Study of Type II epsilon-PL Degrading Enzyme (pldII) in Streptomyces albulus through the CRISPRi System

epsilon-Poly-L-lysine (epsilon-PL) is a widely used antibacterial peptide polymerized of 25-35 L-lysine residues. The antibacterial effect of epsilon-PL is closely related to the polymerization degree. However, the mechanism of epsilon-PL degradation in S. albulus remains unclear. This study utilized the integrative plasmid pSET152-based CRISPRi system to transcriptionally repress the epsilon-PL degrading enzyme (pldII). The expression of pldII is regulated by changing the recognition site of dCas9. Through the epsilon-PL bacteriostatic experiments of repression strains, it was found that the repression of pldII improves the antibacterial effect of the epsilon-PL product. The consecutive MALDI-TOF-MS results confirmed that the molecular weight distribution of the epsilon-PL was changed after repression. The repression strain S1 showed a particular peak with a polymerization degree of 44, and other repression strains also generated epsilon-PL with a polymerization degree of over 40. Furthermore, the homology modeling and substrate docking of pldII, a typical endo-type metallopeptidase, were performed to resolve the degradation mechanism of epsilon-PL in S. albulus. The hydrolysis of epsilon-PL within pldII, initiated from the N-terminus by two amino acid-binding residues, Thr194 and Glu281, led to varying levels of polymerization of epsilon-PL.