Substrate-induced gene-expression screening of environmental metagenome libraries for isolation of catabolic genes

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Abstract
Recent awareness that most microorganisms in the environment are resistant to cultivation has prompted scientists to directly clone useful genes from environmental metagenomes1. Two screening methods are currently available for the metagenome approach, namely, nucleotide sequence–based screening2 and enzyme activity–based screening3. Here we have introduced and optimized a third option for the isolation of novel catabolic operons, that is, substrate-induced gene expression screening (SIGEX). This method is based on the knowledge that catabolic-gene expression is generally induced by relevant substrates and, in many cases, controlled by regulatory elements situated proximate to catabolic genes. For SIGEX to be high throughput, we constructed an operon-trap gfp-expression vector available for shotgun cloning that allows for the selection of positive clones in liquid cultures by fluorescence-activated cell sorting. The utility of SIGEX was demonstrated by the cloning of aromatic hydrocarbon–induced genes from a groundwater metagenome library and subsequent genome-informatics analysis.

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