Department of Molecular Genetics, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, 9747 AG Groningen, The Netherlands.
ACS Synth Biol. 2023 Mar 17;12(3):852-862. doi: 10.1021/acssynbio.2c00661. Epub 2023 Mar 1.
Circularin A is a circular bacteriocin belonging to a subgroup of the ribosomally synthesized and post-translationally modified peptide (RiPP) superfamily. The post-translational biosynthesis of circular bacteriocins primarily consists of leader cleavage, core peptide circularization, and bacteriocin secretion. However, none of these processes have been fully elucidated due to the complex biosynthesis of such bacteriocins and the lack of homology to the functions of other known biosynthetic enzymes. In this study, we investigated the leader- and terminal residue requirements for the biosynthesis of circularin A by systematic mutational analyses, including the mutational effects of variable leader lengths, as well as site-directed substitutions of residues at positions near the leader cleavage site and the circularization site. Results show that the leader with only one Met residue, the shortest leader possible, is sufficient to produce mature circularin A; helix-forming short-sidechain hydrophobic residues are required at positions Val1 and Ala2 of the N-terminus to form active peptide derivatives, indicating the possible steric hindrance effect at these two positions; and an aromatic residue is required at the C-terminal Tyr69 position to produce a mature circular derivative. However, the requirements for residues at position Ala68 are much more relaxed relative to the positions of Val1 and Ala2, since even substitution with the largest possible residue, i.e., tryptophan, still allows the generation of an active Ala68Trp derivative. Our findings provide new perspectives for the biosynthesis of this short-leader circular bacteriocin, which enables the application of circular bacteriocin biosynthesis in rational modified peptide engineering.
Circularin A 是一种环形细菌素，属于核糖体合成和翻译后修饰肽（RiPP）超家族的一个亚群。环形细菌素的翻译后生物合成主要包括前导肽切割、核心肽环化和细菌素分泌。然而，由于这些细菌素的复杂生物合成以及与其他已知生物合成酶功能缺乏同源性，这些过程尚未完全阐明。在这项研究中，我们通过系统的突变分析研究了 circularin A 生物合成的前导和末端残基要求，包括可变前导长度的突变效应，以及在靠近前导切割位点和环化位点的位置处残基的定点取代。结果表明，仅含有一个 Met 残基的最短前导肽足以产生成熟的 circularin A；N 端的 Val1 和 Ala2 位置需要形成活性肽衍生物的短侧链疏水性残基，表明这两个位置可能存在空间位阻效应；C 端的 Tyr69 位置需要一个芳香族残基才能产生成熟的环状衍生物。然而，与 Val1 和 Ala2 位置相比，Ala68 位置的残基要求要宽松得多，因为即使取代最大的残基即色氨酸，仍然可以产生活性 Ala68Trp 衍生物。我们的发现为这种短前导环形细菌素的生物合成提供了新的视角，这使得环形细菌素生物合成能够在合理的修饰肽工程中得到应用。
