Abstract:[Objective] A high yield of ε-poly-L-lysine (ε-PL) producing strain was bred, and the effect of different carbon sources on fermentation performance was studied.[Methods] Genome shuffling and ribosome engineering were used to enhanced strain's productivity, and pH shock strategy was used to fermentation using different carbon sources.[Results] After four rounds of genome shuffling and ribosome engineering, we obtained a high yield mutant Streptomyces albulus GS114 with ε-PL productivity of 3.0 g/L, which was 1.7 folds than that of the initial strain. When we performed fed-batch fermentation using glucose and glycerol as carbon sources in a 5-L fermenter, ε-PL productions reached 43.4 and 45.7 g/L after 192 h fed-batch fermentations, which were increased by 11.0% and 14.9% than that of Streptomyces albulus M-Z18, respectively. Meanwhile, the dry cell weights decreased by 24.0% and 33.2%, and ε-PL yields increased by 34.2% and 30.7%, respectively.[Conclusion] Genome shuffling and ribosome engineering are effective to breed high yield strains.