A novel metagenome-derived viral RNA polymerase and its application in a cell-free expression system for metagenome screening
Creators
- 1. Department of Microbiology and Biotechnology, University of Hamburg, Ohnhorststr. 18, 22609, Hamburg, Germany
- 2. Institute for General Microbiology, Christian-Albrechts-University, 24118, Kiel, Germany Department of Microbiology and Biotechnology, University of Hamburg, Ohnhorststr. 18, 22609, Hamburg, Germany
- 3. Division of Biosciences, Institute of Structural and Molecular Biology, University College London, Gower Street, London, WC1E 6BT, UK
- 4. Novozymes A/S, Microbial Discovery, Bagsværd, Denmark
- 5. Chair of Biotechnology, RWTH Aachen, Worringerweg 3, 52074, Aachen, Germany
Description
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, identifed 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 efcient medium-throughput pipeline and protocol allowing the expression of metagenome-derived genes and the production of proteins in cell-free system is sufcient for the initial testing of the predicted activities. Here, we have successfully identifed and verifed 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.
Files
A novel metagenome-derived viral RNA polymerase and its application in a cell-free expression system for metagenome screening.pdf
Files
(6.5 MB)
| Name | Size | Download all |
|---|---|---|
|
md5:b657b1084a613976f6b2b86307594b9b
|
6.5 MB | Preview Download |