Abstract：
ε-Poly-L-lysine (ε-PL) is a widely used antibacterial peptide polymerized of 25-35 L-lysine residues. The antibacterial effect of ε-PL is closely related to the polymerization degree. However, the mechanism of ε-PL degradation in S. albulus remains unclear. This study utilized the integrative plasmid pSET152-based CRISPRi system to transcriptionally repress the ε-PL degrading enzyme (pldII). The expression of pldII is regulated by changing the recognition site of dCas9. Through the ε-PL bacteriostatic experiments of repression strains, it was found that the repression of pldII improves the antibacterial effect of the ε-PL product. The consecutive MALDI-TOF-MS results confirmed that the molecular weight distribution of the ε-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 ε-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 ε-PL in S. albulus. The hydrolysis of ε-PL within pldII, initiated from the N-terminus by two amino acid-binding residues, Thr194 and Glu281, led to varying levels of polymerization of ε-PL.
摘要：
ε-聚-L-赖氨酸（ε-PL）是一种广泛使用的抗菌肽，由25-35个L-赖氨酸残基聚合而成。ε-PL的抗菌效果与聚合度密切相关。然而,ε-PL降解S.albulus的机理尚不清楚。这项研究利用基于pSET152的整合质粒CRISPRi系统来转录抑制ε-PL降解酶（pldII）。通过改变dCas9的识别位点来调节pldII的表达。通过抑制菌株的ε-PL抑菌实验,发现PLDII的抑制改善了ε-PL产物的抗菌作用。连续的MALDI-TOF-MS结果证实,ε-PL的分子量分布在抑制后改变。抑制菌株S1显示出聚合度为44的特定峰，其他抑制菌株也产生聚合度超过40的ε-PL。此外，PLDII的同源建模和底物对接，一种典型的内型金属肽酶,进行了研究，以解决ε-PL在S.albulus中的降解机理。PldII内ε-PL的水解，由两个氨基酸结合残基从N端启动，Thr194和Glu281导致ε-PL的聚合水平变化。
