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

Abstract

Recent awareness that most microorganisms in the environment are resistant to cultivation has prompted scientists to directly clone useful genes from environmental metagenomes. Two screening methods are currently available for the metagenome approach, namely, nucleotide sequence-based screening and enzyme activity-based screening. 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.

A Gene Cluster Involved in Degradation of Substituted Salicylates viaorthoCleavage inPseudomonassp. Strain MT1 Encodes Enzymes Specifically Adapted for Transformation of 4-Methylcatechol and 3-Methylmuconate

“…The organization of the cat gene cluster of strain MT1 is identical to the organization characteristic for Pseudomonas strains and shows highest similarity to the cluster identified from an environmental metagenome library constructed from petroleumcontaminated groundwater (57). In MT1, the function of C12O catA and MCI catB is to create, together with muconolactone isomerase, a functional catechol branch of the 3-oxoadipate pathway.…”

Contemporary molecular tools in microbial ecology and their application to advancing biotechnology

“…This knowledge was exploited and an operon-trap green fluorescent protein (GFP)-expression vector was constructed for shotgun cloning of metagenomic DNA. In the presence of the substrate, the cloned catabolic gene is expressed resulting in GFP expression, thus facilitating the high-throughput selection of positive clones in liquid cultures by fluorescence-activated cell sorting (Uchiyama, Abe et al 2005). The SIGEX method was developed and tested first time to clone the phenoldegradation operon (pox operon) from Ralstonia eutropha.…”

Selection of biocatalysts for chemical synthesis

“…All of these approaches yielded regulators with altered effector profiles, which is consistent with the notion that structural information can be helpful, but is not required for directed evolution of proteins. In addition, new regulators that respond to chosen effectors have been sought and isolated from bacterial genomes (33,34). Finally, the conserved domain architecture, the typical small-molecule-binding domains and strongly conserved DNA-binding domains serve as markers to retrieve potential regulators from genomic sequence databases (35)(36)(37).…”