Department of Molecular Genetics, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Groningen, The Netherlands.
Department of Molecular Genetics, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Groningen, The Netherlands
Appl Environ Microbiol. 2020 Sep 1;86(18). doi: 10.1128/AEM.01319-20.
Large-scale mass spectrometry-based peptidomics for bioactive-peptide discovery is relatively unexplored because of challenges in intracellular peptide extraction and small-peptide identification. Here, we present an analytical pipeline for large-scale intracellular peptidomics of It entails an optimized sample preparation protocol for , used as an "enzyme complex" to digest β-casein, an extraction method for its intracellular peptidome, and a peptidomics data analysis and visualization procedure. In addition, we proofread the publicly available bioactive-peptide databases and obtained an optimized database of bioactive peptides derivable from bovine β-casein. We used the pipeline to examine cultures of MG1363 and a set of 6 isogenic multiple peptidase mutants incubated with β-casein. We observed a clearly strain-dependent accumulation of peptides with several bioactivities, such as angiotensin-converting enzyme (ACE)-inhibitory, dipeptidyl peptidase 4 (DPP-IV)-inhibitory, and immunoregulatory functions. The results suggest that both the number of different bioactive peptides and the bioactivity diversity can be increased by editing the proteolytic system of This comprehensive pipeline offers a model for discovery of bioactive peptides in combination with other proteins and might be applicable to other bacteria. Lactic acid bacteria (LAB) are very important for the production of safe and healthy human and animal fermented foods and feed and, increasingly more, in the functional food industry. The intracellular peptidomes of LAB are promising reservoirs of bioactive peptides. We show here that targeted genetic engineering of the peptide degradation pathway allows steering the composition of the peptide pool of the LAB and production of peptides with interesting bioactivities. Our work could be used as a guideline for modifying proteolytic systems in other LAB to further explore their potential as cell peptide factories.
基于大规模质谱的生物活性肽发现的肽组学研究相对较少，因为细胞内肽提取和小肽鉴定存在挑战。在这里，我们提出了一种用于大规模细胞内肽组学的分析流程，该流程需要优化用于β-酪蛋白消化的样品制备方案、其细胞内肽组的提取方法以及肽组学数据分析和可视化程序。此外，我们校对了公开的生物活性肽数据库，并获得了源自牛β-酪蛋白的生物活性肽的优化数据库。我们使用该流程来研究 MG1363 培养物和一组 6 种同基因多肽酶突变体与β-酪蛋白孵育的情况。我们观察到几种具有生物活性的肽的积累明显依赖于菌株，例如血管紧张素转换酶（ACE）抑制剂、二肽基肽酶 4（DPP-IV）抑制剂和免疫调节功能。结果表明，通过编辑蛋白酶系统，可以增加不同生物活性肽的数量和生物活性多样性。该综合流程为与其他蛋白质结合发现生物活性肽提供了一个模型，并且可能适用于其他细菌。乳酸菌（LAB）在生产安全和健康的人类和动物发酵食品和饲料以及越来越多的功能性食品工业中非常重要。LAB 的细胞内肽组是生物活性肽的有前途的来源。我们在这里表明，靶向遗传工程肽降解途径允许控制 LAB 中肽库的组成，并生产具有有趣生物活性的肽。我们的工作可以用作在其他 LAB 中修改蛋白水解系统的指南，以进一步探索它们作为细胞肽工厂的潜力。
