CRISPRi-mediated multigene downregulating redirects the metabolic flux to spinosad biosynthesis in Saccharopolyspora spinosa

Microorganisms are often likened to complex production workshops. In Saccharopolyspora spinosa (S. spinosa), the biosynthesis of spinosad is a production line within its intricate workshop. Optimizing the entire production environment and reducing unnecessary metabolic flow is essential to increasing spinosad yield. Pyruvate serves as a crucial precursor for spinosad biosynthesis. Previous studies revealed that the pyc gene is highly expressed in the gluconeogenic pathway, leading to a pyruvate shunt. By downregulating pyc, we enhanced spinosad yield, although the improvement was below expectations. We speculated that most of the accumulated pyruvate following the pyc knockdown entered some synthetic pathways unrelated to spinosad. Through metabolic pathway and qRT-PCR analyses, we found that the expression levels of gltA1 and atoB3 within the pyruvate metabolic tributary, including the TCA cycle and ethylmalonyl-CoA pathway, were significantly increased in the pyc knockdown strain. The combined knockdown of these three genes optimized the spinosad production line, increasing its yield to 633.1 ± 38.6 mg/L, representing a 199.4 % increase. This study identifies three key genes for optimizing spinosad biosynthesis and offers insights into gene screening and the efficient construction of Spinosad-producing strains.