Product-Induced Gene Expression, a Product-Responsive Reporter Assay Used To Screen Metagenomic Libraries for Enzyme-Encoding Genes

A reporter assay-based screening method for enzymes, which we named product-induced gene expression (PIGEX), was developed and used to screen a metagenomic library for amidases. A benzoate-responsive transcriptional activator, BenR, was placed upstream of the gene encoding green fluorescent protein...

Full description

Saved in:
Bibliographic Details
Published in:Applied and Environmental Microbiology 2010-11, Vol.76 (21), p.7029-7035
Main Authors: Uchiyama, Taku, Miyazaki, Kentaro
Format: Article
Language:English
Subjects:
Amidohydrolases - genetics
Amidohydrolases - metabolism
Bacteria - enzymology
Bacteria - genetics
Benzoates - metabolism
Biological and medical sciences
Cloning, Molecular
DNA, Bacterial - genetics
Enzymes
Enzymes - genetics
Escherichia coli
Escherichia coli - enzymology
Escherichia coli - genetics
Fluorescence
Fundamental and applied biological sciences. Psychology
Gene expression
Gene Expression Profiling - methods
Genes
Genomic Library
Genomics
Genotype
Metagenomics - methods
Methods
Microbiology
Molecular Sequence Data
Plasmids - genetics
Polymerase Chain Reaction - methods
Proteins
Substrate Specificity
Citations: Items that this one cites
Items that cite this one
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary:A reporter assay-based screening method for enzymes, which we named product-induced gene expression (PIGEX), was developed and used to screen a metagenomic library for amidases. A benzoate-responsive transcriptional activator, BenR, was placed upstream of the gene encoding green fluorescent protein and used as a sensor. Escherichia coli sensor cells carrying the benR-gfp gene cassette fluoresced in response to benzoate concentrations as low as 10 μM but were completely unresponsive to the substrate benzamide. An E. coli metagenomic library consisting of 96,000 clones was grown in 96-well format in LB medium containing benzamide. The library cells were then cocultivated with sensor cells. Eleven amidase genes were recovered from 143 fluorescent wells; eight of these genes were homologous to known bacterial amidase genes while three were novel genes. In addition to their activity toward benzamide, the enzymes were active toward various substrates, including D- and L-amino acid amides, and displayed enantioselectivity. Thus, we demonstrated that PIGEX is an effective approach for screening novel enzymes based on product detection.
ISSN:0099-2240
1098-5336
1098-6596
DOI:10.1128/AEM.00464-10