Journal
SCIENTIFIC REPORTS
Volume 12, Issue 1, Pages -Publisher
NATURE PORTFOLIO
DOI: 10.1038/s41598-022-22383-x
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Funding
- German Federal Ministry of Education and Research (BMBF) [031B867B, 031B0837B, 031A571A, 031A571B]
- EU's Horizon 2020 project FuturEnzyme [101000327]
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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 active proteins. In this study, researchers identified a novel single-subunit bacteriophage RNA polymerase, EM1 RNAP, from a metagenome data set obtained from an elephant dung microbiome. They developed an efficient medium-throughput pipeline and protocol using EM1 RNAP and a translation-competent Escherichia coli extract to express metagenome-derived genes and produce proteins in a cell-free system, allowing for initial testing of predicted activities.
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.
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