Abstract: Genome shuffling technique was used for breeding of Bacillus licheniformis TJ12 for enhanced bacitracin
production. Firstly, UV light, nitroso-guanidin (NTG) or atmospheric and room temperature plasma (ARTP) was used to
obtain a candidate mutant library. Based on the optimized conditions for protoplast preparation and regeneration, multiparental
whole genome shuffling was carried out with 4 mutant strains (U11, U23, H1, and L71) by polyethylene glycol
(PEG) mediated protoplasts fusion. As a result, the shuffled strain F3 was identified by biparental inactivation method to
show excellently improved antibacterial activity and good genetic stability. The maximum production of bacitracin A of
760 mg/L was obtained when the strain F3 was cultured in shake flasks for 36 h, which was increased 1.7 folds compared with
the initial strain TJ12. The strain F3 moved earlier into the stable growth period, but its final biomass was lower than that of
the wild strain. There was almost no difference in pH during the fermentation process. The production of reducing sugar and
bacitracin by the strain F3 was higher compared with the wild strain. Additionally, the expression of regulatory genes was
upregulated in the strain F3, and the expression of synthetic gene was upregulated more significantly. With a higher production
of bacitracin in each cell of the strain F3, we speculated that the strain had a more powerful mechanism of self-tolerance to
bacitracin and the relevant parts of the synthetic genes might be changed when compared with the initial strain.

Key words: bacitracin, Bacillus licheniformis, genome shuffling, mutagenesis, protoplast