Summary: Current challenge for dynamic pathway control in metabolic engineering is enabling the components of the artificial regulatory system to be tunable. Here, we designed and built a heme-responsive regulatory system containing a heme biosensor HrtR and CRISPRi to regulate chemicals production while maintaining the intracellular heme homeostasis. A series of engineered biosensors with varied sensitivity and threshold were obtained by semi-rational design with site saturated mutation of HrtR. The modified metabolite-binding affinity of HrtR was confirmed by heme titration and molecular dynamic simulation. Dynamic regulation pattern of the system was validated by the fluctuation of gene expression and intracellular heme concentration. The efficiency of this regulatory system was proved by improving the 5-aminolevulinic acid (ALA) production to 5.35g/L, the highest yield in batch fermentation of Escherichia coli. This system was also successfully used in improving porphobilinogen (PBG) and porphyrins biosynthesis and can be applied in many other biological processes.