Intracellular Assembly of Interacting Enzymes Yields Highly‐Active Nanoparticles for Flow Biocatalysis

All‐enzyme hydrogel (AEH) particles with a hydrodynamic diameter of up to 120 nm were produced intracellularly with an Escherichia coli‐based in vivo system. The inCell‐AEH nanoparticles were generated from polycistronic vectors enabling simultaneous expression of two interacting enzymes, the Lactobacillus brevis alcohol dehydrogenase (ADH) and the Bacillus subtilis glucose‐1‐dehydrogenase (GDH), fused with a SpyCatcher or SpyTag, respectively. Formation of inCell‐AEH was analyzed by dynamic light scattering and atomic force microscopy. Using the stereoselective two‐step reduction of a prochiral diketone substrate, we show that the inCell‐AEH approach can be advantageously used in whole‐cell flow biocatalysis, by which flow reactors could be operated for >4 days under constant substrate perfusion. More importantly, the inCell‐AEH concept enables the recovery of efficient catalyst materials for stable flow bioreactors in a simple and economical one‐step procedure from crude bacterial lysates. We believe that our method will contribute to further optimization of sustainable biocatalytic processes.