Thermostable Coenzyme a Ligase for Efficient Biosynthesis of 2-Pyrrolidone via Protein and Fermentation Engineering

2-Pyrrolidone is an important chemical intermediate with broad applications in the materials and pharmaceutical industries. Traditional petrochemical synthesis methods pose significant environmental challenges. In the case of biosynthesis, the limited thermostability of coenzyme A ligase (CaiC) represents a major barrier to industrial-scale production. This study focused on enhancing the thermostability and catalytic efficiency of EcCaiC through protein engineering. Conserved sequences were identified, and flexible regions were targeted for virtual mutagenesis using FoldX and Rosetta. The resulting mutant, M3, exhibited a 7.86-fold increase in half-life(<i>t</i>_1/2) at 55 °C and a <i>T</i>_m of 59.3 °C. Additionally, the catalytic efficiency (<i>k</i>_cat/<i>K</i>_m) of M3 improved by 52.8%, reaching 5.73 mM^-1 s^-1 compared to the wild type. Subsequently, EcCaiC^M3 was introduced into <i>Corynebacterium glutamicum</i> S9114, with targeted knockout of byproduct synthesis genes. Finally, fed-batch fermentation in a 5 L bioreactor achieved a 2-pyrrolidone yield of 58.28 g/L, a glucose conversion rate of 0.32 g/g, and a productivity of 0.97 g/L/h. This work establishes an efficient biosynthetic platform for 2-pyrrolidone, providing a robust foundation for its industrial production.