Novel Reprogramming of Polyketide Synthase for Valerolactam Production

δ-Valerolactam (VL), as an organic compound, is an important precursor chemical for nylon and has a wide range of applications in organic synthesis, pharmaceutical synthesis, polymer materials, and other fields. This study introduces a novel biosynthetic method for producing VL in the engineered strain Escherichia coli BL21 through the reprogramming of polyketide synthases (PKS). Initially, an in vitro multienzyme system was constructed to verify the reliability of the VL synthesis pathway. Subsequently, an optimized biosynthetic pathway was established in E. coli, converting l-aspartate to VL with a yield of 3.66 mg/L in a 250 mL shake flask. Various engineering strategies were then implemented to enhance VL production, including substrate–enzyme affinity modification and multidimensional substrate optimization. These methods resulted in a 3.7-fold increase in VL yield, reaching 13.5 mg/L in shake flask cultures. Further scale-up in a 5 L fed-batch fermenter achieved a VL concentration of 76.2 mg/L. This research provides innovative insights into the optimization of VL production pathways and industrial-scale production.