Abstract: Fabclavines produced by Xenorhabdus budapestensis strain XBD8 exhibit broad-spectrum antimicrobial activity, showing significant potential for application in the control of plant disease. However, their low yield and high application costs have hindered industrial development. This study aimed to construct high-yield Fabclavine-producing strains through a combined strategy of genome reduction and promoter engineering. Genome mining of Xenorhabdus budapestensis strain XBD8 using the bioinformatics tool antiSMASH identified 11 secondary metabolite biosynthetic gene clusters(BGCs) in addition to the Fabclavine BGC. Iterative knockout of core biosynthetic genes from these BGCs generated a streamlined chassis strain, designated FRT12. Notably, gene2204, involved in the biosynthesis of Fabclavines Fcl-7 and Fcl-8, was identified during this genome reduction process. Promoter engineering strategy was applied to replace the promoters of genes fclC and gene2204, leading to the successful construction of the dual-promoter replacement strain MPW9. The yield of Fabclavines was increased from 73.4 mg/L to 121.3 mg/L. By swapping these two promoters in the streamlined chassis strain FRT12, the constructed strain MPW11 demonstrated further improved Fabclavines production,reaching 142.6 mg/L in shake-flask fermentation. These findings establish a foundation for industrial-scale production of Fabclavines.

Key words: fabclavines, Xenorhabdus budapestensis, genome streamlining, promoter engineering