Enhancement of Spinosyn Production by Integrating a Static and Dynamic CRISPRi-Mediated Metabolic Switch in <i>Saccharopolyspora spinosa</i>

Spinosyns, polyketide-derived macrolide insecticides produced by <i>Saccharopolyspora spinosa</i>, have broad market prospects. However, their large-scale production is still challenged by the low titers of industrial strains. Here, we developed an integrated static and dynamic CRISPRi strategy to redirect metabolic flux toward spinosyn overproduction without affecting cell growth. Initially, static CRISPRi-mediated repression of four genes (<i>gltA1</i>, <i>fabH3</i>, <i>fabH4</i>, and <i>glgC</i>) in three key nodes of primary metabolism boosted spinosyn titers by 19-55% without growth impairment. Simultaneous repression of these genes led to a further increase of spinosyn production. However, static repression of two other genes (<i>pyc</i> or <i>gltA2</i>) severely hindered bacterial growth and reduced spinosyn biosynthesis. To address this, two growth phase-dependent promoters from the spinosyn biosynthetic gene cluster (BGC) were characterized and employed for dynamic repression of <i>pyc</i> and <i>gltA2</i>, resulting in increased spinosyn titers by 44-68% while maintaining robust growth. Finally, combined static and dynamic repression of these six targets simultaneously achieved over a 2-fold increase of spinosyn production. Collectively, integrated static and dynamic CRISPRi-based reprogramming of primary metabolic pathways in <i>S. spinosa</i> achieved a balance between growth and secondary metabolism, thereby leading to spinosyn overproduction. This strategy has broad potential applicability in other actinomycetes for natural product overproduction.