Bioinformatic Expansion and Discovery of Thiopeptide Antibiotics

Abstract

Thiopeptides are members of the ribosomally synthesized and post-translationally modified peptide family of natural products. Most characterized thiopeptides display nanomolar potency toward Gram-positive bacteria by blocking protein translation with several being produced at the industrial scale for veterinary and livestock applications. Employing our custom bioinformatics program, RODEO, we expand the thiopeptide family of natural products by a factor of four. This effort revealed many new thiopeptide biosynthetic gene clusters with products predicted to be distinct from characterized thiopeptides and identified gene clusters for previously characterized molecules of unknown biosynthetic origin. To further validate our data set of predicted thiopeptide biosynthetic gene clusters, we isolated and characterized a structurally unique thiopeptide featuring a central piperidine and rare thioamide moiety. Termed saalfelduracin, this thiopeptide displayed potent antibiotic activity toward several drug-resistant Gram-positive pathogens. A combination of whole-genome sequencing, comparative genomics, and heterologous expression experiments confirmed that the thioamide moiety of saalfelduracin is installed post-translationally by the joint action of two proteins, TfuA and YcaO. These results reconcile the previously unknown origin of the thioamide in two long-known thiopeptides, thiopeptin and Sch 18640. Armed with these new insights into thiopeptide chemical-genomic space, we provide a roadmap for the discovery of additional members of this natural product family.

Figure 1

Representative structures of thiopeptides. The number of atoms in the primary macrocycle are given. All known thiopeptides are close structural analogs of these seven natural products.

Figure 2

Analysis of thiopeptide [4+2] enzymes found in GenBank. (A) Thiopeptide [4+2] enzymes constitute ~4% of the Lant_Dehydr_C enzyme superfamily. The majority of [4+2] enzymes closely match the lactazole- or thiomuracin-like pHMMs. (B) Sequence similarity network of thiopeptide [4+2] enzymes (100% identical sequences are conflated, alignment score 43, colored as in panel A). While thiomuracin-, thiostrepton-, and berninamycin-like [4+2] enzymes tend to cluster together, the lactazole-like [4+2] enzymes form numerous unexplored groups at this alignment value. Blue outline nodes co-occur with additional TfuA/YcaO-encoding genes.

Figure 3

Bioinformatic analysis of thiopeptide precursor peptides. (A) Sequence similarity network of precursor peptides (conflated at 100% identity, alignment score of 8). Sequence logos of the top 14 most populous clusters are in Figure S6. Nodes are colored based on Cys content as in panel B. (B) Histogram of Cys content in thiopeptide precursors. (C) Histogram of Ser+Thr content in thiopeptide precursors. (D) Histogram of maximum possible (cyclo)dehydrations excluding critical dehydrations incorporated into pyridine/(dehydro)piperidine. This value is calculated by Cys+Ser+Thr-2 (if the C-terminus is C/S/T, the residue is not counted given these are not known to undergo dehydration).

Figure 4

Discovery of saalfelduracin (A) Saalfelduracin biosynthetic gene cluster and predicted precursor peptide (B) Proposed structure of saalfelduracin with motifs distinct from thiostrepton in red (C) ¹H shift of the Thr12 amide and Cα proton in saalfelduracin (red), compared to thiostrepton (black).

Figure 5

(A) Organization of thiopeptide biosynthetic gene clusters and precursor peptides with core sequences in bold. Whole-genome sequencing of S. tateyamensis and M. arborensis revealed analogous thiopeptide gene clusters with additional ycaO-tfuA genes, denoted by asterisks. (B) Structures of thiopeptides with thioamide analogs (red).

Figure 6

MALDI-TOF MS of S. laurentii integration mutants. Colonies of S. laurentii with chromosomal copies of ycaO-tfuA genes under strong constitutive promotion from M. arborensis (marTY), S. tateyamensis (staTY), and A. saalfeldensis (asaTY) were analyzed by MALDI-TOF MS. Conjugants produced a thioamidated species (m/z 1680), 16 Da greater than unmodified thiostrepton (m/z 1664) and consistent with Sch 18640. High-resolution masses were obtained on an Orbitrap instrument.