Streptomyces are famed producers of secondary metabolites with diverse bioactivities and structures. However, biosynthesis of natural products will consume vast precursors from primary metabolism, and some secondary metabolites are toxic to the hosts. To overcome this circumstance and over-produce secondary metabolites, one of the strategies is to over-express biosynthetic genes under strong promoters specifically expressed during secondary metabolism. For this purpose, here based on Microarray and eGFP reporter assays, we obtained a promoter thlM4p, whose activity was undetectable in the first 2 days of fermentation, but sevenfold higher than the strong promoter ermE*p in the following days. Moreover, when the positive regulator gene scnRII was driven from thlM4p, natamycin yield increased 30% compared to ermE*p. Therefore, we provide a new way to identify promoters, which is silenced during primary metabolism while strongly expressed under secondary metabolism of Streptomyces.