Mixing and Matching of Hybrid Megasynthases is a Hub for the Evolution of Metabolic Diversity in Cyanobacteria

Modular megasynthases, such as polyketide synthases (PKSs) and non‐ribosomal peptide synthetases (NRPSs), are molecular assembly lines that biosynthesize many pharmaceutically and ecologically important natural products. Understanding how these compounds evolve could inspire the artificial evolution of compound diversity by metabolic engineering. Over the past two decades, a number of seminal studies have significantly contributed to our understanding of natural product evolution. However, the evolution of NRPS and PKS assembly lines remains poorly understood, especially for NRPS/PKS hybrids. Here, we provide substantial evidence for a remarkable cluster‐mixing event involving three cyanobacterial biosynthetic gene clusters, resulting in the emergence of novel peptide‐polyketide hybrids that were named minutumamides. By combining retro‐evolutionary analysis with structure‐guided genome mining we could discover a potential evolutionary ancestor that links nostopeptolide and minutumamide biosynthesis. In addition, we were able to trace nostopeptolide‐related module and domain blocks in various other biosynthetic pathways indicating a surprisingly vivid mixing and matching of biosynthesis genes in the evolution of NRPS and cis‐acyltransferase PKS/NRPS pathways, which was previously regarded as a unique feature of trans‐acyltransferase PKS. These remarkable insights into the evolutionary plasticity of NRPS‐PKS assembly lines provide valuable guidance for pathway engineers looking for productive combinations that yield "non‐natural" hybrid natural products.