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 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.
Uchiyama
Taku
T
Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology, Tsukuba, Ibaraki 305-8566, Japan.
Miyazaki
Kentaro
K
D000581:Amidohydrolases / Q000235:genetics / Q000378:metabolism; D001419:Bacteria / Q000201:enzymology / Q000235:genetics*; D001565:Benzoates / Q000378:metabolism; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D004798:Enzymes / Q000235:genetics*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D020869:Gene Expression Profiling / Q000379:methods*; D015698:Genomic Library; D005838:Genotype; D056186:Metagenomics / Q000379:methods*; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods; D013379:Substrate Specificity
D001565:Benzoates; D004269:DNA, Bacterial; D004798:Enzymes; D000581:Amidohydrolases