Thiopeptide antibiotics are a group of highly modified peptide metabolites. The defining scaffold for the thiopeptides is a macrocycle containing a dehydropiperidine or pyridine ring, dehydrated amino acids, and multiple thiazole or oxazole rings. Some members of the thiopeptides, such as thiostrepton, also contain either a quinaldic acid or indolic acid substituent derived from tryptophan. Although the amino acid precursors of these metabolites are well-established, the biogenesis of these complex peptides has remained elusive. Whole-genome scanning of Streptomyces laurentii permitted identification of a thiostrepton prepeptide, TsrA, and involvement of TsrA in thiostrepton biosynthesis was confirmed by mutagenesis. A gene cluster responsible for thiostrepton biosynthesis is reported, and the encoded gene products are discussed. The disruption of a gene encoding an amidotransferase, tsrT, led to the loss of thiostrepton production and the detection of a new metabolite, contributing further support to the identification of the tsr cluster. The tsr locus also appears to possess the gene products needed to convert tryptophan to the quinaldic acid moiety, and an aminotransferase was found to catalyze an early step in this pathway. This work establishes that the thiopeptides are a type of bacteriocin, a family of genetically encoded antimicrobial peptides, and are subjected to extensive posttranslational modification during maturation of the prepeptide.

Thiopeptides, or thiazolylpeptides, are a family of highly modified peptide antibiotics first discovered several decades ago. Dozens of thiopeptides have since been identified, but, until recently, the biosynthetic genes responsible for their production remained elusive. The biosynthetic systems for a handful of thiopeptide metabolites were identified in the first portion of 2009. The surprising finding that these metabolites arise from the enzymatic tailoring of a simple, linear, ribosomally-synthesized precursor peptide led to a renewed appreciation of the architectural complexity accessible by posttranslational modification. This recent progress toward understanding thiopeptide antibiotic biosynthesis benefits the discovery of novel thiopeptides by either directed screening techniques or by mining available microbial genome sequences. Furthermore, access to the biosynthetic machinery now opens an avenue to the biosynthetic engineering of thiopeptide analogs. This Highlight discusses the genetic and biochemical insights revealed by these initial reports of the biosynthetic gene clusters for thiopeptide metabolites.

Thiostrepton A 1, produced by Streptomyces laurentii ATCC 31255 (S. laurentii), is one of the more well-recognized thiopeptide metabolites. Thiostrepton A 1 and other thiopeptides are of great interest due to their potent activities against emerging antibiotic-resistant Gram-positive pathogens. Although numerous lines of evidence have established that the thiopeptides arise from the post-translational modification of ribosomally-synthesized peptides, few details have been revealed concerning this elaborate process. Alteration to the primary amino acid sequence of the precursor peptide provides an avenue to probe the substrate specificity of the thiostrepton post-translational machinery. Due to the difficulties in the genetic manipulation of S. laurentii, the heterologous production of thiostrepton A 1 from an alternate streptomycete host was sought to facilitate the biosynthetic investigations of the peptide metabolite. The production of thiostrepton A 1 from the non-cognate hosts did not lend itself to be as robust as S. laurentii-based production, therefore an alternate strategy was pursued for the production of thiostrepton variants. The introduction of a fosmid used in the heterologous production of thiostrepton A 1, harboring the entire thiostrepton biosynthetic gene cluster, into the tsrA deletion mutant permitted restoration of thiostrepton A 1 production near to that of the wild-type level. The fosmid was then engineered to enable the replacement of wild-type tsrA. Introduction of expression fosmids encoding alternate TsrA sequences into the S. laurentii tsrA deletion mutant led to the production of thiostrepton variants retaining antibacterial activity, demonstrating the utility of this expression platform toward thiopeptide engineering.

Metabolites that harbor a core indane scaffold are found to have diverse biological properties. Indanomycin and related pyrroloketoindanes are ionophores and have demonstrated antiparasitic, insecticidal, and antibacterial activities. To understand the biochemical mechanisms guiding formation of the central indane ring, the biosynthetic gene cluster for indanomycin was identified from Streptomyces antibioticus NRRL 8167 and sequenced to approximately 80 kb; this revealed five genes encoding subunits of a polyketide synthase (PKS) and 18 other open reading frames. The involvement of this cluster in indanomycin biosynthesis was confirmed by deletion mutagenesis. The indanomycin PKS lacks the expected thioesterase at the carboxy terminus of the final module, and instead appears to house an incomplete module containing an unusual cyclase domain. These findings now enable additional detailed genetic and biochemical studies of the mechanisms guiding the generation of pyrroloketoindanes.

The seventh residue of the highly modified peptide antibiotic thiostrepton is predicted to be critical for antibacterial activity. Substitution of Thr7 in the thiostrepton precursor peptide not only disrupts biological activity but also impacts the successful biosynthesis of thiostrepton analogs.