New chemical and biosynthetic methodologies for the study of lanthipeptides

Recent genome sequencing efforts have revealed that a common biosynthetic route to peptide natural products involves ribosomally synthesized and posttranslationally modified peptides (RiPPs). One of the largest classes of RiPPs is the lanthionine-containing peptides (lanthipeptides), which are characterized by intramolecular thioether crosslinks dubbed lanthionine (Lan) and methyllanthionine (MeLan). The evolvability and brevity of lanthipeptide biosynthetic pathways as well as the substrate tolerance of the biosynthetic machinery facilitates the heterologous expression of lanthipeptides and renders the ribosome-derived compounds attractive for bioengineering efforts. A major drawback to the production of lanthipeptides, either in E. coli or in vitro, is the removal of the leader peptide after posttranslational modification to generate the mature natural product. In this thesis, Chapter 2 will discuss a method to introduce a photolabile linker between lanthipeptide leader and core regions. Posttranslational modification of the lanthipeptide by its cognate synthetase in vitro, followed by UV-light mediated removal of the leader peptide, yielded the mature lanthipeptide. Chapter 3 will spotlight a novel way to engineer the ribosomal machinery to incorporate a hydroxy acid into the peptide thus generating an ester linkage directly between the lanthipeptide leader and core regions, which is selectively hydrolyzed under mild alkaline conditions. Labeling of natural products with biophysical probes has greatly contributed to investigations of their modes of action and has provided tools for visualization of their targets. However, the mode of action of only a few lantibiotics has been determined thus far. A general challenge is the availability of a suitable functional group for chemoselective modification. Chapter 4 will discuss novel methodology to introduce an N-terminal ketone into various lanthipeptides by the generation of a cryptic N-terminal dehydro amino acid by the cognate biosynthetic enzymes. Spontaneous hydrolysis of the N-terminal enamines after leader peptide removal results in α-ketoamides that site-specifically react with an aminooxy-derivatized alkyne or fluorophore. The fluorescently-modified lantibiotics were added to bacteria, and their cellular localization was visualized by confocal fluorescence microscopy as a means to determine their modes of action