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

Microorganisms are often likened to complex production workshops. In <i>Saccharopolyspora spinosa</i> (<i>S. spinosa</i>), 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 <i>pyc</i> gene is highly expressed in the gluconeogenic pathway, leading to a pyruvate shunt. By downregulating <i>pyc</i>, we enhanced spinosad yield, although the improvement was below expectations. We speculated that most of the accumulated pyruvate following the <i>pyc</i> knockdown entered some synthetic pathways unrelated to spinosad. Through metabolic pathway and qRT-PCR analyses, we found that the expression levels of <i>gltA1</i> and <i>atoB3</i> within the pyruvate metabolic tributary, including the TCA cycle and ethylmalonyl-CoA pathway, were significantly increased in the <i>pyc</i> 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.