Transcriptional Regulation of Congocidine (Netropsin) Biosynthesis and Resistance.

The production of specialized metabolites by Streptomyces bacteria is usually temporally regulated. This regulation is complex and frequently involves both global and pathway-specific mechanisms. Streptomyces ambofaciens ATCC23877 produces several specialized metabolites, including spiramycins, stambomycins, kinamycins and congocidine. The production of the first three molecules has been shown to be controlled by one or several cluster-situated transcriptional regulators. However, nothing is known regarding the regulation of congocidine biosynthesis. Congocidine (netropsin) belongs to the family of pyrrolamide metabolites, which also includes distamycin and anthelvencins. Most pyrrolamides bind into the minor groove of DNA, specifically in A/T-rich regions, which gives them numerous biological activities, such as antimicrobial and antitumoral activities. We previously reported the characterization of the pyrrolamide biosynthetic gene clusters of congocidine (cgc) in S. ambofaciens ATCC23877, distamycin (dst) in Streptomyces netropsis DSM40846, and anthelvencins (ant) in Streptomyces venezuelae ATCC14583. The three gene clusters contain a gene encoding a putative transcriptional regulator, cgc1, dst1, and ant1, respectively. Cgc1, Dst1, and Ant1 present a high percentage of amino acid sequence similarity. We demonstrate here that Cgc1, an atypical orphan response regulator, activates the transcription of all cgc genes in the stationary phase of S. ambofaciens growth. We also show that the cgc cluster is constituted of eight main transcriptional units. Finally, we show that congocidine induces the expression of the transcriptional regulator Cgc1 and of the operon containing the resistance genes (cgc20 and cgc21, coding for an ABC transporter), and propose a model for the transcriptional regulation of the cgc gene cluster. IMPORTANCE Understanding the mechanisms of regulation of specialized metabolite production can have important implications both at the level of specialized metabolism study (expression of silent gene clusters) and at the biotechnological level (increase of the production of a metabolite of interest). We report here a study on the regulation of the biosynthesis of a metabolite from the pyrrolamide family, congocidine. We show that congocidine biosynthesis and resistance are controlled by Cgc1, a cluster-situated regulator. As the gene clusters directing the biosynthesis of the pyrrolamides distamycin and anthelvencin encode a homolog of Cgc1, our findings may be relevant for the biosynthesis of other pyrrolamides. In addition, our results reveal a new type of feed-forward induction mechanism, in which congocidine induces its own biosynthesis through the induction of the transcription of cgc1.

Dynamics of the compartmentalized Streptomyces chromosome during metabolic differentiation.

Bacteria of the genus Streptomyces are prolific producers of specialized metabolites, including antibiotics. The linear chromosome includes a central region harboring core genes, as well as extremities enriched in specialized metabolite biosynthetic gene clusters. Here, we show that chromosome structure in Streptomyces ambofaciens correlates with genetic compartmentalization during exponential phase. Conserved, large and highly transcribed genes form boundaries that segment the central part of the chromosome into domains, whereas the terminal ends tend to be transcriptionally quiescent compartments with different structural features. The onset of metabolic differentiation is accompanied by a rearrangement of chromosome architecture, from a rather 'open' to a 'closed' conformation, in which highly expressed specialized metabolite biosynthetic genes form new boundaries. Thus, our results indicate that the linear chromosome of S. ambofaciens is partitioned into structurally distinct entities, suggesting a link between chromosome folding, gene expression and genome evolution.

Revised Structure of Anthelvencin A and Characterization of the Anthelvencin Biosynthetic Gene Cluster.

Anthelvencins A and B are pyrrolamide metabolites produced by Streptomyces venezuelae ATCC 14583 and 14585. Isolated in 1965, they were reported to exhibit anthelmintic and moderate antibacterial activities. In this study, we revise the structure of anthelvencin A and identify a third anthelvencin metabolite, bearing two N-methylated pyrrole groups, which we named anthelvencin C. We sequenced the genome of S. venezuelae ATCC 14583 and identified a gene cluster predicted to direct the biosynthesis of anthelvencins. Functional analysis of this gene cluster confirmed its involvement in anthelvencin biosynthesis and allowed us to propose a biosynthetic pathway for anthelvencins. In addition to a nonribosomal peptide synthetase (NRPS), the assembly of anthelvencins involves an enzyme from the ATP-grasp ligase family, Ant23. We propose that Ant23 uses a PCP-loaded 4-aminopyrrole-2-carboxylate as substrate. As observed for the biosynthesis of the other pyrrolamides congocidine (produced by Streptomyces ambofaciens ATCC 25877) and distamycin (produced by Streptomyces netropsis DSM 40846), the NRPS assembling anthelvencins is composed of stand-alone domains only. Such NRPSs, sometimes called type II NRPSs, are less studied than the classical multimodular NRPSs. Yet, they constitute an interesting model to study protein-protein interactions in NRPSs and are good candidates for combinatorial biosynthesis approaches.

Study of bicyclomycin biosynthesis in Streptomyces cinnamoneus by genetic and biochemical approaches.

The 2,5-Diketopiperazines (DKPs) constitute a large family of natural products with important biological activities. Bicyclomycin is a clinically-relevant DKP antibiotic that is the first and only member in a class known to target the bacterial transcription termination factor Rho. It derives from cyclo-(L-isoleucyl-L-leucyl) and has an unusual and highly oxidized bicyclic structure that is formed by an ether bridge between the hydroxylated terminal carbon atom of the isoleucine lateral chain and the alpha carbon of the leucine in the diketopiperazine ring. Here, we paired in vivo and in vitro studies to complete the characterization of the bicyclomycin biosynthetic gene cluster. The construction of in-frame deletion mutants in the biosynthetic gene cluster allowed for the accumulation and identification of biosynthetic intermediates. The identity of the intermediates, which were reproduced in vitro using purified enzymes, allowed us to characterize the pathway and corroborate previous reports. Finally, we show that the putative antibiotic transporter was dispensable for the producing strain.

Modular and Integrative Vectors for Synthetic Biology Applications in Streptomyces spp.

With the development of synthetic biology in the field of (actinobacterial) specialized metabolism, new tools are needed for the design or refactoring of biosynthetic gene clusters. If libraries of synthetic parts (such as promoters or ribosome binding sites) and DNA cloning methods have been developed, to our knowledge, not many vectors designed for the flexible cloning of biosynthetic gene clusters have been constructed. We report here the construction of a set of 12 standardized and modular vectors designed to afford the construction or the refactoring of biosynthetic gene clusters in Streptomyces species, using a large panel of cloning methods. Three different resistance cassettes and four orthogonal integration systems are proposed. In addition, FLP recombination target sites were incorporated to allow the recycling of antibiotic markers and to limit the risks of unwanted homologous recombination in Streptomyces strains when several vectors are used. The functionality and proper integration of the vectors in three commonly used Streptomyces strains, as well as the functionality of the Flp-catalyzed excision, were all confirmed. To illustrate some possible uses of our vectors, we refactored the albonoursin gene cluster from Streptomyces noursei using the BioBrick assembly method. We also used the seamless ligase chain reaction cloning method to assemble a transcription unit in one of the vectors and genetically complement a mutant strain.IMPORTANCE One of the strategies employed today to obtain new bioactive molecules with potential applications for human health (for example, antimicrobial or anticancer agents) is synthetic biology. Synthetic biology is used to biosynthesize new unnatural specialized metabolites or to force the expression of otherwise silent natural biosynthetic gene clusters. To assist the development of synthetic biology in the field of specialized metabolism, we constructed and are offering to the community a set of vectors that were intended to facilitate DNA assembly and integration in actinobacterial chromosomes. These vectors are compatible with various DNA cloning and assembling methods. They are standardized and modular, allowing the easy exchange of a module by another one of the same nature. Although designed for the assembly or the refactoring of specialized metabolite gene clusters, they have a broader potential utility, for example, for protein production or genetic complementation.

Draft Genome Sequence of Streptomyces sp. M1013, a Close Relative of Streptomyces ambofaciens and Streptomyces coelicolor.

We report the draft genome sequence of Streptomyces sp. M1013, a strain isolated from the Medicago arborea rhizosphere in Izmir, Turkey. An average nucleotide identity (ANI) analysis reveals that this strain belongs to the same species as Streptomyces canus ATCC12647 and is closely related to Streptomyces ambofaciens and Streptomyces coelicolor.

Complete genome sequence of Streptomyces ambofaciens ATCC 23877, the spiramycin producer.

Streptomyces ambofaciens ATCC23877 is a soil bacterium industrially exploited for the production of the macrolide spiramycin which is used in human medicine as an antibacterial and anti-toxoplasmosis chemical. Its genome consists of a 8.3 Mbp linear chromosome and a 89 kb circular plasmid. The complete genome sequence reported here will enable us to investigate Streptomyces genome evolution and to discover new secondary metabolites with potential applications notably in human medicine.

Natural combinatorial biosynthesis involving two clusters for the synthesis of three pyrrolamides in Streptomyces netropsis.

The pyrrolamides constitute a small family of secondary metabolites that are known for their ability to bind noncovalently to the DNA minor groove with some sequence specificity. To date, only a single pyrrolamide biosynthetic gene cluster has been reported, directing the synthesis of congocidine (netropsin) in Streptomyces ambofaciens. In this study, we improve our understanding of pyrrolamide biosynthesis through the identification and characterization of the gene cluster responsible for the production of distamycin in Streptomyces netropsis DSM40846. We discover that the strain produces two other pyrrolamides, the well-characterized congocidine and a congocidine/distamycin hybrid that we named disgocidine. S. netropsis DSM40846 genome analysis led to the identification of two distinct pyrrolamide-like biosynthetic gene clusters. We show here that these two clusters are reciprocally dependent for the production of the three pyrrolamide molecules. Furthermore, based on detailed functional analysis of these clusters, we propose a biosynthetic route to congocidine and distamycin and an updated model for pyrrolamide assembly. The synthesis of disgocidine, the distamycin/congocidine hybrid, appears to constitute the first example of "natural combinatorial biosynthesis" between two related biosynthetic pathways. Finally, we analyze the genomic context of the two biosynthetic gene clusters and suggest that the presently interdependent clusters result from the coevolution of two ancestral independent pyrrolamide gene clusters.

Genome mining of Streptomyces ambofaciens.

Since the discovery of the streptomycin produced by Streptomyces griseus in the middle of the last century, members of this bacterial genus have been largely exploited for the production of secondary metabolites with wide uses in medicine and in agriculture. They have even been recognized as one of the most prolific producers of natural products among microorganisms. With the onset of the genomic era, it became evident that these microorganisms still represent a major source for the discovery of novel secondary metabolites. This was highlighted with the complete genome sequencing of Streptomyces coelicolor A3(2) which revealed an unexpected potential of this organism to synthesize natural products undetected until then by classical screening methods. Since then, analysis of sequenced genomes from numerous Streptomyces species has shown that a single species can carry more than 30 secondary metabolite gene clusters, reinforcing the idea that the biosynthetic potential of this bacterial genus is far from being fully exploited. This review highlights our knowledge on the potential of Streptomyces ambofaciens ATCC 23877 to synthesize natural products. This industrial strain was known for decades to only produce the drug spiramycin and another antibacterial compound, congocidine. Mining of its genome allowed the identification of 23 clusters potentially involved in the production of other secondary metabolites. Studies of some of these clusters resulted in the characterization of novel compounds and of previously known compounds but never characterized in this Streptomyces species. In addition, genome mining revealed that secondary metabolite gene clusters of phylogenetically closely related Streptomyces are mainly species-specific.

Post-PKS tailoring steps of the spiramycin macrolactone ring in Streptomyces ambofaciens.

Spiramycins are clinically important 16-member macrolide antibiotics produced by Streptomyces ambofaciens. Biosynthetic studies have established that the earliest lactonic intermediate in spiramycin biosynthesis, the macrolactone platenolide I, is synthesized by a type I modular polyketide synthase (PKS). Platenolide I then undergoes a series of post-PKS tailoring reactions yielding the final products, spiramycins I, II, and III. We recently characterized the post-PKS glycosylation steps of spiramycin biosynthesis in S. ambofaciens. We showed that three glycosyltransferases, Srm5, Srm29, and Srm38, catalyze the successive attachment of the three carbohydrates mycaminose, forosamine, and mycarose, respectively, with the help of two auxiliary proteins, Srm6 and Srm28. However, the enzymes responsible for the other tailoring steps, namely, the C-19 methyl group oxidation, the C-9 keto group reduction, and the C-3 hydroxyl group acylation, as well as the timing of the post-PKS tailoring reactions, remained to be established. In this study, we show that Srm13, a cytochrome P450, catalyzes the oxidation of the C-19 methyl group into a formyl group and that Srm26 catalyzes the reduction of the C-9 keto group, and we propose a timeline for spiramycin-biosynthetic post-PKS tailoring reactions.

The nonribosomal synthesis of diketopiperazines in tRNA-dependent cyclodipeptide synthase pathways.

We review here work on the biosynthesis of diketopiperazines (DKPs), a large class of natural products with noteworthy biological activities, focusing on the biosynthetic pathways involving cyclodipeptide synthases (CDPSs), a newly defined family of enzymes. Distinct from nonribosomal peptide synthetases (NRPSs), the other family of enzymes synthesizing DKPs, CDPSs bridge the primary and secondary metabolic pathways by hijacking aminoacyl-tRNAs to produce DKPs. This review includes a comprehensive description of the state of the art for CDPS-dependent pathways, and highlights the ways in which this knowledge could be used to increase the diversity of natural DKPs by pathway engineering.

Glycosylation steps during spiramycin biosynthesis in Streptomyces ambofaciens: involvement of three glycosyltransferases and their interplay with two auxiliary proteins.

Streptomyces ambofaciens synthesizes spiramycin, a 16-membered macrolide antibiotic used in human medicine. The spiramycin molecule consists of a polyketide lactone ring (platenolide) synthesized by a type I polyketide synthase, to which three deoxyhexoses (mycaminose, forosamine, and mycarose) are attached successively in this order. These sugars are essential to the antibacterial activity of spiramycin. We previously identified four genes in the spiramycin biosynthetic gene cluster predicted to encode glycosyltransferases. We individually deleted each of these four genes and showed that three of them were required for spiramycin biosynthesis. The role of each of the three glycosyltransferases in spiramycin biosynthesis was determined by identifying the biosynthetic intermediates accumulated by the corresponding mutant strains. This led to the identification of the glycosyltransferase responsible for the attachment of each of the three sugars. Moreover, two genes encoding putative glycosyltransferase auxiliary proteins were also identified in the spiramycin biosynthetic gene cluster. When these two genes were deleted, one of them was found to be dispensable for spiramycin biosynthesis. However, analysis of the biosynthetic intermediates accumulated by mutant strains devoid of each of the auxiliary proteins (or of both of them), together with complementation experiments, revealed the interplay of glycosyltransferases with the auxiliary proteins. One of the auxiliary proteins interacted efficiently with the two glycosyltransferases transferring mycaminose and forosamine while the other auxiliary protein interacted only with the mycaminosyltransferase.

Cyclodipeptide synthases are a family of tRNA-dependent peptide bond-forming enzymes.

Cyclodipeptides and their derivatives belong to the diketopiperazine (DKP) family, which is comprised of a broad array of natural products that exhibit useful biological properties. In the few known DKP biosynthetic pathways, nonribosomal peptide synthetases (NRPSs) are involved in the synthesis of cyclodipeptides that constitute the DKP scaffold, except in the albonoursin (1) pathway. Albonoursin, or cyclo(alpha,beta-dehydroPhe-alpha,beta-dehydroLeu), is an antibacterial DKP produced by Streptomyces noursei. In this pathway, the formation of the cyclo(Phe-Leu) (2) intermediate is catalyzed by AlbC, a small protein unrelated to NRPSs. We demonstrated that AlbC uses aminoacyl-tRNAs as substrates to catalyze the formation of the DKP peptide bonds. Moreover, several other bacterial proteins, presenting moderate similarity to AlbC, also use aminoacyl-tRNAs to synthesize various cyclodipeptides. Therefore, AlbC and these related proteins belong to a newly defined family of enzymes that we have named cyclodipeptide synthases (CDPSs).

An iterative nonribosomal peptide synthetase assembles the pyrrole-amide antibiotic congocidine in Streptomyces ambofaciens.

Congocidine (netropsin) is a pyrrole-amide (oligopyrrole, oligopeptide) antibiotic produced by Streptomyces ambofaciens. We have identified, in the right terminal region of the S. ambofaciens chromosome, the gene cluster that directs congocidine biosynthesis. Heterologous expression of the cluster and in-frame deletions of 8 of the 22 genes confirm the involvement of this cluster in congocidine biosynthesis. Nine genes can be assigned specific functions in regulation, resistance, or congocidine assembly. In contrast, the biosynthetic origin of the precursors cannot be easily inferred from in silico analyses. Congocidine is assembled by a nonribosomal peptide synthetase (NRPS) constituted of a free-standing module and several single-domain proteins encoded by four genes. The iterative use of its unique adenylation domain, the utilization of guanidinoacetyl-CoA as a substrate by a condensation domain, and the control of 4-aminopyrrole-2-carboxylate polymerization constitute the most original features of this NRPS.

MbtH-like protein-mediated cross-talk between non-ribosomal peptide antibiotic and siderophore biosynthetic pathways in Streptomyces coelicolor M145.

MbtH-like proteins are a family of small proteins encoded by genes found in many, but not all, non-ribosomal peptide synthetase-encoding gene clusters that direct the biosynthesis of peptide antibiotics and siderophores. Studies published to date have not elucidated the function of MbtH-like proteins, nor have they clarified whether they are required for metabolite biosynthesis. Here it is shown that only one of two genes (cdaX or cchK) encoding MbtH-like proteins in Streptomyces coelicolor is required for biosynthesis of the peptide siderophore coelichelin and the calcium-dependent peptide antibiotic (CDA). The cdaX and cchK genes can functionally complement each other in trans, suggesting that CdaX and CchK can cross-talk with the coelichelin and CDA biosynthetic pathways, respectively. Transcriptional analyses of wild-type S. coelicolor and a double cchK/cdaX replacement mutant indicate that CchK and CdaX may not be involved in transcriptional regulation of coelichelin and CDA biosynthetic gene clusters.

Multiple biosynthetic and uptake systems mediate siderophore-dependent iron acquisition in Streptomyces coelicolor A3(2) and Streptomyces ambofaciens ATCC 23877.

Siderophore-mediated iron acquisition has been well studied in many bacterial pathogens because it contributes to virulence. In contrast, siderophore-mediated iron acquisition by saprophytic bacteria has received relatively little attention. The independent identification of the des and cch gene clusters that direct production of the tris-hydroxamate ferric iron-chelators desferrioxamine E and coelichelin, respectively, which could potentially act as siderophores in the saprophyte Streptomyces coelicolor A3(2), has recently been reported. Here it is shown that the des cluster also directs production of desferrioxamine B in S. coelicolor and that very similar des and cch clusters direct production of desferrioxamines E and B, and coelichelin, respectively, in Streptomyces ambofaciens ATCC 23877. Sequence analyses of the des and cch clusters suggest that components of ferric-siderophore uptake systems are also encoded within each cluster. The construction and analysis of a series of mutants of S. coelicolor lacking just biosynthetic genes or both the biosynthetic and siderophore uptake genes from the des and cch clusters demonstrated that coelichelin and desferrioxamines E and B all function as siderophores in this organism and that at least one of these metabolites is required for growth under defined conditions even in the presence of significant quantities of ferric iron. These experiments also demonstrated that a third siderophore uptake system must be present in S. coelicolor, in addition to the two encoded within the cch and des clusters, which show selectivity for coelichelin and desferrioxamine E, respectively. The ability of the S. coelicolor mutants to utilize a range of exogenous xenosiderophores for iron acquisition was also examined, showing that the third siderophore-iron transport system has broad specificity for tris-hydroxamate-containing siderophores. Together, these results define a complex system of multiple biosynthetic and uptake pathways for siderophore-mediated iron acquisition in S. coelicolor and S. ambofaciens.

Discovery of a new peptide natural product by Streptomyces coelicolor genome mining.

Analyses of microbial genome sequences reveal numerous examples of gene clusters encoding proteins typically involved in complex natural product biosynthesis but not associated with the production of known natural products. In Streptomyces coelicolor M145 there are several gene clusters encoding new nonribosomal peptide synthetase (NRPS) systems not associated with known metabolites. Application of structure-based models for substrate recognition by NRPS adenylation domains predicts the amino acids incorporated into the putative peptide products of these systems, but the accuracy of these predictions is untested. Here we report the isolation and structure determination of the new tris-hydroxamate tetrapeptide iron chelator coelichelin from S. coelicolor using a genome mining approach guided by substrate predictions for the trimodular NRPS CchH, and we show that this enzyme, which lacks a C-terminal thioesterase domain, together with a homolog of enterobactin esterase (CchJ), are required for coelichelin biosynthesis. These results demonstrate that accurate prediction of adenylation domain substrate selectivity is possible and raise intriguing mechanistic questions regarding the assembly of a tetrapeptide by a trimodular NRPS.

A new approach to bioconjugates for proteins and peptides ("pegylation") utilising living radical polymerisation.

The synthesis of protein-polymer bioconjugates is reported using N-succinimidyl ester functionalised polymers from transition metal mediated living radical polymerisation.