Enhancement of spinosad production in Saccharopolyspora spinosa by overexpression of the complete 74-kb spinosyn gene cluster.

BACKGROUND

Spinosad, a secondary metabolite produced by Saccharopolyspora spinosa, is a polyketide macrolide insecticide with low toxicity and environmental friendliness. Owing to the high level of DNA methylation and unclear regulatory mechanisms, gene engineering to increase spinosad production is challenging. Limited improvements in yield have been observed with heterologous expression or partial overexpression of the 74-kb spinosyn gene cluster (spn), and research on the overexpression of the complete spinosyn gene cluster is lacking.

RESULTS

The plasmid pCM265-spn was constructed using CRISPR/Cas9-mediated Transformation-Associated Recombination cloning to enable the overexpression of the complete spn gene cluster in Sa. spinosa. The engineered strain Sa. spinosa-spn achieved a 124% increase in spinosad yield (693 mg/L) compared to the wild type (309 mg/L). The overexpression of the spn gene cluster also delayed spore formation and reduced hyphal compartmentalization by influencing the transcription of related genes (bldD, ssgA, whiA, whiB, and fstZ). Transcriptional analysis revealed significant upregulation of genes in the spn gene cluster, thereby enhancing secondary metabolism. Additionally, optimization of the fermentation medium through response surface methodology further increased spinosad production to 920 mg/L.

CONCLUSIONS

This study is the first to successfully overexpress the complete spn gene cluster in Sa. spinosa, significantly enhancing spinosad production. These findings have significance for further optimization of spinosad biosynthesis.