A General Method for Fluorescent Labeling of the N‑Termini of Lanthipeptides and Its Application to Visualize their Cellular Localization

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. A general challenge is the availability of a suitable functional group for chemoselective modification. We demonstrate here that an N-terminal ketone is readily introduced 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 results in α-ketoamides that site-specifically react with an aminooxy-derivatized alkyne or fluorophore. The methodology was successfully applied to prochlorosins 1.7 and 2.8, as well as the lantibiotics lacticin 481, haloduracin α, and haloduracin β. The fluorescently modified lantibiotics were added to bacteria, and their cellular localization was visualized by confocal fluorescence microscopy. Lacticin 481 and haloduracin α localized predominantly at sites of new and old cell division as well as in punctate patterns along the long axis of rod-shaped bacilli, similar to the localization of lipid II. On the other hand, haloduracin β was localized nonspecifically in the absence of haloduracin α, but formed specific patterns when coadministered with haloduracin α. Using two-color labeling, colocalization of both components of the two-component lantibiotic haloduracin was demonstrated. These data with living cells supports a model in which the α component recognizes lipid II and then recruits the β-component

Facile Removal of Leader Peptides from Lanthipeptides by Incorporation of a Hydroxy Acid

The biosynthesis of ribosomally synthesized and post-translationally modified peptide (RiPP) natural products typically involves a precursor peptide which contains a leader peptide that is important for the modification process, and that is removed in the final step by a protease. Genome mining efforts for new RiPPs are often hampered by the lack of a general method to remove the leader peptides. We describe here the incorporation of hydroxy acids into the precursor peptides in <i>E. coli</i> which results in connection of the leader peptide via an ester linkage that is readily cleaved by simple hydrolysis. We demonstrate the method for two lantibiotics, lacticin 481 and nukacin ISK-1

An Engineered Lantibiotic Synthetase That Does Not Require a Leader Peptide on Its Substrate

Ribosomally synthesized and post-translationally modified peptides are a rapidly expanding class of natural products. They are typically biosynthesized by modification of a C-terminal segment of the precursor peptide (the core peptide). The precursor peptide also contains an N-terminal leader peptide that is required to guide the biosynthetic enzymes. For bioengineering purposes, the leader peptide is beneficial because it allows promiscuous activity of the biosynthetic enzymes with respect to modification of the core peptide sequence. However, the leader peptide also presents drawbacks as it needs to be present on the core peptide and then removed in a later step. We show that fusing the leader peptide for the lantibiotic lacticin 481 to its biosynthetic enzyme LctM allows the protein to act on core peptides without a leader peptide. We illustrate the use of this methodology for preparation of improved lacticin 481 analogues containing non-proteinogenic amino acids