Improved production of class I lanthipeptides in .

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 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 through co-expression of the producing organism's glutamyl-tRNA synthetase (GluRS) and tRNA pair in the vector pEVOL.

-Elimination of glutamylated threonine in lanthipeptide biosynthesis.

Methyllanthionine (MeLan) containing macrocycles are key structural features of lanthipeptides. They are formed typically by -elimination of L-Thr residues followed by cyclization of L-Cys residues onto the ()-dehydrobutyrine (Dhb) intermediates. In this report we demonstrate that the biosynthesis of lanthipeptides containing the D--L-MeLan macrocycle such as the morphogenetic lanthipeptide SapT proceeds through ()-Dhb intermediates formed by net -elimination of L-Thr.

A scalable platform to discover antimicrobials of ribosomal origin.

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a promising source of new antimicrobials in the face of rising antibiotic resistance. Here, we report a scalable platform that combines high-throughput bioinformatics with automated biosynthetic gene cluster refactoring for rapid evaluation of uncharacterized gene clusters. As a proof of concept, 96 RiPP gene clusters that originate from diverse bacterial phyla involving 383 biosynthetic genes are refactored in a high-throughput manner using a biological foundry with a success rate of 86%.

Class V Lanthipeptide Cyclase Directs the Biosynthesis of a Stapled Peptide Natural Product.

Lanthipeptides are a class of cyclic peptides characterized by the presence of one or more lanthionine (Lan) or methyllanthionine (MeLan) thioether rings. These cross-links are produced by α,β-unsaturation of Ser or Thr residues in peptide substrates by dehydration, followed by a Michael-type conjugate addition of Cys residues onto the dehydroamino acids. Lanthipeptides may be broadly classified into at least five different classes, and the biosynthesis of classes I-IV lanthipeptides requires catalysis by LanC cyclases that control both the site-specificity and the stereochemistry of the conjugate addition.

Divergent Evolution of Lanthipeptide Stereochemistry.

The three-dimensional structure of natural products is critical for their biological activities and, as such, enzymes have evolved that specifically generate active stereoisomers. Lanthipeptides are post-translationally modified peptidic natural products that contain macrocyclic thioethers featuring lanthionine (Lan) and/or methyllanthionine (MeLan) residues with defined stereochemistry. In this report, we compare two class I lanthipeptide biosynthetic gene clusters (BGCs), and , that represent two families of lanthipeptide gene clusters found in Actinobacteria.

Unexpected Methyllanthionine Stereochemistry in the Morphogenetic Lanthipeptide SapT.

Lanthipeptides are polycyclic peptides characterized by the presence of lanthionine (Lan) and/or methyllanthionine (MeLan). They are members of the ribosomally synthesized and post-translationally modified peptides (RiPPs). The stereochemical configuration of (Me)Lan cross-links is important for the bioactivity of lanthipeptides.

l-methionine Adenosylation: Radical Intermediates and the Catalytic Competence of the 5'-Deoxyadenosyl Radical.

Radical -adenosyl-l-methionine (SAM) enzymes employ a [4Fe-4S] cluster and SAM to initiate diverse radical reactions via either H-atom abstraction or substrate adenosylation. Here we use freeze-quench techniques together with electron paramagnetic resonance (EPR) spectroscopy to provide snapshots of the reaction pathway in an adenosylation reaction catalyzed by the radical SAM enzyme pyruvate formate-lyase activating enzyme on a peptide substrate containing a dehydroalanine residue in place of the target glycine. The reaction proceeds via the initial formation of the organometallic intermediate Ω, as evidenced by the characteristic EPR signal with = 2.

Substrate Sequence Controls Regioselectivity of Lanthionine Formation by ProcM.

Lanthipeptides belong to the family of ribosomally synthesized and post-translationally modified peptides (RiPPs). The (methyl)lanthionine cross-links characteristic to lanthipeptides are essential for their stability and bioactivities. In most bacteria, lanthipeptides are maturated from single precursor peptides encoded in the corresponding biosynthetic gene clusters.

Structural Analysis of Class I Lanthipeptides from NL19 Reveals an Unusual Ring Pattern.

Lanthipeptides are ribosomally synthesized and post-translationally modified peptide natural products characterized by the presence of lanthionine and methyllanthionine cross-linked amino acids 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 as a cosubstrate to glutamylate Ser/Thr followed by glutamate elimination. A vast majority of lanthipeptides identified from class I synthase systems have been from Gram-positive bacteria.