Thiopeptide is a kind of typical ribosomally synthesized and post-translationally modified peptides (RiPPs), which has more than 100 members. Biosynthesis studies of thiopeptides not only contribute to the development of new drug seeds, but also facilitate the understandings in complex post-translational modifications (PTMs) of proteins and/or peptides. Among all the thiopeptide members, thiostrepton (TSR) possesses the most complex structure and diverse biological activities, which draws researchers’ great intersects.
The significance of the relative endogenous concentrations of the two compounds and of the relative extent of the incorporation of exogenously added labeled material into thiostrepton are discussed in terms of the biosynthetic pathway linking tryptophan and 4-(1-hydroxyethyl)quinoline-2-carboxylate in S.
Functional assignment of the remaining orfs within the tsr cluster allowed the proposal for tailoring intermediate 3 into thiostrepton. Nine genes, tsrFAEBDUPQI, serve as candidates encoding enzymes responsible for the quinaldic acid formation, affording the 27-membered side ring moiety ( and S2). They encode a methyltransferase (TsrF), a pyridoxal phosphate (PLP)-dependent aminotransferase (TsrA), an acyl-CoA α/β dehydrogenase-like protein (TsrE), a hydrolase (TsrB), a polyketide cyclase-like enzyme (TsrD), a dehydrogenase/reductase (TsrU), a P450 epoxidase (TsrP), an acyl-CoA synthetase (TsrQ), and a hydrolase/esterase (TsrI). Together, these enzyme functions are consistent with the previously proposed pathway for the quinaldic acid moiety (4) formation from Trp (; ; ): the biosynthetic process may require methylation, desamination and oxidation, followed by imine ring opening and recyclization, epoxidation and carboxyl group activation. The attachment of 4 onto Ile42 (via amination) and Thr53 (via esterification) may lead to the closure of the side macrocyclic ring with the concomitant release of the 41-aa LP. Finally, tsrR and tsrC, encode a putative P450 hydroxylase (TsrR) and an asparagine synthase-like protein (TsrC), respectively, serving as candidates for the oxidations of Ile51 and C-terminal amide bond formation to furnish thiostrepton (). While the precise timing for many steps remains to be determined, the available tsr gene cluster and proposed pathway for thiostrepton biosynthesis have now set the stage for experimental validation.
HEQ is a discrete intermediate in 1 biosynthesis, and can be activated as the adenylate by S. laurentii cell-free extracts., The exact sequence of events governing formation of Loop 2 in 1 awaits elucidation, and its proposed biosynthetic pathway is included in . First, HEQ is tethered to the Thr12 side chain in 7. Epoxidation to 8, proteolysis of the leader peptide, and ultimately a nucleophilic attack from the newly liberated Ile1 N-terminal amine, could then forge the quinaldic acid-containing macrocycle ()., For TsrA Thr7Ala, this process appears to derail at an advanced stage of thiostrepton maturation, likely preceding the proposed epoxidation of the 7 HEQ. Although the HEQ moiety is most likely enzymatically installed even for the shunt product 4, the adventitious and non-enzymatic attachment of an adenylated HEQ to the Thr12 side chain of an advanced intermediate cannot yet be definitively ruled out. Following peptidolysis of the leader peptide and the two N-terminal proteinogenic amino acids, a dehydroalanine is transiently exposed at the N-terminus in 9 prior to tautomerization and hydrolysis to provide the shunt product 4 ().
To examine if the newly unveiled thiostrepton pathway is common for the thiopeptide biosynthesis in general, we next cloned and sequenced the biosynthetic gene cluster of siomycin A (deposited into GenBank under the accession number FJ436355), a naturally occurring analog of thiostrepton (; ) (), from S. sioyaensis ATCC 13989 for comparative analysis. Remarkably, within the sio gene cluster, sioH encodes a 61-aa precursor peptide SioH containing a 17-aa SP that is nearly identical to that of TsrH (). The only exception is the N-terminal Val45-Ser46 residues, which is consistent with their structural difference in a Val-dehydroalanine unit of siomycin A in place of an Ile-Ala unit of thiostrepton. The seven genes, sioJKLMNOS, highly conserved relatives to tsrJKLMNOS in both sequence and organization (), were proposed to encode the comparable enzymatic activities for the similar thiopeptide core formation as that in thiostrepton biosynthesis ( and 3). The inactivation of the putative cyclodehydratase gene sioO completely abolished the production (), confirming its indispensability to the siomycin A biosynthesis. These findings support a common paradigm for the thiopeptide biosynthesis. Thus, a bacterial thiopeptide biosynthetic machinery should minimally be characterized by: 1) a ribosomally synthesized precursor peptide whose SP is Cys and Ser/Thr-rich, matching the aa sequence of the resultant thiopeptide backbone (); 2) a cyclodehydratase/dehydrogenase complex (i.g. TsrOM and SioOM) to catalyze the formation of the multiple thiazole and thiazoline rings; 3) a dehydratase pair (i.g. TsrJK and SioJK) to generate the dehydroamino acid residues; and 4) at least a TsrN or SioN-like protein to furnish the 6-membered nitrogen heterocycle via putative [4 + 2] cycloaddition. These enzymes are unique to thiopeptide biosynthesis. As exemplified by phylogenetic analysis of the thiopeptide cyclodehydratases, they are distinct from the polythiazole synthetases in both bacteriocin and cyanobactin biosynthesis (; ; ; ) ().
Thiostrepton (; ; ) (), often referred to as the parent compound in this family, was chosen to be the model molecule for accessing the genetic basis of thiopeptide biosynthesis. Cloning and sequencing of the tsr gene cluster (deposited into GenBank under the accession number FJ436358) from Streptomyces laurentii ATCC 31255 revealed 21 open reading frames (orfs), whose deduced gene products supported a new paradigm for thiopeptide biosynthesis featuring a ribosomally synthesized precursor pepetide and conserved posttranslational modifications ( and ). The 58-aa precursor peptide TsrH contains a 41-aa leading peptide (LP) and a 17-aa structural peptide (SP). The SP sequence IASASCTTCICTCSCSS is in perfect agreement with the amino acids constituting the thiostrepton peptide backbone, unveiling for the first time the ribosomal origin of thiostrepton (). Central to the tsr gene cluster are the seven orfs, tsrJKLMNOS, the deduced products of which presumably act on the precursor peptide TsrH to afford the characteristic thiostrepton macrocyclic core structure ( and ). TsrO, with sequence similarity to the cyclodehydratase PatD (38% similarity and 22% identity) in the patellamide biosynthesis (), might be functionally associated with the putative dehydrogenase TsrM and able to catalyze the nucleophilic attack of each Cys side chain onto the proceeding carbonyl group followed by dehydration and optional dehydrogenation to afford the thiazoline and thiazole moieties characteristic to 1. TsrJ and TsrK, homologous to the N- (25% similarity and 11% identity) and C-terminal (30% similarity and 15% identity) sequences of SpaB in the subtilin biosynthesis (), respectively, presumably are responsible for multiple dehydrations of Ser or Thr residues, yielding the dehydroamino acids featured in 2 as those in lantibiotics. The existence of the additional dehydratase TsrS (also homologous to the N-terminal SpaB, 24% similarity and 11% identity) suggests a putative residue or region-dependent dehydration pattern in the thiostrepton biosynthesis. The resultant linear intermediate 2 might then be forced into a conformation that can readily undergo an intramolecular [4 + 2] cycloaddition reaction to afford the 6-membered heterocyclic ring and complete the biosynthesis of the 26-membered macrocyclic system, giving the intermediate 3 for further modifications. TsrN and TsrL, showing no significant sequence homology to any proteins of known functions, could serve as candidates to catalyze this process. To provide experimental evidence supporting the above bioinformatics-based proposal, selected genes were inactivated to validate their indispensability. As exemplified by the in-frame deletion of tsrJ, the resulting mutant strain completely lost the ability to produce thiostrepton (), clearly confirming its involvement in the thiostrepton biosynthesis.
Thiopeptides are a class of polythiazolyl antibiotics (). The clinical interest in this family was recently renewed since many members show potent activity against various drug-resistant pathogens, including methicillin-resistant Staphylococcus aureus (MRSA), penicillin-resistant Streptococcus pneumoniae (PRSP), and vancomycin-resistant enterococci (VRE). Thiopeptides share a characteristic macrocyclic core, consisting of multiple thiazoles, dehydroamino acids, and a 6-membered, tri- or tetra-substituted nitrogen heterocycle, with side chain(s) appending additional structural diversity ( and S1). The complex architectures pose a tremendous challenge to chemical synthesis (; ). Although previous isotope-labeled experiments, which aimed at the elucidation of the biosynthetic origins of a few members, established that all moieties exclusively derive from proteinogenic amino acids (; ; ; ), the biosynthetic pathways of thiopeptides remain elusive. Here, we set out to investigate their biosynthesis by exploiting the genetic basis. Cloning, sequencing and characterization of the thiostrepton and siomycin A gene clusters unveiled a new biosynthetic paradigm for the thiopeptide specific core formation, featuring ribosomally synthesized precursor peptides and conserved posttranslational modifications. 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, validated that this paradigm is common in thiopeptide biosynthesis.
In summary, we successfully generated variants of TsrA at the biologically critical Thr7 residue. Although impaired in production titers, the thiostrepton variants were subjected to structural characterization and assessment of their antibacterial activities. The Thr7Ala and Thr7Val substitutions impaired biological activity, confirming key interactions between Thr7 of 1 and the ribosome. Furthermore, the identification of 4 grants insight into the late stages of thiostrepton biosynthesis, suggesting that epoxidation of HEQ and quinaldic acid loop closure occur late during thiostrepton maturation.
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 new 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 novel thiopeptides by prediction at the genetic level and to generate structural diversity by applying combinatorial biosynthesis methods.
SignificanceIn this study, we have uncovered a common paradigm for thiopeptide biosynthesis featuring ribosomally synthesized precursor peptides and conserved posttranslational modifications. We discovered this new pathway by first cloning, sequencing, and characterizing the thiostrepton biosynthetic gene cluster from S. laurentii ATCC 31255, subsequently validating its generality by cloning, sequencing, and characterizing the siomycin A biosynthetic gene cluster from S. sioyaensis ATCC 13989, and finally demonstrating its applicability as a new paradigm for thiopeptide biosynthesis by genome mining and ultimate confirmation of thiocillin I production in B. cereus ATCC 14579, a strain that was previously unknown as a thiopeptide producer. The newly discovered pathway is remarkably concise and efficient, in contrast to the heroic efforts of chemical synthesis of these natural products. Sequence permutations to the precursor peptides followed by the diverse tailoring modifications on the resulting thiopeptide scaffold could be a very attractive strategy to access thiopeptide structural diversity. The findings reported here now set the stage to accelerate the discovery of novel thiopeptides by genome mining and to generate structural diversity by applying combinatorial biosynthesis methods.