A novel metagenome-derived viral RNA polymerase
25. Oktober 2022, von Dr. Yuchen Han
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, EM1 RNAP, identified from a metagenome data set obtained from an elephant dung microbiome. EM1 RNAP and its promoter sequence are distantly related to T7 RNA polymerase. Using EM1 RNAP 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.
A novel metagenome-derived viral RNA polymerase and its application in a cell-free expression system for metagenome screening
Published on 25 October 2022 in Scientific Reports
The protocol of the metagenomic screening via in vitro expression system can be found in the book Metagenomics: Methods in Molecular Biology (vol 2555. Humana, New York).
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