Liu Fangfang, van Heel Auke J, Chen Jingqi, Kuipers Oscar P
Department of Molecular Genetics, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Groningen, Netherlands.
Front Microbiol. 2022 Nov 23;13:1026290. doi: 10.3389/fmicb.2022.1026290. eCollection 2022.
Circular bacteriocins, also known as bacterial head-to-tail cyclized peptides, are a subgroup of ribosomally synthesized and post-translationally modified peptides (RiPPs). Compared with their conventional linear counterparts, circular bacteriocins are highly stable over a broad temperature and pH range, and circularization decreases proteolytic degradation by exopeptidases. These features render them great potential as scaffold candidates to withstand strident conditions in food- and pharmaceutical applications. However, the biosynthesis and bioactivity of circular bacteriocins still remain largely unknown. To investigate and gain more insights into the biosynthesis of circular bacteriocins and to achieve efficient production and characterization of bacteriocin variants, we developed an efficient cloning and heterologous expression system for clostridial circularin A and successfully produced this circular peptide in NZ9000. We report three system formats with single plasmid or plasmid combinations to achieve successful cloning and functional production of circularin A in . These systematic varieties enabled us to choose the appropriate method to efficiently obtain various constructs with desired properties. With the established heterologous systems in , we performed several mutagenesis studies in the precursor peptide to study its structure/function relationships. The overlay activity assay revealed that these mutant variants had variable effects on different indicator strains: lysine substitution for certain glutamine residue(s) greatly decreased its bioactivity against and NZ9000, and alanine replacement for the cationic residues significantly reduced the activity against ATCC 15521, whereas alanine substitution for the aromatic residues decreased its bioactivity against all three testing strains dramatically. Moreover, the conditions for bacteriocin production were optimized. Results show that supplementing the minimal medium with extra glucose (or sucrose) and immediate nisin-induction improved the peptide yield significantly. Briefly, we developed an excellent system for the production of circularin A and a wide range of variant peptides in a convenient host, as well as a method for fast detection of peptide production and activity. This system facilitated our mutagenesis studies which provided valuable insights into the effects of mutating specific residues on its biosynthesis and bioactivity, and will eventually enable more complex research into the biosynthesis of circularin A.
环状细菌素，也被称为细菌头尾环化肽，是核糖体合成及翻译后修饰肽（RiPPs）的一个亚群。与传统的线性同类物相比，环状细菌素在很宽的温度和pH范围内都高度稳定，并且环化作用减少了外肽酶引起的蛋白水解降解。这些特性使其作为支架候选物在食品和制药应用中抵御严苛条件方面具有巨大潜力。然而，环状细菌素的生物合成和生物活性在很大程度上仍然未知。为了研究并更深入了解环状细菌素的生物合成，以及实现细菌素变体的高效生产和表征，我们开发了一种用于梭菌环形菌素A的高效克隆和异源表达系统，并在NZ9000中成功生产了这种环状肽。我们报告了三种系统形式，包括单质粒或质粒组合，以在NZ9000中成功克隆并功能性生产环形菌素A。这些系统变体使我们能够选择合适的方法来高效获得具有所需特性的各种构建体。利用在NZ9000中建立的异源系统，我们对前体肽进行了多项诱变研究，以研究其结构/功能关系。覆盖活性测定表明，这些突变变体对不同指示菌株有不同影响：某些谷氨酰胺残基被赖氨酸取代大大降低了其对乳酸乳球菌和NZ9000的生物活性，阳离子残基被丙氨酸取代显著降低了对金黄色葡萄球菌ATCC 15521的活性，而芳香族残基被丙氨酸取代则显著降低了其对所有三种测试菌株的生物活性。此外，还优化了细菌素的生产条件。结果表明，在基本培养基中添加额外的葡萄糖（或蔗糖）并立即进行乳链菌肽诱导可显著提高肽产量。简而言之，我们开发了一个出色的系统，用于在便捷的宿主中生产环形菌素A和多种变体肽，以及一种快速检测肽生产和活性的方法。该系统促进了我们的诱变研究，为特定残基突变对其生物合成和生物活性的影响提供了有价值的见解，并最终将使对环形菌素A生物合成的更复杂研究成为可能。
