Multifaceted metabolic engineering of Streptomyces hygroscopicus for milbemycins overproduction.

Milbemycins, 16-membered macrolide antibiotics structurally similar to avermectins, have significant applications in agricultural and veterinary pest control. However, their industrial production is hindered by low fermentation yields. To address this, we employed an iterative, multi-step strategy to engineer Streptomyces hygroscopicus SIPI-054 for milbemycin overproduction. First, deletion of three competitive pathways increased the titer of milbemycin A3/A4 by 34.2%. Second, integrating one to three additional copies of the milbemycin biosynthetic gene cluster (BGC) via site-specific recombination further boosted the titers by 71.1% to 112.8%. Finally, a dynamic regulatory strategy using the quorum sensing (QS)-responsive promoter (sbbAp) to drive CRISPRi was employed to control key primary metabolic nodes, including the citrate synthases (gltA) in the tricarboxylic acid cycle and β-ketoacyl-ACP synthases (fabH) in the fatty acid synthesis pathway. The optimal engineered strain achieved the highest reported shake-flask titer to date for milbemycin A3/A4 (6.14 g/L), representing a 3.4-fold increase over the starting strain. Collectively, this work demonstrates a powerful engineering strategy that combines competitive pathway elimination, gene dosage amplification, and dynamically regulated CRISPRi to effectively reprogram microbial metabolism, offering a generalizable framework for the industrial overproduction of Streptomyces natural products.