Engineering bacteriophages through deep mining of metagenomic motifs

Bacteriophages can adapt to new hosts by altering sequence motifs through recombination or convergent evolution. Where such motifs exist and what fitness advantage they confer remains largely unknown. We report a new method, Metagenomic Sequence Informed Functional Scoring (Meta-SIFT), to discover sequence motifs in metagenomic datasets that can be used to engineer phage activity. Meta-SIFT uses experimental deep mutational scanning data to create sequence profiles to enable deep mining of metagenomes for functional motifs which are otherwise invisible to searches. We experimentally tested over 17,000 Meta-SIFT derived sequence motifs in the receptor-binding protein of the T7 phage. The screen revealed thousands of T7 variants with novel host specificity with functional motifs sourced from distant families. Position, substitution and location preferences dictated specificity across a panel of 20 hosts and conditions. To demonstrate therapeutic utility, we engineered active T7 variants against foodborne pathogen E. coli O121. Meta-SIFT is a powerful tool to unlock the functional potential encoded in phage metagenomes to engineer bacteriophages.