Aim The aim of the present work was to investigate the overexpression of thewysRgene inStreptomyces albulusvar. wuyiensis strain CK-15 based on the Delta wysR3mutant strain including the effect on morphological development, wuyiencin production and antibacterial activity. At the same time, we report a new rapid method for producing genetically engineered strains for industrial production of wuyiencin. Methods and Results We developed a method to create awysRoverexpression strain based on the Delta wysR3mutant strain by direct transformation. In this method, the desired gene fragment to be overexpressed was amplified by polymerase chain reaction (PCR) using Phusion High Fidelity DNA polymerase and fused with the linearized pSETC integrative plasmid by Gibson assembly. The resulting recombinant plasmid was transformed into Delta wysR3mutant strain by the intergeneric conjugation method. The plasmid was then integrated into the chromosome and the resulting apramycin-resistant overexpression strain was confirmed by PCR using the Apra-F and Apra-R primers. Finally, we successfully screened the genetically engineered strain with overexpression ofwysRgene in Delta wysR3mutant. Conclusion We can conclude that overexpression ofwysRgene in Delta wysR3mutant strain proved to be an effective strategy for significantly increasing wuyiencin production together with faster morphological development. Quantitative real-time RT-PCR analysis showed thatwysRregulated wuyiencin biosynthesis by modulating other putative regulatory genes andbld,whi,chp,rdlandramfamily genes are crucial for the morphological development. Significance and Impact of the Study Overexpression ofwysRgene in the Delta wysR3mutant strain named OoWysR strain may increase the efficiency in the industrial fermentation processes for wuyiencin production. The mechanism by whichwysRoverexpression promotes rapid sporulation and a high yield of wuyiencin production is likely related to modulation of other putative regulatory genes.