Thirteen posttranslational modifications convert a 14-residue peptide into the antibiotic thiocillin

Abstract

The thiazolylpeptides are a family of >50 bactericidal antibiotics that block the initial steps of bacterial protein synthesis. Here, we report a biosynthetic gene cluster for thiocillin and establish that it, and by extension the whole class, is ribosomally synthesized. Remarkably, the C-terminal 14 residues of a 52-residue peptide precursor undergo 13 posttranslational modifications to give rise to thiocillin, making this antibiotic the most heavily posttranslationally-modified peptide known to date.biosynthesis ͉ natural products ͉ thiazolylpeptides

Recombinant thiopeptides containing noncanonical amino acids

“…Similar intolerance to mutation has been previously reported with natural amino acids at the V6 position, supporting the latter hypothesis (7,28). Because methanolic extraction from the cell material was used to isolate variants as previously described (7,19,28), it is unknown whether these new ncAA-modified variants are substrates for the ABC transporter tclW or the efflux pump tclX of the endogenous thiocillin gene cluster. No overt toxicity in the producing host was observed, suggesting the copies of ribosomal L11 proteins in the thiocillin cluster encoded by tclQ and tclT were still able to protect the host against new variants that retained antimicrobial activity.…”

“…2A). To test for expression of wild-type thiocillin, transformed B. cereus ΔtclE-H + pRIPP-tclE(wt) cultures were grown in LB media with IPTG induction for 60 h and pelleted cell material was extracted with methanol as previously described (7,19,28). Wild-type thiocillin and congeners were validated in methanolic extracts by HPLC purification and LC-MS analysis (SI Appendix, Fig.…”

“…These positions were chosen based on previously demonstrated tolerance to mutation with natural amino acids (7). In total, 15 separate expressions were carried out with the potential for production of 96 novel compounds [including all possible stochastic modifications found for wild-type thiocillin (19)]. The incorporation of at least one ncAA was tolerated at every site, as demonstrated by the detection of at least one variant for each site by LC-MS. Only 2 of the 15 expressions failed to yield any detectable thiocillins: T4BocK and V6AlocK (Table 1 and SI Appendix, Figs.…”

“…Using this method, we demonstrate the production of variants of the mature full-length thiopeptide thiocillin containing ncAAs at defined sites. Thiocillins are naturally derived from a ribosomally synthesized 52-residue precursor peptide that undergoes 13 posttranslational modifications to yield the mature thiopeptide antibiotic in B. cereus 14579 (19). To illustrate the utility of the expression of thiopeptides with an expanded genetic repertoire, ncAAs with bioorthogonal chemical reactivity were incorporated into thiocillin and subsequently modified with fluorescent and photoaffinity probes.…”

Catalytic promiscuity in the biosynthesis of cyclic peptide secondary metabolites in planktonic marine cyanobacteria

“…Recent genomeenabled studies have shown that many classes of peptide natural products initially suspected to be of nonribosomal origin are in fact gene-encoded (e.g., refs. [2][3][4][5][6] and that structural motifs previously thought to be found only in nonribosomal secondary metabolites are also found in ribosomally synthesized compounds (7). Clearly, synthesis of secondary metabolites via the ribosome turns out to be much more widespread than originally anticipated.…”