Improved production of class I lanthipeptides in

• Published in: Chemical science (Cambridge) Vol. 14; no. 1; pp. 2537 - 2546
• Main Authors: Lee, Hyunji, Wu, Chunyu, Desormeaux, Emily K, Sarksian, Raymond, van der Donk, Wilfred A
• Format: Journal Article
• Published: 08.03.2023
• ISSN: 2041-6520; 2041-6539
• DOI: 10.1039/d2sc06597e

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. Expression of Glu-tRNA and its synthetase from lanthipeptide encoding bacteria using pEVOL improves production in E. coli . Often-observed glutathionylation can be reversed using LanCL enzymes.