Pathway optimization plays an important role in fine-tuning metabolic pathways. In most conditions, more than three genes are involved in the biosynthesis pathway of a specific target product. To improve the titer of products, rational regulation of a group of genes by a series of promoters with different strengths is essential. On the basis of a series of RNA-Seq data, a set of 66 native promoters was chosen to fine-tune gene expression in . Promoter strength was characterized by measuring the fluorescence strength of the enhanced green fluorescent protein through fluorescence-activated cell sorting. The expressions of P, P, P, P, and P were stronger than that of P, whereas those of another 15 promoters were stronger than that of P. Then, 30 promoters were chosen to optimize the biosynthesis pathway of (2)-naringenin from -coumaric acid. With a high-throughput screening method, the highest titer of (2)-naringenin in a 5 L bioreactor reached 1.21 g/L from -coumaric acid, which is the highest titer according to the currently available reports.