Whole-Genome Analysis of Bacillus Licheniformis Ali5 and Synthesis of Lichenysin via Genome Shuffling

Whole-genome sequencing of Bacillus licheniformis Ali5 was performed via MGI-seq PE150 and Nanopore single-molecule real-time sequencing. The strain has a 4,114,664 bp circular genome encoding 4030 protein-coding genes. Functional annotation across NR, COG, GO, KEGG, CARD, BacMet, and CAZy databases identified 4025, 2812, 988, 1242, 72, 69, and 94 corresponding genes, respectively, and antiSMASH 6.0 revealed multiple antimicrobial biosynthetic gene clusters, including intact lichenysin and lichenicidin VK21 A1/A2 gene clusters. Three rounds of recursive protoplast fusion-based genome shuffling, paired with a dual-index screening system, significantly improved strain growth and lichenysin biosynthesis. Recombinants exhibited shortened lag phase, enhanced proliferation, improved stationary-phase stability, and higher diauxic peak biomass. PP3-176 and PP3-186 showed 4.6%-8.1% higher 12-h shake-flask titer and 3.1%-4.0% higher maximum titer than the parental average, with excellent fermentation stability. 1-L bioreactor validation confirmed strong scale-up potential. PP3-186 achieved 27.2% and 31.6% titer increases at 12 h and 20 h, while PP3-176 yielded 20.4% and 14.6% improvements with robust metabolic performance. This study validates genome shuffling as an effective strategy for enhancing lichenysin production, providing candidate strains and technical support for industrial application.