PUBLICATION
A metagenomic library cloning strategy that promotes high-level expression of captured genes to enable efficient functional screening
- Authors
- Rich, M.H., Sharrock, A.V., Mulligan, T.S., Matthews, F., Brown, A.S., Lee-Harwood, H.R., Williams, E.M., Copp, J.N., Little, R.F., Francis, J.J.B., Horvat, C.N., Stevenson, L.J., Owen, J.G., Saxena, M.T., Mumm, J.S., Ackerley, D.F.
- ID
- ZDB-PUB-231029-72
- Date
- 2023
- Source
- Cell chemical biology 30(12): 1680-1691.e6 (Journal)
- Registered Authors
- Mulligan, Tim, Mumm, Jeff, Saxena, Meera T.
- Keywords
- FatI restriction cloning, bacterial start codons, environmental DNA screening, functional metagenome screening, metronidazole, niclosamide, nitroreductase, synthetic biology, targeted cell ablation
- MeSH Terms
-
- Animals
- Cloning, Molecular
- Metagenome
- Metronidazole*/metabolism
- Metronidazole*/pharmacology
- Nitroreductases/genetics
- Zebrafish*/genetics
- Zebrafish*/metabolism
- PubMed
- 37898120 Full text @ Cell Chem Biol
Citation
Rich, M.H., Sharrock, A.V., Mulligan, T.S., Matthews, F., Brown, A.S., Lee-Harwood, H.R., Williams, E.M., Copp, J.N., Little, R.F., Francis, J.J.B., Horvat, C.N., Stevenson, L.J., Owen, J.G., Saxena, M.T., Mumm, J.S., Ackerley, D.F. (2023) A metagenomic library cloning strategy that promotes high-level expression of captured genes to enable efficient functional screening. Cell chemical biology. 30(12):1680-1691.e6.
Abstract
Functional screening of environmental DNA (eDNA) libraries is a potentially powerful approach to discover enzymatic "unknown unknowns", but is usually heavily biased toward the tiny subset of genes preferentially transcribed and translated by the screening strain. We have overcome this by preparing an eDNA library via partial digest with restriction enzyme FatI (cuts CATG), causing a substantial proportion of ATG start codons to be precisely aligned with strong plasmid-encoded promoter and ribosome-binding sequences. Whereas we were unable to select nitroreductases from standard metagenome libraries, our FatI strategy yielded 21 nitroreductases spanning eight different enzyme families, each conferring resistance to the nitro-antibiotic niclosamide and sensitivity to the nitro-prodrug metronidazole. We showed expression could be improved by co-expressing rare tRNAs and encoded proteins purified directly using an embedded His6-tag. In a transgenic zebrafish model of metronidazole-mediated targeted cell ablation, our lead MhqN-family nitroreductase proved ∼5-fold more effective than the canonical nitroreductase NfsB.
Genes / Markers
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Orthology
Engineered Foreign Genes
Mapping