Thiopeptide biosynthesis featuring ribosomally synthesized precursor peptides and conserved posttranslational modifications

Abstract

Thiopeptides, with potent activity against various drug-resistant pathogens, contain a characteristic macrocyclic core consisting of multiple thiazoles, dehydroamino acids, and a 6-membered nitrogen heterocycle. Their biosynthetic pathways remain elusive, in spite of great efforts by in vivo feeding experiments. Here, cloning, sequencing, and characterization of the thiostrepton and siomycin A gene clusters unveiled a biosynthetic paradigm for the thiopeptide specific core formation, featuring ribosomally synthesized precursor peptides and conserved posttranslational modifications. The paradigm generality for thiopeptide biosynthesis was supported by genome mining and ultimate confirmation of the thiocillin I production in Bacillus cereus ATCC 14579, a strain that was previously unknown as a thiopeptide producer. These findings set the stage to accelerate the discovery of thiopeptides by prediction at the genetic level and to generate structural diversity by applying combinatorial biosynthesis methods.

Figure 1

Structures of thiostrepton, siomycin A and thiocillin I (whose ¹H-¹H COSY, HMBC, and selected ROESY correlations in this study were labeled).

Figure 2

Gene cluster and proposed biosynthetic pathway. (A) Organization of the tsr biosynthetic genes, the deduced functions of which are labeled in color and summarized in Table 1. (B) Biosynthetic hypothesis for the thiostrepton (or siomycin A) framework and modifications. Color coding indicates the thiazole/thiazoline (red), dehydroamino acids (blue), and 6-membered nitrogen heterocycle (orange).

Figure 3

Comparison and validation of the thiopeptide framework formation. (A) Peptide precursors for thiostrepton (TsrH), siomycin A (SioH), thiocillin I (TclB_1–4), and two putative thiopeptides (Sare_2569 and SGR_4418). The SP sequences are labeled in bold (asterisk indicates the proposed SPs). The SP amino acids difference of SioH from TsrH is underlined. (B) Organization of the thiopeptide framework-forming genes identified from the producers of thiostrepton (tsr), siomycin A (sio), and thiocillin I (tcl), and two uncharacterized producers Salinispora arenicola CNS-205 (sare) and S. griseus subsp. Griseus NBRC 13350 (sgr). (C) HPLC analysis of the thiopeptide production in the wild type strain of the thiostrepton (solid dot) producer S. laurentii ATCC 31255 (I), tsrJ mutant S. laurentii strain SL1001 (II), wild type strain of the siomycin A (solid star) producer S. sioyaensis ATCC 13989 (III), sioO mutant S. sioyaensis strain SL2001 (IV), wild type strain of the thiocillin I (solid triangle) producer B. cereus ATCC 14579 (V), and tclE mutant B. cereus strain SL3001 (VI).