Biosynthetic Insights into Linaridin Natural Products from Genome Mining and Precursor Peptide Mutagenesis

Abstract

Linaridin is a small class of peptide natural products belonging to the ribosomally synthesized and post-translationally modified peptides (RiPPs) superfamily. By an extensive genome-wide survey of linaridin biosynthetic genes, we show that this class of natural products is widespread in nature and possesses vast structural diversity. The linaridin precursor peptides are relatively conserved in the N-termini but have diverse sequences in the core region, which appear to have coevolved with the biosynthetic enzymes. Using the prototypic linaridin cypemycin as a model, we have explored the structure-activity relationships involved in precursor peptide maturation and generated a diverse set of novel cypemycin variants, among which the T2S variant exhibits enhanced activity against Micrococcus luteus. Our results reveal valuable insights into linaridin biosynthesis and highlight the potential to explore this class of natural products by genome mining and by biosynthetic engineering studies.

Substrate specificity of the cypemycin decarboxylase CypD

“…As with most RiPPs, linaridins are produced from a precursor peptide, of which an N-terminal leader peptide is finally removed by proteolysis, and a C-terminal core region is posttranslationally modified to the mature product. Although only three members of linaridin family have been structurally characterized [ [8] , [9] , [10] ], a recent genome mining study showed that this RiPP family is widespread in nature and the members are structurally diverse [ 11 ].…”

“…dehydrobutyrine or dehydroalanine) is usually an important characteristic of lanthipeptides, a large and well-studied class of RiPPs [ [15] , [16] , [17] ]. However, biosynthesis of cypemycin dehydroamino acids involves a unique set of enzymes with unclear functions and mechanisms [ 8 , 11 ]. AviCys moieties are also found in several lanthipeptides such as epidermin, mersacidin, NAI-107 [ 18 , 19 ], and microvionine [ 20 ].…”

Biosynthesis of the nosiheptide indole side ring centers on a cryptic carrier protein NosJ

“…Ribosomally synthesized and posttranslationally modified peptides (RiPPs) are a major class of natural products, as revealed by the genome-sequencing programs of the past decade 1 , 2 . These compounds are found in all three domains of life, possessing vast structural diversity ranging from relatively simple and linear structures as exemplified by linaridins 3 , 4 to highly modified and complex macrocyclic scaffolds. Among the most extensively modified RiPPs are thiopeptides, a class of sulfur-rich, polyazole-containing macrocyclic peptides featuring a central six-membered nitrogen-containing heterocycle 5 .…”

Convergent evolution of the Cys decarboxylases involved in aminovinyl‐cysteine (AviCys) biosynthesis

“…A). Biosynthesis of the AviCys moiety involves a flavoprotein, which is generically termed LanD when it is involved in lanthipeptide biosynthesis , or LinD in linaridin biosynthesis . This flavoprotein catalyzes an oxidative decarboxylation of the C‐terminal Cys to form a thioenol (Fig.…”

“…1A). Biosynthesis of the AviCys moiety involves a flavoprotein, which is generically termed LanD when it is involved in lanthipeptide biosynthesis [18], or LinD in linaridin biosynthesis [19]. This Abbreviations Dha, dehydroalanine; Dhb, dehydrobutyrine; FAD, flavin adenine dinucleotide; LanD, lanthipeptide decarboxylase; LinD, linaridin decarboxylase; MCMC, Markov chain Monte Carlo; PPC, 4 0 -phosphopantothenoylcysteine; RiPPs, ribosomally synthesized and post-translationally modified peptides; SAXS, synchrotron small-angle X-ray scattering; SeMet, selenium methionine.…”