Data_Sheet_1_Engineering circular bacteriocins: structural and functional effects of α-helix exchanges and disulfide introductions in circularin A.PDF

Circular bacteriocins form a distinct group of antimicrobial peptides (AMPs) characterized by their unique head-to-tail ligated circular structure and functional properties. They belong to the ribosomally synthesized and post-translationally modified peptide (RiPP) family. The ribosomal origin of these peptides facilitates rapid diversification through mutations in the precursor genes combined with specific modification enzymes. In this study, we primarily explored the bacteriocin engineering potential of circularin A, a circular bacteriocin produced by Clostridium beijerinckii ATCC 25752. Specifically, we employed strategies involving α-helix replacements and disulfide bond introductions to investigate their effects on both biosynthesis and bioactivity of the bacteriocin. The results show the feasibility of peptide engineering to introduce certain structural properties into circularin A through carefully designed approaches. The introduction of cysteines for potential disulfide bonds resulted in a substantial reduction in bacteriocin biosynthesis and/or bioactivity, indicating the importance of maintaining dynamic flexibility of α-helices in circularin A, while reduction of the potential disulfide in one case increased the activity. The 5 α-helices of circularin A were respectively replaced by corresponding helices from another circular peptide, enterocin AS-48, and modestly active peptides were obtained in a few cases. Overall, this study provides valuable insights into the engineering potential of circular bacteriocins as antimicrobial agents, including their structural and functional restrictions and their suitability as peptide engineering scaffolds. This helps to pave the way for the development of novel antimicrobial peptides with tailored properties based on circular bacteriocins.

环状细菌素构成一类独特的抗菌肽（AMPs），其特征在于其独特的头尾连接的环状结构和功能特性。它们属于由核糖体合成的和翻译后修饰的肽（RiPP）家族。这些肽的核糖体起源通过前体基因中的突变与特定的修饰酶相结合，促进了其快速多样化。在本研究中，我们主要探讨了由产气荚膜梭菌ATCC 25752产生的环状细菌素环状素A的细菌素工程潜力。具体而言，我们采用了涉及α-螺旋替换和二硫键引入的策略，以研究其对细菌素生物合成和生物活性的影响。结果表明，通过精心设计的方法，通过肽工程将某些结构特性引入环状素A是可行的。引入半胱氨酸以形成潜在的二硫键导致细菌素生物合成和/或生物活性显著降低，这表明维持环状素A中α-螺旋的动态灵活性至关重要，而在一种情况下，减少潜在的二硫键反而增加了活性。环状素A的5个α-螺旋分别被来自另一种环状肽肠球菌素AS-48的相应螺旋所替换，在少数情况下获得了具有一定活性的肽。总体而言，本研究为环状细菌素作为抗菌剂的工程潜力提供了宝贵的见解，包括它们的结构和功能限制以及作为肽工程支架的适用性。这有助于为基于环状细菌素的具有定制特性的新型抗菌肽的开发铺平道路。