Hijacking a Linaridin Biosynthetic Intermediate for Lanthipeptide Production

Abstract

Linaridins and lanthipeptides are two classes of natural products belonging to the ribosomally synthesized and posttranslationally modified peptide (RiPP) superfamily. Although these two RiPP classes share similar structural motifs such as dehydroamino acids and thioether-based cross-links, the biosynthesis of linaridins and lanthipeptides involved distinct sets of enzymes. Here, we report the identification of a novel lanthipeptide cypepeptin from a recombinant strain of Streptomyces lividans, which harbors most of the cypemycin (a prototypic linaridin) biosynthetic gene cluster but lacks the decarboxylase gene cypD. In contrast to the generally believed structure of cypemycin, multiple d-amino acids and Z-dehydrobutyrines were observed in both cypepeptin and cypemycin, and the stereochemistry of each amino acid was established by the extensive structural analysis in combination with genetic knockout and mutagenesis studies. Comparative analysis of cypemycin and cypepeptin showed that the aminovinyl-cysteine (AviCys) moiety of cypemycin plays an essential role in disrupting the cell integrity of M. luteus, which cannot be functionally substituted by the structurally similar lanthionine moiety.

Reconstitution of the Linaridin Pathway Provides Access to the Family-Determining Activity of Two Membrane-Associated Proteins in the Formation of Structurally Underestimated Cypemycin

“…In this study, we demonstrated that cypemycin is a previously uncharacterized, d -amino acid-rich linaridin. A similar conclusion was reported by Zhang et al during the manuscript review process . In particular, we provide access to the family-determining activity of CypH and CypL, which are two membrane-associated proteins unique to linaridin formation, based on heterologous reconstitution of the cypemycin pathway.…”

“…A similar conclusion was reported by Zhang et al during the manuscript review process. 20 In particular, we provide access to the familydetermining activity of CypH and CypL, which are two membrane-associated proteins unique to linaridin formation, based on heterologous reconstitution of the cypemycin pathway. As in S. coelicolor, the cooccurrence of CypH and CypL is necessary and sufficient for the formation of a highly modified linaridin RiPP, by transforming the CypA precursor peptide through three types, 16 PTMs in total in its CP sequence, that is, 11 epimerization reactions, 4 Thr dehydrations, and 1 Cys dethiolation, followed by hydrolysis by removal of the LP sequence to achieve linaridin 5.…”

syn-Elimination of glutamylated threonine in lanthipeptide biosynthesis

“…S5 and S6, ESI†) and linaridins. 32,33 Although we cannot rule out that during purification minor products may have been removed that could contain ( Z )-Dhb, these results clearly demonstrate that the predominant product of mSptA (1–37)trypsin contains an ( E )-Dhb and not a ( Z )-Dhb residue. To our knowledge, this represents the first example of detection of a peptide containing an ( E )-Dhb residue with ribosomal origin.…”

“…Cypemycin had been proposed to contain an ( E )-Dhb, but that proposal was recently shown to be incorrect. 32 Therefore, the GL SptB b in SapT biosynthesis catalyzes net syn -elimination of glutamylated Thr residues to form ( E )-Dhb residues. Subsequent anti -addition of l -Cys across the Si face of the ( E )-Dhb residue by SptC then forms d - allo - l -MeLan macrocycles found in SapT.…”

Genome Mining of Linaridins Provides Insights into the Widely Distributed LinC Oxidoreductases

“…OH-4156 in 1993 and is structurally characterized by the presence of four Dhb residues, a 2-aminovinyl-cysteine (AviCys) moiety, and an N , N -dimethylalanine. − Recently, the structure of cypemycin was reinvestigated by two independent studies through heterologous expression of the cyp BGC in S . lividans 66 and S. coelicolor , respectively, and was found to contain 12 epimerized amino acid residues (Figure A, bottom structure).…”