CRISPR/Cas9-Mediated Polyketide Synthase Replacement for High-Yield Biosynthesis and Biological Activity of Milbemycin D

Milbemycin D is a promising 16-membered macrolide insecticide with reported superior efficacy, but its commercial development has been hindered by extremely low natural yields. This study aimed to construct a high-yielding microbial platform for milbemycin D production using combinatorial biosynthesis and advanced genome editing. An optimized CRISPR/Cas9-AcrIIA4 system was employed to seamlessly replace the aveA3 polyketide synthase (PKS) gene in the ivermectin B1b-producing strain Streptomyces avermitilis HU501 with the heterologous milA3 PKS from S. bingchenggensis. The engineered strain was validated genetically and metabolically, followed by high-throughput screening and fermentation optimization in various media. The biosynthesized compound was structurally confirmed by spectroscopy. Bioactivity was evaluated against Bursaphelenchus xylophilus, Hyphantria cunea, and Plutella xylostella. The engineered strain S. avermitilis HU501-M successfully shifted its major product to milbemycin D, reaching a final titer of 679.03 mg/L. Bioassays revealed that milbemycin D exhibited significantly enhanced potency, with LC50 values 8–24% lower than those of milbemycin A3/A4. This work demonstrates an efficient CRISPR/Cas9-mediated PKS replacement strategy to achieve the high-yield production of milbemycin D, offering a promising microbial source and a generalizable framework for engineering complex polyketide pathways. This proof-of-concept establishes a foundation for future process development toward potential commercial application.