Modular multi-enzyme cascades enable green and sustainable synthesis of non-canonical amino acids from glycerol

Non-canonical amino acids (ncAAs) bearing diverse functional groups hold transformative potential in drug discovery, protein engineering, and biomaterial science. However, their industrial-scale production remains constrained by the inefficiency, high cost, and environmental burden of conventional chemical and enzymatic methods. Here, we present a modular multi-enzyme cascade platform that leverages glycerol—an abundant and sustainable byproduct of biodiesel production—as a low-cost substrate for ncAAs synthesis. Directed evolution of O-phospho-l-serine sulfhydrylase (OPSS) enhances the catalytic efficiency of C–N bond formation by 5.6-fold, enabling the efficient synthesis of triazole-functionalized ncAAs. By integrating a plug-and-play enzymatic strategy, our system enables gram- to decagram-scale production of 22 ncAAs with C–S, C–Se, and C–N side chains and can be readily scaled up to a 2 liter reaction system. Notably, water is the sole byproduct, and all products exhibit an atomic economy of >75%, highlighting the environmental compatibility of this platform. This work establishes a green, cost-effective, and industrially viable approach to expanding amino acid diversity, providing a versatile toolkit for applications in pharmaceuticals, synthetic biology, and next-generation biomaterials.