Efficient glycine synthesis from CO2-derived oxalate via an in vitro multi-enzyme cascade

Glycine, the simplest amino acid with versatile physiological functions, is widely used in pharmaceuticals, animal feed, and the food industry. However, conventional production methods are limited by sustainability, safety, and environmental concerns. Here, we develop an efficient and green one-pot in vitro multi-enzyme cascade that converts oxalate into glycine under mild conditions. Upon optimization of the reaction conditions and integration of a cofactor recycling system, nearly complete conversion (100%) of oxalate to glycine was achieved in a 1-mL reaction system within 7 hours. After 4 hours of reaction in a 10 mL system, the glycine yield reached 95.7%, with a corresponding synthesis rate of 179.6 mg L-1 h-1. This work demonstrates that a C2-fixing multi-enzyme cascade circumvents the inherent challenge of C-C bond formation, achieving efficient conversion of CO2-derived oxalate to glycine, and presents a practical and conceptually innovative strategy for carbon-negative chemical synthesis.