Protein engineering of lantibiotics

Abstract

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 he 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 lantibioties 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.

Discovery and in vitro biosynthesis of haloduracin, a two-component lantibiotic

“…The biological activity of lantibiotic peptides depends upon the formation of the correct ring structures (24)(25)(26)(27)(28) and the removal of the N-terminal leader sequence of the modified peptides (5,6). To investigate the biological activity of the haloduracin peptides prepared in vitro, the leader sequence of each product must therefore be removed.…”

Cloning and engineering of the cinnamycin biosynthetic gene cluster from Streptomyces cinnamoneus cinnamoneus DSM 40005

“…Because the biosynthetic gene cluster for nisin (17,18) comprises only 11 genes, and that for the type B lantibiotic mersacidin (13,19) contains only 10, it is unlikely that all 21 of the genes described here are involved in the production of cinnamycin.…”

Structure and Biological Activity of Chemically Modified Nisin A Species

“…The observation that the presence of a 2-hydroxyalanine residue at position 5 reduces the growth-inhibiting effect of nisin seems to be in contrast with the observation of Liu and Hansen (1992) that [AlaS]subtilin, a bacteriocin closely related to nisin, exhibits an unimpaired growth-inhibiting activity but a strongly reduced spore-killing effect. In the case of nisin, [AlaSInisin and [Ala5, Ala33lnisin were reported to have an unimpaired growth-inhibiting activity (Dodd and Gasson, 1994;Kuipers et al, 1996). The three-dimensional structure of nisin in water and in membrane-mimicking micelles shows a clearly amphipathic character for the Nterminal part of the molecule (van de Ven et al, 1991 ;van den Hooven et al, 1996a).…”

“…This indicates that Dha5 or an intact ring A is essential for the functional properties of nisin. It has been reported that [AlaSInisin and [ A M , Ala331nisin have normal biological activities (Dodd and Gasson, 1994 ;Kuipers et al, 1996). [Ala5]subtilin, a bacteriocin structurally very similar to nisin, was reported to have an unimpaired growth-inhibiting activity but a strongly reduced spore-killing effect (Liu and Hansen, 1992).…”