Xu Yanli, Reuvekamp Roos, Kuipers Oscar P
Department of Molecular Genetics, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Groningen 9747 AG, The Netherlands.
ACS Synth Biol. 2024 Dec 20;13(12):4209-4217. doi: 10.1021/acssynbio.4c00650. Epub 2024 Dec 11.
Lacticin 481, a ribosomally synthesized and post-translationally modified peptide (RiPP), exhibits antimicrobial activity, for which its characteristic lanthionine and methyllanthionine ring structures are essential. The post-translational introduction of (methyl)lanthionines in lacticin 481 is catalyzed by the enzyme LctM. In addition to macrocycle formation, various other post-translational modifications can enhance and modulate the chemical and functional diversity of antimicrobial peptides. The incorporation of noncanonical amino acids, occurring in many nonribosomal peptides (NRPs), is a valuable strategy to improve the properties of antimicrobial peptides. Ornithine, a noncanonical amino acid, can be integrated into RiPPs through the conversion of arginine residues by the newly characterized peptide arginase OspR. Recently, a flexible expression system was described for engineering lanthipeptides using the post-translational modification enzyme SyncM, which has a relaxed substrate specificity. This study demonstrates that SyncM is able to catalyze the production of active lacticin 481 by recognition of a designed hybrid leader peptide, which enables the incorporation of both ornithine and (methyl)lanthionine. Utilizing this hybrid leader peptide, the functional order was established for the production of active ornithine-containing lacticin 481 analogues at positions 8 and 12 . Furthermore, this study demonstrates that prior lanthionine (Lan) and methyllanthionine (MeLan) formation may preclude ornithine incorporation at specific sites of lacticin 481. The antibacterial activity of ornithine-containing lacticin 481 analogues was evaluated using as the indicator strain. Overall, the synthetic biology pathway constructed here helped to elucidate aspects of the substrate preferences of OspR and SyncM, offering practical guidance to combine these modifications for further lantibiotic bioengineering.
乳酸链球菌素481是一种核糖体合成并经翻译后修饰的肽（RiPP），具有抗菌活性，其特征性的羊毛硫氨酸和甲基羊毛硫氨酸环结构对此至关重要。乳酸链球菌素481中（甲基）羊毛硫氨酸的翻译后引入由LctM酶催化。除了大环形成外，各种其他翻译后修饰可以增强和调节抗菌肽的化学和功能多样性。许多非核糖体肽（NRP）中出现的非标准氨基酸的掺入是改善抗菌肽特性的一种有价值的策略。鸟氨酸是一种非标准氨基酸，可以通过新鉴定的肽精氨酸酶OspR将精氨酸残基转化而整合到RiPP中。最近，描述了一种灵活的表达系统，用于使用具有宽松底物特异性的翻译后修饰酶SyncM对羊毛硫肽进行工程改造。这项研究表明，SyncM能够通过识别设计的杂交前导肽来催化活性乳酸链球菌素481的产生，这使得鸟氨酸和（甲基）羊毛硫氨酸都能掺入。利用这种杂交前导肽，确定了在第8位和第12位产生含鸟氨酸的活性乳酸链球菌素481类似物的功能顺序。此外，这项研究表明，先前的羊毛硫氨酸（Lan）和甲基羊毛硫氨酸（MeLan）形成可能会阻止鸟氨酸掺入乳酸链球菌素481的特定位点。使用作为指示菌株评估了含鸟氨酸的乳酸链球菌素481类似物的抗菌活性。总体而言，这里构建的合成生物学途径有助于阐明OspR和SyncM的底物偏好方面，为结合这些修饰进行进一步的羊毛硫抗生素生物工程提供了实际指导。
