Improving butenyl-spinosyn production in Saccharopolyspora pogona through combination of metabolic engineering and medium optimization

Butenyl-spinosyn is a high-quality biological insecticide produced by <i>Saccharopolyspora pogona</i> that effectively targets a broad range of insect pests. However, the large-scale production of this insecticide is hindered by its low yield. Herein, based on prior comparative genomic analysis, five mutations were individually overexpressed in aG6. Subsequently, the combinatorial overexpression of <i>sp1322</i> (encoding NAD-glutamate dehydrogenase) and <i>sp6746</i> (encoding dTDP-glucose 4,6-dehydratase) in aG6 resulted in strain O1322-6746. The production of butenyl-spinosyn in O1322-6746 was 77.1% higher than that in aG6. Comparative targeted metabolomic analysis uncovered that O1322-6746 exhibited increased metabolic flux toward butenyl-spinosyn precursors. Furthermore, single-factor experiments, Plackett-Burman analysis and response surface methodology were performed to optimize the fermentation medium for O1322-6746. Ultimately, butenyl-spinosyn production was enhanced to 298.5 mg/L in a 5-L bioreactor, marking the highest yield ever reported. This work demonstrated that combining metabolic engineering with medium optimization is an effective strategy to improve butenyl-spinosyn production.