In this study, we carried out genome-scale CRISPRi modulation combining fluorescence-activated cell sorting (FACS) and next-generation sequencing (NGS) to identify genetic determinants for FFAs overproduction in Escherichia coli. The pcnBi (AP) strain that repressed the expression of pcnB produced 2992 mg l FFAs, which was 4.0-fold of the CF (A) strain. To analyze the underlying mechanism of FFAs overproduction, the engineered strain pcnBi and the control strain CF were applied to the transcriptomic and proteomic analyses.