| Thiopeptide antibiotics are an important class of natural products. Although their biosynthesis, especially construction of unusual amino acid residues, attracted organic chemists for many years, few experimental data on the enzymatic polypeptide formation has been obtained.Cyclothiazomycin (CLT), isolated as a novel and selective inhibitor of human plasma rennin, is a unique bridged macrocyclic thiopeptide, whose stereo structure was recently revealed as containing a dehydroserine, two dehydrothreonine residues, three thiazolines, three thiazoles, and a tri-substituted pyridine. Compared with common thiopeptides, it lacks the characteristic 2- and 3-azole substituent on the central pyridine domain, but instead, possesses an alanine-derived heterocyclic residue with (R)-configuration and a quaternary sulfide to exhibits two macro-cyclic peptide loops.Streptomyceshygroscopicus var. yingchengensis 10-22 (10-22), isolated by Zhou et al in 1980s', can produce three antifungal antibiotics, 5102-I, 5102-II and 5102-III. Known as a polypeptide containing Thr, Ser and Cysantibiotic, antibiotic 5102-II can protect against leaf spot of corn (disease caused by Cochliobolus heterostrophus). However, the lack of detailed structural information and the unsuccessful cloning strategy based on non-ribosomal peptide hypothesis have hampered the cloning of its biosynthesis gene cluster for years.Previously failures raised the possibility that the polypeptide antibiotic 5102-II is synthesized by ribosomal pathway followed by posttranslational modifications. A pair of degenerated primers, designed based on the conserved cyclodehydratase, was used to clone a 700 bp fragment from the genomic DNA of S. hygroscopicus 10-22. Based on this nucleotide sequence, a pair of specific nestle primers was designed to screen the S. hygroscopicus 10-22 genomic library. One of the positive cosmids, 14E6, with a 35 kb insertion, was sequenced (Accession number:FJ472825) and 27 ORFs were predicted in this sequence. Significantly, a 180-nt orf, named as cltA, in the sequence was found to encode a 60-amino acid precursor peptide, of which the C terminal sequence is perfectly consistent with the structural peptide of cyclothiazomycin. Moreover, both the bioassay and Q-TOF analysis idenfied that the polypeptide antiobiotic produced by 10-22 was indeed CLT.According to the organization of ORFs, a profile of heterologous expression was successfully taken in S. lividans 1326, which is the first time for thiopeptides. Therefore, a minimized gene cluster responsible for the production of CLT has been confirmed to contain 15 ORFs and 9 of them are suggested to be directly involved into the biosynthesis.Based on the heterologous expression study and the bioinformatic analysis on the minimized intact gene cluster, we proposed an eight-enzyme involved biosynthetic scheme for the posttranslational modifications of cyclothiazomycin. In this scheme, three proteins, CltC, CltD and CltN were suggested to be responsible for the characteristic structural elements: the selectively dehydrogenated thiazolines, tri-substituted central pyridine and tertiary thioether. Importantly, the LP cleavage was deduced to be catalyzed by the C-terminal domain of CltD during the pyridine formation. The other two enzymes encoded by a putative operon, cltMN, were supposed to participate in tailoring step to generate the tertiary thioether, leading to the final cyclization of the bridged macrocyclic structure.Until early in this year, three groups cloned three different thiopeptides biosynthetases almost at the same time. Here we reported the first cloning and heterlogous expression of the biosynthetic gene cluster of CLT. Remarkably, this rigorous bioinformatic analysis based on the heterologous expression results presented a precise biosynthetic scheme for cyclothiazomycin, which may offer a short-cut access to the biosynthesis of other 'unnatural' products generated by posttranslational modification. |
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