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A Study of Type II ɛ-PL Degrading Enzyme (pldII) in Streptomyces albulus through the CRISPRi System
Qinyu Li1,2,3, Xiaojia Chen1,2,3, Yuanjie Wu4
1Lab of Biorefinery, Shanghai Advanced Research Institute, Chinese Academy of Sciences, No. 99 Haike Road, Shanghai 201210, China.
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.
Insights
Repressing the ε-PL degrading enzyme (pldII) in S. albulus enhances the antibacterial activity of ε-Poly-L-lysine (ε-PL). This study reveals pldII
Area of Science:
- Microbiology
- Molecular Biology
- Biochemistry
Background:
- ε-Poly-L-lysine (ε-PL) is a peptide with significant antibacterial properties, influenced by its polymerization degree.
- The precise mechanism by which ε-PL is degraded in *S. albulus* has not been fully elucidated.
- Understanding ε-PL degradation is crucial for optimizing its antibacterial applications.
Purpose of the Study:
- To investigate the role of the ε-PL degrading enzyme (pldII) in *S. albulus*.
- To elucidate the degradation mechanism of ε-PL by pldII.
- To enhance the antibacterial efficacy of ε-PL by modulating pldII expression.
Main Methods:
- Utilized a pSET152-based CRISPRi system to transcriptionally repress the expression of the *pldII* gene.
- Conducted ε-PL bacteriostatic experiments with engineered *S. albulus* strains.
- Analyzed ε-PL molecular weight distribution using MALDI-TOF-MS.
- Performed homology modeling and substrate docking of the pldII enzyme.
Main Results:
- Repression of *pldII* significantly improved the antibacterial effect of ε-PL.
- MALDI-TOF-MS analysis showed altered molecular weight distribution of ε-PL in repression strains.
- Specific repression strains produced ε-PL with higher polymerization degrees (e.g., 44 in S1).
- pldII was identified as a metallopeptidase that hydrolyzes ε-PL from the N-terminus.
Conclusions:
- The ε-PL degrading enzyme (pldII) plays a key role in reducing the antibacterial activity of ε-PL in *S. albulus*.
- Targeting pldII expression is a viable strategy to enhance ε-PL's efficacy.
- The study provides insights into the molecular mechanism of ε-PL degradation by pldII.
