[Background] The final step of the peptide nucleoside antibiotic Blasticidin S (BS) biosynthesis pathway is the leucylblasticidin S (LBS) hydrolyzation to BS. The intact cells of BS native producer Streptomyces griseochromogenes and the heterologous producer Streptomyces lividans WJ2 can catalyze this step. Earlier work showed that both strains encode three PepN homologues, of which PepN1 from each strain is mainly responsible for the hydrolysis of LBS and leucyldemethylblasticidin S (LDBS) at comparable catalytic efficiency. However, comparing with the native producer that only synthesizes BS, WJ2 can also generate LBS and LDBS. This result indicates that the competence of PepN1 is different in these two producers. [Objective] Investigation of the effects of the expression level and localization of PepN1 on the productivity and components of BS and derivatives in two producers. [Methods] The concentration of PepN1 in the cell lysate and in the cell culture medium of two BS producers at different growth time were tracked and compared by Western blot using PepN1 polyclonal antibody. The protein in the culture medium of the two strains was collected by ammonium sulfate precipitation, and the activity of hydrolyzed LDBS was assayed in vitro. [Results] On one hand, the content of PepN1 in the native strain didn’t change from the 2nd to 6th day, whereas PepN1 started to diminish from the 4th day and totally disappeared on the 6th day in the heterologous strain. On the other hand, PepN1 was detected in the culture medium of native producer rather than the heterologous strain, which was consistent with the different activity of two strain’s cell culture medium in hydrolyzing LDBS. [Conclusion] The BS native producer can continuously express PepN1 and export part of PepN1 outside of the cell. On the contrary, the heterologous producer WJ2 cease to synthesize PepN1 from the 4th day and it can’t be detected in the culture medium, leading to the accumulation of leucylated intermediates. Given that intact cells of two strains are capable of hydrolyzing LDBS and LBS, PepN1 that diminished from the cell lysate might secrete to the cell wall, but not release into the medium, this part of activity accounts for the generation of DBS and BS in WJ2.