries for Hydrogenase Enzymes

Methods Mol Biol. 2017:1539:261-270. doi: 10.1007/978-1-4939-6691-2_17.

Authors

Nicole Adam¹, Mirjam Perner²

Affiliations

¹ Molecular Biology of Microbial Consortia, Biocenter Klein Flottbek, University of Hamburg, Ohnhorststr. 18, 22609, Hamburg, Germany.
² Molecular Biology of Microbial Consortia, Biocenter Klein Flottbek, University of Hamburg, Ohnhorststr. 18, 22609, Hamburg, Germany. mirjam.perner@uni-hamburg.de.

PMID: 27900696
DOI: 10.1007/978-1-4939-6691-2_17

Abstract

Here we outline how to identify hydrogenase enzymes from metagenomic libraries through an activity-based screening approach. A metagenomic fosmid library is constructed in E. coli and the fosmids are transferred into a hydrogenase deletion mutant of Shewanella oneidensis (ΔhyaB) via triparental mating. If a fosmid exhibits hydrogen uptake activity, S. oneidensis' phenotype is restored and hydrogenase activity is indicated by a color change of the medium from yellow to colorless. This new method enables screening of 48 metagenomic fosmid clones in parallel.

Keywords: Function-based screen; Hydrogen uptake; Hydrogenase; Metagenome.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Enzyme Activation
Gene Expression
Gene Library*
Genetic Vectors / genetics
Hydrogen / metabolism
Hydrogenase / genetics*
Hydrogenase / metabolism*
Metagenome*
Metagenomics* / methods

Substances

Hydrogen
Hydrogenase