Guo Longcheng, Kuipers Oscar P, Broos Jaap
Department of Molecular Genetics, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Groningen 9747 AG, The Netherlands.
ACS Synth Biol. 2025 Sep 19;14(9):3568-3577. doi: 10.1021/acssynbio.5c00353. Epub 2025 Aug 12.
Glycosylation, a widespread post-translational modification, is present in all kingdoms of life. Despite the extensive structural diversity found in ribosomally synthesized and post-translationally modified peptides (RiPPs), only a few glycosylated bacteriocins, known as glycocins, have been identified. Notably, glycocins such as glycocin F, ASM1, and enterocin F4-9, exhibit antimicrobial properties and distinct glycoactivity, indicating that glycosylation is crucial for their bioactivity. The development of practical, and widely applicable systems for glycosylation of RiPPs is therefore highly desirable. In this study, we introduce an expression system that utilizes as a host for the efficient incorporation of the noncanonical amino acid homopropargylglycine (Hpg) into the well-studied RiPP nisin, and some structurally related variants. Hpg, which has an alkyne functional group, allows for further chemical modifications with azido-sugar containing substrates through click chemistry. We reveal that glycosylated nisin at position 17 shows strong activity against strains, but its activity against other pathogens such as , , and is reduced. Moreover, mode of action studies show that the addition of sugar diminishes its typical pore-forming ability of nisin against while preserving its lipid II binding ability. Interestingly, the addition of a hydrophilic sugar significantly enhances its water solubility around 4-fold at neutral pH, indicating potential for improved drug applications. These findings highlight the potential of this methodology for glycosylation of RiPPs, leading to the creation of new antimicrobial products with varied characteristics. This also broadens the toolkit for enhancing and discovering peptide-based drugs.
糖基化是一种广泛存在的翻译后修饰，存在于所有生命王国中。尽管在核糖体合成和翻译后修饰的肽（RiPPs）中发现了广泛的结构多样性，但仅鉴定出少数糖基化的细菌素，即糖菌素。值得注意的是，诸如糖菌素F、ASM1和肠菌素F4-9等糖菌素具有抗菌特性和独特的糖活性，表明糖基化对其生物活性至关重要。因此，非常需要开发实用且广泛适用的RiPPs糖基化系统。在本研究中，我们引入了一种表达系统，该系统利用作为宿主，将非天然氨基酸高炔丙基甘氨酸（Hpg）高效掺入到经过充分研究的RiPP乳链菌肽以及一些结构相关的变体中。具有炔基官能团的Hpg允许通过点击化学与含叠氮糖的底物进行进一步的化学修饰。我们发现，17位糖基化的乳链菌肽对菌株具有很强的活性，但其对其他病原体如、和的活性降低。此外，作用方式研究表明，糖的添加降低了乳链菌肽对的典型成孔能力，同时保留了其与脂质II的结合能力。有趣的是，添加亲水性糖在中性pH下显著提高了其水溶性约4倍，表明在改善药物应用方面具有潜力。这些发现突出了这种RiPPs糖基化方法的潜力，从而能够创造出具有不同特性的新型抗菌产品。这也拓宽了增强和发现基于肽的药物的工具包。
