dTSR enables efficient improvement of heterologous production of spinosad in .

Spinosad (spinosyns A and D) is a highly effective and environmentally friendly insecticide widely used for pest control. However, the difficulty of genetic manipulation in the original strain, , has hindered improvements in fermentation yields using synthetic biology methods. Additionally, there is a lack of simple and effective methods for enhancing the production of polyketide natural products derived from slow-growing rare actinomycetes. In this study, we developed a doubly transposition and site-specific recombination (dTSR) approach to insert bacterial attachment sites () and two copies of spinosad biosynthetic gene cluster ( BGC) into various chromosomal locations of , thereby generating heterologous production strains for screening of spinosyn producers with improved yields. Engineered strains from the first round of TSR breeding produced spinosad at levels ranging from 5.6 to 30.5 mg/L. The second round of TSR breeding produced engineered strains with increased yields, with the highest spinosad production reaching 137.1 ± 10.9 mg/L. These results indicated that (1) the dTSR approach could efficiently generate initial heterologous strains with significantly improved spinosad production, and (2) the dTSR approach enabled random integration of a second copy of BGC into various chromosomal locations in , thereby further increasing heterologous spinosad production to high levels. This study provides a simple, rational, and efficient approach to improve the heterologous production of polyketide natural products in rare actinomycetes.