Hierarchically Engineered Multi‐Enzyme Nanoreactors for in vitro Drug Biosynthesis and Pathway Transplantation Into Cells

Even though most proteins evolved to function within multi-protein systems such as metabolic networks or signaling pathways, most technical protein applications are based on isolated proteins, for example therapeutic antibodies or industrial enzymes. Reliable methods for the stabilization, in vitro operation and intracellular delivery of multi-protein systems could unlock new applications in green biotechnology, diagnostics, and medical therapy. We demonstrate that the entire violacein biosynthesis pathway, consisting of up to six separately purified enzymes, can be infiltrated into a hierarchically etched MIL-101(Fe) (eMIL) metal organic framework. eMIL nanoreactors reshaped pathway dynamics and reaction flows, multiplied violacein yield in vitro and enabled pathway reuse, lyophilization and storage. Moreover, eMIL nanoreactors delivered the six-protein system into mammalian cells, where it produced violacein from cell-provided substrates and cofactors. These findings pave the way for the design of "smart" stimuli-responsive multi-enzyme nanoreactors for biotechnological and medical applications.