Unusual Class I Lanthipeptides from the Marine Bacteria Thalassomonas viridans

Abstract

A novel class I lanthipeptide produced by the marine bacterium Thalassomonas viridans XOM25T was identified using genome mining. The putative lanthipeptides were heterologously coexpressed in Escherichia coli as GFP–prepeptide fusions along with the operon-encoded class I lanthipeptide modification machinery VdsCB. The core peptides, VdsA1 and VdsA2, were liberated from GFP using the NisP protease, purified, and analyzed by collision-induced tandem mass spectrometry. The operon-encoded cyclase and dehydratase, VdsCB, exhibited lanthipeptide synthetase activity via post-translational modification of the VdsA1 and VdsA2 core peptides. Modifications were directed by the conserved double glycine leader containing prepeptides of VdsA1 and VdsA2.

Improved production of class I lanthipeptides in Escherichia coli

“…Furthermore, groups of the partially dehydrated peptides were observed by MALDI-TOF MS with multiple increases of 307 Da, which suggests the addition of glutathione (GSH) to the reactive dehydroamino acids of the dehydrated peptides. 30,34,35 This result showed that, like the observations with epilancin 15X, the LanB dehydratases from these BGCs cannot effectively utilize the tRNA Glu /GluRS system from E. coli to activate Ser or Thr residues by glutamylation. Indeed, the tRNA Glu sequence in E. coli at the critical recognition positions is divergent compared to those of Chryseobacterium and Runella (Fig.…”

“…Glutathionylation of reactive dehydroamino acids in lanthipeptides is often observed during heterologous expression in E. coli, 30,34,35 impeding further structural and biological investigations. Efforts to optimize expression conditions to prevent glutathionylation, such as using different E. coli strains or changing the expression temperature, failed to prevent glutathionylation.…”

“…Although heterologous expression of a number of class I lanthipeptides has been successful in E. coli , 31–37 many class I dehydratases fail to fully modify their substrates in this heterologous host. Previous studies have demonstrated that including GluRS and tRNA Glu from the native lanthipeptide producing organism in the heterologous production system improves the production of the fully modified peptide.…”

“…30 While the expression of class II lanthipeptide BGCs exhibited the highest success rate of all RiPPs investigated and resulted in the discovery of new compounds active against ESKAPE pathogens, the expression of class I lanthipeptide BGCs encountered two major challenges that hampered further structural and biological studies. 30 Although heterologous expression of a number of class I lanthipeptides has been successful in E. coli, [31][32][33][34][35][36][37] many class I dehydratases fail to fully modify their substrates in this heterologous host. Previous studies have demonstrated that including GluRS and tRNA Glu from the native lanthipeptide producing organism in the heterologous production system improves the production of the fully modied peptide.…”

“…One of the other challenges during heterologous expression of class I lanthipeptides in E. coli is the formation of glutathione (GSH) adducts. 30,34,35 Glutathione is a thiol-containing tripeptide that is involved in a variety of physiological functions in both mammals and bacteria. 40,41 Due to the relatively high concentration of GSH in E. coli, which can accumulate to concentrations exceeding 10 mM, 42,43 the thiol of GSH sometimes reacts with electrophilic dehydroamino acids in lanthipeptides.…”