Maky Mohamed Abdelfattah, Ishibashi Naoki, Nakayama Jiro, Zendo Takeshi
Department of Food Hygiene and Control, Faculty of Veterinary Medicine, South Valley University, Qena 83522, Egypt.
Laboratory of Microbial Technology, Division of Systems Bioengineering, Department of Bioscience and Biotechnology, Faculty of Agriculture, Graduate School, Kyushu University, Fukuoka 819-0395, Japan.
Microorganisms. 2021 Nov 1;9(11):2276. doi: 10.3390/microorganisms9112276.
Enterocin F4-9 belongs to the glycocin family having post-translational modifications by two molecules of -acetylglucosamine β--linked to Ser37 and Thr46. In this study, the biosynthetic gene cluster of enterocin F4-9 was cloned and expressed in JH2-2. Production of glycocin by the JH2-2 expression strain was confirmed by expression of the five genes. The molecular weight was greater than glycocin secreted by the wild strain, F4-9, because eight amino acids from the N-terminal leader sequence remained attached. This N-terminal extension was eliminated after treatment with the culture supernatant of strain F4-9, implying an extracellular protease from . F4-9 cleaves the N-terminal sequence. Thus, leader sequences cleavage requires two steps: the first via the EnfT protease domain and the second via extracellular proteases. Interestingly, the long peptide, with N-terminal extension, demonstrated advanced antimicrobial activity against Gram-positive and Gram-negative bacteria. Furthermore, was responsible for glycosylation, a necessary step prior to secretion and cleavage of the leader peptide. In addition, was found to grant self-immunity to producer cells against enterocin F4-9. This report demonstrates specifications of the minimal gene set responsible for production of enterocin F4-9, as well as a new biosynthetic mechanism of glycocins.
肠球菌素F4-9属于糖肽家族，其在翻译后会被两个与Ser37和Thr46通过β-连接的N-乙酰葡糖胺分子修饰。在本研究中，肠球菌素F4-9的生物合成基因簇被克隆并在JH2-2中表达。通过五个基因的表达证实了JH2-2表达菌株产生了糖肽。该糖肽的分子量大于野生菌株F4-9分泌的糖肽，因为N端前导序列的八个氨基酸仍附着在其上。用菌株F4-9的培养上清液处理后，这种N端延伸被消除，这意味着F4-9存在一种细胞外蛋白酶可切割N端序列。因此，前导序列的切割需要两个步骤：第一步通过EnfT蛋白酶结构域，第二步通过细胞外蛋白酶。有趣的是，带有N端延伸的长肽对革兰氏阳性和革兰氏阴性细菌表现出更强的抗菌活性。此外，该基因负责糖基化，这是前导肽分泌和切割之前的必要步骤。另外，还发现该基因赋予产生菌细胞对肠球菌素F4-9的自身免疫性。本报告展示了负责产生肠球菌素F4-9的最小基因集的特性，以及糖肽的一种新的生物合成机制。
