Multiple bacterial natural products including the pepteridine virulence factors and the therapeutically-relevant antibiotic virginiamycin M, are biosynthesized at the intersection between primary and specialized metabolism. In these cases, primary metabolic α-ketoacid dehydrogenase complexes (KADHs) provide essential acyl building blocks to multienzyme complexes of specialized metabolism, including modular polyketide synthases (PKSs) and nonribosomal peptide synthetases (NRPSs). More remarkably, in certain pathways, the KADH components are fully integrated into the PKS/NRPS megaenzymes. At present, nothing is known about the sequence, mechanistic and architectural adaptations that were required relative to the ancestral KADHs to afford such chimaeras – information which is necessary for creating novel types of hybrids in the laboratory by genetic engineering. In this context, the present German-French collaborative project aims to investigate this type of system in detail. Specifically, we will: (i) generate an exhaustive catalog of specialized metabolic pathways that incorporate KADH machinery using genome mining; (ii) structurally characterize the products of newly-identified systems by heterologous expression; (iii) use ancestral protein reconstruction to propose a reasonable evolutionary trajectory to present day KADH enzymes; (iv) deploy an integrative structural biology approach to elucidate key architectural features of multienzyme-integrated KADHs (i.e. oligomerization state, stoichiometry of KADH component binding, and interactions with partner domains within the multienzymes); and (v) exploit the obtained fundamental insights to genetically engineer biosynthetic systems, towards the goal of generating bespoke natural product analogs bearing KADH-derived moieties. The proposed project follows on from previous successful collaboration between three of the partner laboratories, and is fully anchored in all groups’ strong, highly-complementary expertise.