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Summary  Heterologous expression of the biosynthetic gene cluster (BGC) is important for studying the microbial natural products (NPs), especially for those kept in silent or poorly expressed in their original strains. Here, we cloned the spinosad BGC through the Cas9‐Assisted Targeting of Chromosome segments and amplified it to five copies through a ZouA‐dependent DNA amplification system in  Streptomyces coelicolor  M1146. The resulting strain produced 1253.9 ± 78.2 μg l −1  of spinosad, which was about 224‐fold compared with that of the parent strain carrying only one copy of the spinosad BGC. Moreover, we further increased spinosad to 1958.9 ± 73.5 μg l −1  by the dynamic regulation of intracellular triacylglycerol degradation. Our study indicates that tandem amplification of the targeted gene cluster is particularly suitable to enhance the heterologous production of valuable NPs with efficiency and simplicity.

Multiplying the heterologous production of spinosad through tandem amplification of its biosynthetic gene cluster in Streptomyces coelicolor