Biosynthesis and engineering of lanthipeptides

The emergence of antibiotic-resistant bacterial strains is a growing concern as antimicrobial drug discovery is not proceeding at the same pace as the growth of resistance. Genome sequencing techniques have advanced in the last 20 years and have revealed the hidden capacity of microbes to generate natural products including antibiotics. Most of these compounds were not detected with the traditional techniques due to conditional expression and low production. One group of natural products that is much larger than anticipated based on the genomes identified is the ribosomally-synthesized and post-translational modified peptides (RiPPs). Lanthipeptides are a class of RiPPs with pharmaceutically valuable properties including antimicrobial activity against clinically-relevant bacterial pathogens, even including drug-resistant strains. These peptides are characterized by thioether crosslinks making them more resistant to degradation under physiological conditions. A detailed understanding of the biosynthesis and mode of action could allow engineering of these peptides for better pharmacological properties. In this thesis, biosynthesis of several lanthipeptides belonging to the cinnamycin group of peptides is described, and the biosynthetic enzymes of cinnamycin were studied in detail. In Chapter II, the biosynthesis of cinnamycin was investigated by reconstitution of the cinnamycin biosynthetic enzyme activities in vitro, and through heterologous expression of the cinnamycin biosynthetic genes in E. coli. These two approaches employed to produce cinnamycin have their own advantages. For instance, in vitro reconstitution of the lantibiotic synthetase CinM and the tailoring enzyme CinX allowed for detailed studies of these enzymes as described in Chapter III. On the other hand, through production of cinnamycin in E. coli, the activity of a new enzyme called cinorf7 was discovered, which could not be achieved in the in vitro experiments. Therefore, Chapter III also contains our attempts to identify the catalytically important residues of cinorf7 by mutational studies. These experiments on cinnamycin biosynthesis set the stage for the biosynthesis of other lanthipeptides in the cinnamycin-group of peptides, which is described in Chapter IV. Lanthipeptide synthetases have been shown to have relaxed substrate specificity, creating new possibilities in the engineering of these molecules for desired functions. Peptide libraries have been commonly used for engineering and selection of pharmaceutically valuable molecules. Lanthipeptides have great potential for drug development compared to linear peptides as they are chemically and metabolically more stable because of the thioether crosslinks. Therefore, large molecular libraries of lanthipeptides with designed ring structure would be useful. In Chapter V, phage display of the well known lanthipeptide nisin is described, which will form a platform for the production and selection of lanthipeptide libraries