The biosynthesis of Iturin A, a compound with potent antimicrobial properties, has attracted considerable interest. However, challenges persist in achieving high yields from wild-type strains and optimizing Bacillus amyloliquefaciens for industrial-scale production. This study investigates the genetic foundations of Iturin A biosynthesis, emphasizing the roles of endogenous plasmids and Rap phosphatases. By optimizing and updating CRISPR/Cas9-based gene-editing tools, the modifications were successfully applied to the genetic manipulation of Bacillus amyloliquefaciens. The deletion of the endogenous plasmid plas1 from B. amyloliquefaciens HM618 significantly enhances Iturin A production, likely due to the removal of the Rap phosphatase gene located on plas1. Additionally, the targeted inactivation of the rapC, rapF, and rapH genes within the chromosomal DNA further increases Iturin A yield and specific productivity, although this is accompanied by reduced cell growth. By strategically combining plasmid deletion with targeted gene inactivation, a balance between cellular growth and Iturin A production is established. The engineered strain subsequently produced 849.9 mg/L of Iturin A within 48 hours, demonstrating a significant improvement in production efficiency. These findings underscore the crucial role of microbial genetics in optimizing Iturin A biosynthesis and offer a novel approach for enhancing the industrial production of this valuable antimicrobial compound in B. amyloliquefaciens.