<jats:title>Abstract</jats:title> <jats:p>Natamycin is an important polyene macrolide antifungal agent produced by several Streptomyces strains and is widely used as a food preservative and fungicide in food, medicinal and veterinary products. In order to increase the yield of natamycin, this study aimed at cloning and overexpressing a natamycin-positive regulator, slnM2, with different promoters in the newly isolated strain Streptomyces lydicus A02, which is capable of producing natamycin. The slnM gene in S. lydicus is highly similar to gene pimM (scnRII), the pathway-specific positive regulator of natamycin biosynthesis in S. natalensis and S. chattanoogensis, which are PAS-LuxR regulators. Three engineered strains of S. lydicus, AM01, AM02 and AM03, were generated by inserting an additional copy of slnM2 with an ermEp* promoter, inserting an additional copy of slnM2 with dual promoters, ermEp* and its own promoter, and inserting an additional copy of slnM2 with its own promoter, respectively. No obvious changes in growth were observed between the engineered and wild-type strains. However, natamycin production in the engineered strains was significantly enhanced, by 2.4-fold in strain AM01, 3.0-fold in strain AM02 and 1.9-fold in strain AM03 when compared to the strain A02 in YEME medium without sucrose. These results indicated that the ermEp* promoter was more active than the native promoter of slnM2. Overall, dual promoters displayed the highest transcription of biosynthetic genes and yield of natamycin.