De Novo Biosynthesis of Valinomycin From Glucose Using In Vitro Reconstituted Hybrid Pathways

In vitro biotransformation mediated by cell‐free biosynthetic systems provides a flexible biomanufacturing platform that enables the reconstitution of hybrid metabolic pathways for complex natural product biosynthesis from simple substrates. Here, we report the total biosynthesis of valinomycin from glucose using in vitro reconstituted hybrid pathways consisting of three catalytic modules. First, a four‐enzyme short glycolytic pathway was integrated to convert glucose to pyruvate, one of the key precursors of valinomycin. Subsequently, a second pathway for L‐valine biosynthesis catalyzes the conversion of pyruvate to α‐ketoisovalerate and L‐valine, which serve as the other two precursors. Finally, de novo biosynthesis of valinomycin is achieved through the third module, valinomycin synthetase, which assembles the three precursors generated from the first two modules. Overall, we demonstrate the successful reconstitution of in vitro hybrid pathways for valinomycin biosynthesis using glucose as the sole input substrate. This synthetic strategy provides a modular framework for designing easy‐to‐use enzymatic pathways to produce value‐added complex natural products from simple and inexpensive substrates.