Improved production of class I lanthipeptides in Escherichia coli

Lanthipeptides are ribosomally synthesised and post-translationally modified peptides containing lanthionine (Lan) and methyllanthionine (MeLan) residues that are formed by dehydration of Ser/Thr residues followed by conjugate addition of Cys to the resulting dehydroamino acids. Class I lanthipeptide dehydratases utilize glutamyl-tRNA(Glu) as a co-substrate to glutamylate Ser/Thr followed by glutamate elimination. Here we report a new system to heterologously express class I lanthipeptides in Escherichia coli through co-expression of the producing organism's glutamyl-tRNA synthetase (GluRS) and tRNA(Glu) pair in the vector pEVOL. In contrast to the results in the absence of the pEVOL system, we observed the production of fully-dehydrated peptides, including epilancin 15X, and peptides from the Bacteroidota Chryseobacterium and Runella. A second common obstacle to production of lanthipeptides in E. coli is the formation of glutathione adducts. LanC-like (LanCL) enzymes were previously reported to add glutathione to dehydroamino-acid-containing proteins in Eukarya. Herein, we demonstrate that the LanCL enzymes can remove GSH adducts from C-glutathionylated peptides with dl- or ll-lanthionine stereochemistry. These two advances will aid synthetic biology-driven genome mining efforts to discover new lanthipeptides.

Automated summary

This study reports a new system for heterologous expression of class I lanthipeptides in Escherichia coli by co-expressing the producing organism's glutamyl-tRNA synthetase (GluRS) and tRNA(Glu) pair. The system allows for the production of fully-dehydrated peptides, including epilancin 15X and peptides from Bacteroidota Chryseobacterium and Runella strains. Additionally, LanCL enzymes were shown to remove glutathione adducts from C-glutathionylated peptides with dl or ll-lanthionine stereochemistry. These advancements will facilitate the discovery of new lanthipeptides through synthetic biology-driven genome mining efforts.