Untersuchungen zur Struktur und Stabilität neuer, durch Genome Mining identifizierter Lassopeptide

Das zunehmende Auftreten multiresistenter Bakterien macht die Suche nach neuen antibakteriellen Wirkstoffen notwendiger denn je. Naturstoffe stellen dabei eine sehr reichhaltige Quelle dar, da sie über strukturelle und chemische Diversität verfügen, die teilweise nicht oder nur unter großem Aufwand...

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Bibliographic Details
Main Author: Zimmermann, Marcel
Contributors: Marahiel, Mohamed A. (Prof. Dr.) (Thesis advisor)
Format: Doctoral Thesis
Published: Philipps-Universität Marburg 2013
Online Access:PDF Full Text
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Table of Contents: The increasing occurrence of multi-resistant bacteria makes it more necessary than ever to search for new antibacterial compounds. For this, natural products are a rich source, since they feature structural and chemical diversity, which is only hardly or not at all accessible by synthetic chemistry. An interesting method for the identification of new natural products is genome mining, which gains more and more importance as the number of sequenced genomes has risen enormously in the past few years due to the strong reduction in sequencing costs. The lasso peptides are a special family of ribosomally assembled and post-translationally modified peptides (RiPPs), which owe their name to their unique three-dimensional fold. This fold is responsible for their chemical, thermal and proteolytic stability and in some instances for their bioactivities despite the relatively low degree of chemical modification. In this work nearly 100 potential lasso peptide biosynthetic gene clusters could be identified through genome mining. To validate the suitability of this approach, one cluster was selected and different truncated variants of the heat labile lasso peptide astexin-1 were isolated. They were analyzed for thermal and proteolytic stability and the 3D-structure of astexin-1(19) was elucidated via NMR spectroscopy. An extensive mutagenesis study was performed to investigate the specificity of the biosynthetic machinery and to identify important residues for the lasso fold. Through the rational incorporation of specific mutations, it was possible to transform the native heat labile natural product into a heat-stable lasso peptide. In the second part of this work two biosynthetic gene clusters from Caulobacter sp. K31, which were identified by genome mining, were selected and it was possible to isolate four new lasso peptides, caulonodins IV - VII. These four caulonodins feature serine or alanine at position 1 of the lasso peptide sequence and therefore redefine class II of the lasso peptides, since all formerly identified peptides of this class feature glycine at this position. The caulonodins were investigated for thermal and proteolytic stability revealing them to be heat labile lasso peptides. The subsequent mutagenesis study showed the altered specificity of the biosynthetic machinery for position 1 and made it possible to predict the plug amino acids for all four caulonodins. This prediction was confirmed through elucidation of the 3D-structure of caulonodin V through NMR techniques. A mutagenesis study of the leader peptide could confirm that there are more residues involved in the maturation by the biosynthetic machinery than just Thr-2.