Kuipers O P, Bierbaum G, Ottenwälder B, Dodd H M, Horn N, Metzger J, Kupke T, Gnau V, Bongers R, van den Bogaard P, Kosters H, Rollema H S, de Vos W M, Siezen R J, Jung G, Götz F, Sahl H G, Gasson M J
Department of Biophysical Chemistry, Netherlands Institute for Dairy Research.
Antonie Van Leeuwenhoek. 1996 Feb;69(2):161-69. doi: 10.1007/BF00399421.
Whereas protein engineering of enzymes and structural proteins nowadays is an established research tool for studying structure-function relationships of polypeptides and for improving their properties, the engineering of posttranslationally modified peptides, such as the lantibiotics, is just coming of age. The engineering of lantibiotics is less straightforward than that of unmodified proteins, since expression systems should be developed not only for the structural genes but also for the genes encoding the biosynthetic enzymes, immunity protein and regulatory proteins. Moreover, correct posttranslational modification of specific residues could in many cases be a prerequisite for production and secretion of the active lantibiotic, which limits the number of successful mutations one can apply. This paper describes the development of expression systems for the structural lantibiotic genes for nisin A, nisin Z, gallidermin, epidermin and Pep5, and gives examples of recently produced site-directed mutants of these lantibiotics. Characterization of the mutants yielded valuable information on biosynthetic requirements for production. Moreover, regions in the lantibiotics were identified that are of crucial importance for antimicrobial activity. Eventually, this knowledge will lead to the rational design of lantibiotics optimally suited for fighting specific undesirable microorganisms. The mutants are of additional value for studies directed towards the elucidation of the mode of action of lantibiotics.
如今，酶和结构蛋白的蛋白质工程是研究多肽结构-功能关系以及改善其性质的成熟研究工具，而翻译后修饰肽（如羊毛硫抗生素）的工程才刚刚兴起。羊毛硫抗生素的工程化比未修饰蛋白质的工程化更为复杂，因为不仅要为结构基因开发表达系统，还要为编码生物合成酶、免疫蛋白和调节蛋白的基因开发表达系统。此外，在许多情况下，特定残基的正确翻译后修饰可能是活性羊毛硫抗生素产生和分泌的先决条件，这限制了可应用的成功突变的数量。本文描述了乳酸链球菌素A、乳酸链球菌素Z、加里德明、表皮菌素和Pep5等羊毛硫抗生素结构基因表达系统的开发，并给出了这些羊毛硫抗生素最近产生的定点突变体的实例。对突变体的表征产生了关于生产的生物合成要求的有价值信息。此外，还确定了羊毛硫抗生素中对抗菌活性至关重要的区域。最终，这些知识将导致合理设计出最适合对抗特定有害微生物的羊毛硫抗生素。这些突变体对于旨在阐明羊毛硫抗生素作用方式的研究具有额外价值。
