Department of Biosciences and Bioengineering, Indian Institute of Technology Roorkee, Roorkee 247667, Uttarakhand, India.
Department of Biosciences and Bioengineering, Indian Institute of Technology Roorkee, Roorkee 247667, Uttarakhand, India; Centre for Nanotechnology, Indian Institute of Technology Roorkee, Roorkee 247667, Uttarakhand, India.
Int J Biol Macromol. 2022 Nov 30;221:212-223. doi: 10.1016/j.ijbiomac.2022.09.012. Epub 2022 Sep 6.
Bioactive proteins are often overexpressed in different host systems for biotechnological/biomedical applications. Endolysins are natural bactericidal proteins that cleave the bacterial peptidoglycan membrane, and have the potential to be the next-generation enzybiotics. Therefore, the present study aims to elucidate the impact of two endolysins (T4L, T7L) overexpression on metabolic fingerprint of E. coli using NMR spectroscopy. The H NMR-based metabolomics analysis revealed global metabolite profiles of E. coli in response to endolysins. The study has identified nearly 75 metabolites, including organic acids, amino acids, sugars and nucleic acids. RNA Polymerase (RNAP) has been considered as reference protein for marking the specific alterations in metabolic pathways. The data suggested downregulation of central carbon metabolic pathway in both endolysins overexpression, but to a different extent. Also, the endolysin overexpression have highlighted the enhanced metabolic load and stress generation in the host cells, thus leading to the activation of osmoregulatory pathways. The overall changes in metabolic fingerprint of E. coli highlights the enhanced perturbations during the overexpression of T4L as compared to T7L. These untargeted metabolic studies shed light on the regulation of molecular pathways during the heterologous overexpression of these lytic enzymes that are lethal to the host.
生物活性蛋白通常在不同的宿主系统中过表达，用于生物技术/生物医学应用。内溶素是天然的杀菌蛋白，能够切割细菌的肽聚糖膜，具有成为下一代酶制剂的潜力。因此，本研究旨在利用 NMR 光谱阐明两种内溶素（T4L、T7L）过表达对大肠杆菌代谢指纹的影响。基于 1H NMR 的代谢组学分析揭示了内溶素作用下大肠杆菌的全局代谢物图谱。该研究鉴定出近 75 种代谢物，包括有机酸、氨基酸、糖和核酸。RNA 聚合酶（RNAP）被认为是标记代谢途径特定变化的参考蛋白。数据表明，两种内溶素过表达均下调了中心碳代谢途径，但程度不同。此外，内溶素过表达强调了宿主细胞中代谢负荷和应激的增强，从而导致渗透调节途径的激活。大肠杆菌代谢指纹的整体变化突出了 T4L 过表达过程中增强的扰动，而 T7L 过表达则没有。这些非靶向代谢研究阐明了在这些对宿主具有致死性的溶菌酶的异源过表达过程中，分子途径的调控。
