A metagenomic library cloning strategy that promotes high-level expression of captured genes to enable efficient functional screening

Abstract

Functional screening of environmental DNA (eDNA) libraries is a potentially powerful approach to2 discover enzymatic “unknown unknowns”, but is usually heavily biased toward the tiny subset of3 genes preferentially transcribed and translated by the screening strain. We have overcome this by4 preparing an eDNA library via partial digest with restriction enzyme FatI (cuts CATG), causing a5 substantial proportion of ATG start codons to be precisely aligned with strong plasmid-encoded6 promoter and ribosome-binding sequences. Whereas we were unable to select nitroreductases from7 standard metagenome libraries, our FatI strategy yielded 21 nitroreductases spanning eight different8 enzyme families, each conferring resistance to the nitro-antibiotic niclosamide and sensitivity to the9 nitro-prodrug metronidazole. We showed expression could be improved by co-expressing rare tRNAs10 and encoded proteins purified directly using an embedded His6-tag. In a transgenic zebrafish model11 of metronidazole-mediated targeted cell ablation, our lead MhqN-family nitroreductase proved ~5-12 fold more effective than the canonical nitroreductase NfsB.