Non-ribosomal peptides are a diverse and medically important group of natural products. They are biosynthesised by modular non-ribosomal peptide synthetase (NRPS) assembly-lines in which domains from each module act in concert to incorporate a specific amino acid into a peptide. This modular biosynthesis has driven efforts to generate new peptide analogues by substituting amino acid specifying domains. Rational NRPS engineering has increasingly focused on using evolutionarily favoured recombination sites for domain substitution. Here, we present an alternative approach inspired by evolution, which involves large-scale diversification and screening. By adopting a metagenomic approach of amplifying amino acid specifying domains from metagenomic DNA derived from soil, we were able to substitute over 1,000 unique domains into a pyoverdine NRPS. To identify functional domain substitutions, we employed fluorescence and mass spectrometry screening techniques, followed by sequencing. This comprehensive screening process successfully identified more than 100 functional domain substitutions, resulting in the production of 16 distinct pyoverdines as major products. The significance of this metagenomic approach lies in its ability to shift the focus of engineering non-ribosomal peptide biosynthesis. Instead of relying on a high success rate of individual domain substitution, we have developed effective methods that enable the exploration of a broader range of substitutions. This opens new possibilities for the discovery and production of novel non-ribosomal peptides with diverse biological activities.