Expression and characterisation of two novel lanthipeptides and their biosynthetic gene cluster

Abstract

Lanthipeptides belong to the group of ribosomally synthesised and post-translationally modified peptide natural products. The steps of lanthipeptide biosynthesis include the dehydration and cyclisation of specific amino acids to form the characteristic lanthionine residues. Lanthipeptides are divided into four different classes depending on the enzymes catalysing these post-translational modifications. The final step of their biosynthesis involves a proteolytic cleavage that generates the biologically active compound. Lanthipeptides are best known for their antibacterial activity but have also been reported to exhibit antifungal, antiviral and antinociceptive bioactivities, which makes them of commercial interest. This thesis describes the discovery of the first lanthipeptide biosynthesis gene cluster from a marine proteobacterium, the endosymbiont Pseudoalteromonas sp. HM-SA03. In silico analyses of the gene cluster revealed characteristics of class I as well as class II lanthipeptides suggesting the formation of a new (sub-)class that may be specific to Gram-negative bacteria. Multiple sequence alignments of precursor peptides showed that the leader peptides lacked the conserved FNLD/FDLD motif, which was reported to be essential for enzyme recognition. The identified class I modification enzymes were observed to incorporate dehydrated residues and lanthionines in vivo in Escherichia coli. Additionally, three potential proteolytic cleavage sites were identified in the precursor peptides. Mass spectrometric analyses of crude extracts from Pseudoalteromonas sp. HM-SA03 confirmed a double-glycine motif to present the preferred cleavage site which is characteristic for the type of class II bifunctional transporter/protease that was encoded within the lanthipeptide gene cluster. Furthermore, the activity of putative regulators of the gene cluster was analysed in luciferase reporter assays. A two-component system was found to be a positive regulator for the transcription of the structural and self-immunity genes, whereas an unknown protein was demonstrated to function as a repressor for the transcription of the entire gene cluster. The findings of this study identified a novel lanthipeptide biosynthetic pathway with unusual characteristics that will enhance the current knowledge and contribute to the expansion of the current classification system. Future studies of the described lanthipeptides are anticipated to reveal potentially valuable bioactivities.