Identification, Cloning and Heterologous Expression of the Gene Cluster Directing RES-701-3, -4 Lasso Peptides Biosynthesis from a Marine Streptomyces Strain

Abstract

RES-701-3 and RES-701-4 are two class II lasso peptides originally identified in the fermentation broth of Streptomyces sp. RE-896, which have been described as selective endothelin type B receptor antagonists. These two lasso peptides only differ in the identity of the C-terminal residue (tryptophan in RES-701-3, 7-hydroxy-tryptophan in RES-701-4), thus raising an intriguing question about the mechanism behind the modification of the tryptophan residue. In this study, we describe the identification of their biosynthetic gene cluster through the genome mining of the marine actinomycete Streptomyces caniferus CA-271066, its cloning and heterologous expression, and show that the seven open reading frames (ORFs) encoded within the gene cluster are sufficient for the biosynthesis of both lasso peptides. We propose that ResE, a protein lacking known putatively conserved domains, is likely to play a key role in the post-translational modification of the C-terminal tryptophan of RES-701-3 that affords RES-701-4. A BLASTP search with the ResE amino acid sequence shows the presence of homologues of this protein in the genomes of eight other Streptomyces strains, which also harbour the genes encoding the RES-701-3, -4 precursor peptide, split-B proteins and ATP-dependent lactam synthetase required for the biosynthesis of these compounds.

Recent Advances and Perspectives on Expanding the Chemical Diversity of Lasso Peptides

“…The recent application of high-throughput elicitor screening with imaging MS to lasso peptides is also anticipated to bypass these hurdles (Xu et al, 2019). Lastly, bioinformatics approaches have dramatically expanded the known varieties of lasso peptides and have resulted in the discovery of previously unknown tailoring enzymes that can be used to add functionality to an array of lasso peptides (Maksimov et al, 2012;Hegemann et al, 2015;Zhu et al, 2016b;Zhu et al, 2016c;Tietz et al, 2017;Kaweewan et al, 2018;Zong et al, 2018;Oves-Costales et al, 2020).…”

Cell-Free Biosynthesis to Evaluate Lasso Peptide Formation and Enzyme–Substrate Tolerance

“…The threaded conformation, as opposed to an unthreaded “branched-cyclic” conformation, is maintained by a large, steric locking residue located below the ring (in the tail), and is occasionally further stabilized by disulfide bonds. An additional conformational constraint is sometimes provided by a large residue above the plane of the ring (in the loop). − Additional post-translational modifications have been observed in lasso peptides, such as glycosylation, phosphorylation, acetylation, deimination, methylation, epimerization, and hydroxylation. , …”

“…8−10 Additional post-translational modifications have been observed in lasso peptides, such as glycosylation, phosphorylation, acetylation, deimination, methylation, epimerization, and hydroxylation. 1,11 The distinctive and rigid globular shape of lasso peptides typically imparts substantial resistance to thermal and proteolytic degradation, 2,12 which are unusual traits for a peptide. Although nearly 80 lasso peptides have been described, less than 30 have a reported biological activity.…”

Reactivity-Based Screening for Citrulline-Containing Natural Products Reveals a Family of Bacterial Peptidyl Arginine Deiminases

“…As with other RiPP classes, post-translational modifications (PTMs) beyond the class-defining, threaded macrolactam are known, among them are disulfide formation (e.g., BI-32169, etc. ), C-terminal O -methylation (lassomycin), Asp β-hydroxylation (canucin A), Lys ε-acylation (albusnodin), Trp 7-hydroxylation (RES-701–2), , epimerization to d -Trp (MS-271), Ser O -phosphorylation (paeninodin), and Arg deimination (citrulassin A) . Citrulassin A was discovered along with several other new lasso peptides using the Rapid ORF Description and Evaluation Online (RODEO) automated genome-mining tool .…”