H 2 ‐Driven Whole‐Cell Conversion of Diamines to N‐Heterocycles by Recombinant Cupriavidus necator

ABSTRACT Saturated N‐heterocycles such as pyrrolidines and piperidines are core motifs in many agrochemicals and pharmaceutical agents, yet established chemical syntheses often rely on precious metals and face limitations in selectivity, scalability, and sustainability. Biocatalytic approaches offer high chemo‐ and stereoselectivity, but in vitro cascades require enzyme purification and external cofactor recycling, while whole‐cell systems typically depend on carbohydrate feedstocks. Here, we report the engineering of the lithoautotrophic bacterium Cupriavidus necator for the hydrogen‐driven conversion of linear diamines into saturated cyclic amines. Co‐expression of diamine oxidases (DAOs) and an imine reductase (IRED) enabled an intracellular oxidation–reduction cascade powered exclusively by H 2 . Notably, eukaryotic Cu/topaquinone‐dependent DAOs were functionally produced alongside a bacterial oxidase, demonstrating efficient aerobic maturation of complex copper enzymes in a hydrogen‐oxidizing host. Endogenous hydrogenases provide continuous NAD(P)H regeneration, decoupling reductive power from organic carbon metabolism. Process analysis identified gas–liquid mass transfer and redox balance as key determinants of productivity. This platform enables whole‐cell synthesis of pyrrolidine‐ and piperidine‐type products and represents, to our knowledge, the first H 2 ‐powered whole‐cell route from diamines to saturated N‐heterocycles, providing a tunable blueprint for sustainable cyclic amine synthesis.