Here we first improved the epsilon-PL productivity in five species of wild-type strains in Streptomyces (Streptomyces padanus, Streptomyces griseofuscus, Streptomyces graminearus, Streptomyces hygroscopicus, and Streptomyces albulus) by genome shuffling. Then all the shuffled strains were suffered from an interspecific hybridization through stochastic protoplast fusion. One hybrid designated FEEL-1 was selected by morphology and spore color with epsilon-PL production of 1.12 g/L in shake flask, about 2.75-fold higher than that in wild types. The epsilon-PL production of FEEL-1 was then obtained as 24.5 g/L in fed-batch fermentation, which was 63-81 % higher than those in shuffled strains. Random amplified polymorphic DNA revealed that FEEL-1 was probably hybridized from S. padanus, S. griseofuscus, and S. albulus. Activities of several enzymes in FEEL-1 (hexokinase, phosphoenolpyruvate carboxylase, aspartokinase, and citrate synthase) were more active than those in shuffled strains, which was a possible reason for the enhancement of epsilon-PL production. This research highlights the importance of genome shuffling along with interspecific hybridization as a new breeding strategy for improving phenotype of industrial strains.