A cell-free expression system using a novel bacteriophage RNA polymerase for metagenome screening

Abstract The mining of genomes from non-cultivated microorganisms using metagenomics is a powerful tool to discover novel proteins and other valuable biomolecules. However, function-based metagenome searches are often limited by the time-consuming expression of the active proteins in various heterologous host systems. We here report the initial characterization of novel single-subunit bacteriophage RNA polymerase, RNAP_E, identified from a metagenome data set obtained from an elephant dung microbiome. RNAP_E and its promoter sequence are distantly related to T7 RNA polymerase. Using RNAP_E and a translation-competent Escherichia coli extract, we have developed an efficient medium throughput pipeline and protocol allowing the expression of metagenome-derived genes and the production of proteins in cell-free system is sufficient for the initial testing of the predicted activities. Here, we have successfully identified and verified 12 enzymes acting on bis(2-hydroxyethyl) terephthalate (BHET) in a completely clone-free approach and proposed an in vitro high-throughput metagenomic screening method with RNAP_E.