A novel metagenome-derived viral RNA polymerase and its application in a cell-free expression system for metagenome screening

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 hos...

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Bibliographic Details
Published in:	Scientific reports Vol. 12; no. 1; p. 17882
Main Authors:	Han, Yuchen, Kinfu, Birhanu M., Blombach, Fabian, Cackett, Gwenny, Zhang, Hongli, Pérez-García, Pablo, Krohn, Ines, Salomon, Jesper, Besirlioglu, Volkan, Mirzaeigarakani, Tayebeh, Schwaneberg, Ulrich, Chow, Jennifer, Werner, Finn, Streit, Wolfgang R.
Format:	Journal Article
Language:	English
Published:	London Nature Publishing Group UK 25-10-2022 Nature Publishing Group Nature Portfolio
Subjects:	631/1647/2163 631/337/572 631/61/185 Amino acids Cell-free system Cell-Free System - metabolism Cloning DNA-directed RNA polymerase DNA-Directed RNA Polymerases - genetics DNA-Directed RNA Polymerases - metabolism Dung E coli Enzymes Escherichia coli - genetics Escherichia coli - metabolism Genes Genomes Host systems Humanities and Social Sciences Metagenome Metagenomics Metagenomics - methods Microbiomes Microorganisms Mitochondrial DNA multidisciplinary Nucleotide sequence Phages Protein expression Proteins RNA phages RNA polymerase RNA, Viral - metabolism Science Science (multidisciplinary) Viral Replicase Complex Proteins
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Summary:	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.
Bibliography:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23
ISSN:	2045-2322 2045-2322
DOI:	10.1038/s41598-022-22383-x