Natural diversifying evolution of nonribosomal peptide synthetases in a defensive symbiont reveals nonmodular functional constraints

The modular architecture of nonribosomal peptide synthetases (NRPSs) has inspired efforts to study their evolution and engineering. In this study, we analyze in detail a unique family of NRPSs from the defensive intracellular bacterial symbiont, <i>Candidatus</i> Endobryopsis kahalalidifaciens (<i>Ca.</i> E. kahalalidifaciens). We show that intensive and indiscriminate recombination events erase trivial sequence covariations induced by phylogenetic relatedness, revealing nonmodular functional constraints and clear recombination units. Moreover, we reveal unique substrate specificity determinants for multiple enzymatic domains, allowing us to accurately predict and experimentally discover the products of an orphan NRPS in <i>Ca.</i> E. kahalalidifaciens directly from environmental samples of its algal host. Finally, we expanded our analysis to 1,531 diverse NRPS pathways and revealed similar functional constraints to those observed in <i>Ca.</i> E. kahalalidifaciens' NRPSs. Our findings reveal the sequence bases of genetic exchange, functional constraints, and substrate specificity in <i>Ca.</i> E. kahalalidifaciens' NRPSs, and highlight them as a uniquely primed system for diversifying evolution.