Genome shuffling was adopted to breed a recombinant strain named Streptomyces sp.Feel-1 for producing ε-poly-L-lysine(ε-PL) in this research.First Feel-1 was mutated by mutagen EMS and spread on agar plate supplemented with Gly and AEC(S-2-ammonia ethyl-L-homocysteine) as resistance markers.Four improved mutants were obtained in shake-flask fermentation with ε-PL production as 1.78 g/L,1.89 g/L,1.85 g/L,1.86 g/L,respectively,which was increased by 11.3%,18.1%,15.6% and 16.3% compared with Feel-1(1.6 g/L).Then these four positive mutants were used as starting population and subjected for three rounds of genome shuffling by fusing the inactivated protoplasts.Finally three recombinants were proved to give a high yield of ε-PL production as 2.42 g/L,2.35 g/L and 2.37 g/L,respectively,which was over Feel-1 by 51.3%,46.9% and 48.1%,respectively.Fed-batch fermentation by one of the recombinants G3-67 was carried out and the ε-PL production accumulated to 35 g/L,which was the highest reported level in China at present.