epsilon-poly-L-lysine (epsilon-PL) is an excellent antimicrobial agent and produced by Streptomyces albulus. To improve the epsilon-PL production of S. albulus M-Z18, a modified method of ribosome engineering with continuous introduction of streptomycin resistance was adopted in this study. The highest-yield mutant S. albulus SS-62 was generated from 1631 mutants screening, and achieved epsilon-PL production of 3.04 g/L in shake-flask fermentation, which was 1.79-fold higher than the parent strain. In order to reveal the high-yield mechanism of S. albulus SS-62, mutation sites, global protein expression, key enzymes activities and the corresponding gene transcriptions, intracellular flux distribution and cofactors in the epsilon-PL biosynthesis pathway were investigated. We found continuous introduction of streptomycin resistance caused mutations in rpsL (encoding ribosomal protein S12) and rsmG (encoding 16S rRNA methyltransferase). Moreover, the expression changes of regulators and molecular chaperones related to transcription and translation process were occurred, and some key enzymes activities and metabolic flux in the pathway of epsilon-PL biosynthesis were increased. It could be reasonably speculated that these changes eventually led to a significant increase of epsilon-PL production in S. albulus SS-62. Taken together, the present study not only enhanced the epsilon-PL production of S. albums M-Z18 but also provided an insight into the high-yield mechanism of S. albulus SS-62.