Metagenomic domain substitution for the high-throughput modification of nonribosomal peptides

The modular nature of nonribosomal peptide biosynthesis has driven efforts to generate peptide analogs by substituting amino acid-specifying domains within nonribosomal peptide synthetase (NRPS) enzymes. Rational NRPS engineering has increasingly focused on finding evolutionarily favored recombination sites for domain substitution. Here we present an alternative evolution-inspired approach that involves large-scale diversification and screening. By amplifying amino acid-specifying domains en masse from soil metagenomic DNA, we substitute more than 1,000 unique domains into a pyoverdine NRPS. Initial fluorescence and mass spectrometry screens followed by sequencing reveal more than 100 functional domain substitutions, collectively yielding 16 distinct pyoverdines as major products. This metagenomic approach does not require the high success rates demanded by rational NRPS engineering but instead enables the exploration of large numbers of substitutions in parallel. This opens possibilities for the discovery and production of nonribosomal peptides with diverse biological activities.