Han, Yuchen;
Kinfu, Birhanu M;
Blombach, Fabian;
Cackett, Gwenny;
Zhang, Hongli;
Pérez-García, Pablo;
Krohn, Ines;
... Streit, Wolfgang R; + view all
(2022)
A novel metagenome-derived viral RNA polymerase and its application in a cell-free expression system for metagenome screening.
Scientific Reports
, 12
(1)
, Article 17882. 10.1038/s41598-022-22383-x.
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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, 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.
| Type: | Article |
|---|---|
| Title: | A novel metagenome-derived viral RNA polymerase and its application in a cell-free expression system for metagenome screening |
| Location: | England |
| Open access status: | An open access version is available from UCL Discovery |
| DOI: | 10.1038/s41598-022-22383-x |
| Publisher version: | https://doi.org/10.1038/s41598-022-22383-x |
| Language: | English |
| Additional information: | This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third-party material in this article are included in the Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ |
| Keywords: | Metagenome, Viral Replicase Complex Proteins, Cell-Free System, RNA, Viral, DNA-Directed RNA Polymerases, Metagenomics, Escherichia coli |
| UCL classification: | UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > Div of Biosciences > Structural and Molecular Biology UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences UCL UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > Div of Biosciences |
| URI: | https://discovery.ucl.ac.uk/id/eprint/10158612 |
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