Endonucleases play a crucial role as reagents in laboratory research and diagnostics. Here, metagenomics was used to functionally screen a fosmid library for endonucleases. A fosmid library was constructed using metagenomic DNA isolated from soil sampled from the unique environment of the Kogelberg Nature Reserve in the Western Cape of South Africa. The principle of acquired immunity against phage infection was used to develop a plate-based screening technique for the isolation of restriction endonucleases from the library. Using next-generation sequencing and bioinformatics tools, sequence data were generated and analysed, revealing 113 novel open reading frames (ORFs) encoding putative endonuclease genes and ORFs of unknown identity and function. One endonuclease designated Endo52 was selected from the putative endonuclease ORFs and was recombinantly produced in Escherichia coli Rosetta™ (DE3) pLysS. Endo52 was purified by immobilised metal affinity chromatography and yielded 0.437 g per litre of cultivation volume. Its enzyme activity was monitored by cleaving lambda DNA and pUC19 plasmid as substrates, and it demonstrated non-specific endonuclease activity. In addition to endonuclease-like genes, the screen identified several unknown genes. These could present new phage resistance mechanisms and are an opportunity for future investigations.
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The work was supported by the Department of Science and Technology, the Technology Innovation Agency and the Council of Scientific and Industrial Research (CSIR). Many thanks to CapeNature for permitting us to access the Kogelberg Biosphere Reserve for soil sampling.
This study was funded by The Department of Science and Technology (DST), South Africa (ZA), the Technology Innovation Agency (TIA, South Africa (ZA) and the Council of Science and Industrial Research, South Africa (ZA) to Dr. Tsepo L. Tsekoa.
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Mtimka, S., Pillay, P., Rashamuse, K. et al. Functional screening of a soil metagenome for DNA endonucleases by acquired resistance to bacteriophage infection. Mol Biol Rep 47, 353–361 (2020). https://doi.org/10.1007/s11033-019-05137-3
- Fosmid library
- Functional screening
- Kogelberg nature reserve restriction endonuclease
- Restriction enzymes
- Soil metagenome