Engineering Phosphoenolpyruvate Carboxylase with Improved Activity and Stability for High‐Efficiency Carbon Dioxide Fixation

The extraction of excess CO₂ from the environment requires favorable carbon sequestration methods, among which biochar sequestration is one of the most effective approaches. Therefore, to meet the growing demand for high‐efficiency carbon fixation and enhanced metabolic flux in biological carbon sequestration, the development of high‐performance phosphoenolpyruvate carboxylase (PEPC) is of significant importance. In this study, a high‐activity PEPC from Cannabis sativa (CsPEPC) is identified. However, the enzyme exhibits poor thermal stability, with a half‐life of only 13 min at 50 °C. To improve CsPEPC performance, diverse strategies, including molecular dynamics, FoldX energy calculations, and PROSS‐based prediction, are integrated to screen beneficial variants. The resulting mutant CsPEPC^C886R shows a twofold increase in activity (249.2 U mg^–1 at 50 °C) and fivefold greater stability. Furthermore, the addition of 25% glycerol as a protein stabilizer significantly extends the half‐life of CsPEPC^C886R by 31‐fold, ultimately increasing the half‐life to 2178 min. In vitro production experiments show that the optimal mutant CsPEPC^C886R generates 116.8 mM oxaloacetate within 270 min. This work provides a promising PEPC variant for biological CO₂ fixation, which holds great potential for improving carbon assimilation and supports the development of efficient, carbon‐neutral biosynthetic pathways.