Identification of the Biosynthetic Gene Cluster and Regulatory Cascade for the Synergistic Antibacterial Antibiotics Griseoviridin and Viridogrisein in Streptomyces griseoviridis

Griseoviridin (GV) and viridogrisein (VG, also referred to as etamycin), produced by Streptomyces griseoviridis, are two chemically unrelated compounds belonging to the streptogramin family. Both of these natural products demonstrate broad‐spectrum antibacterial activity and constitute excellent candidates for future drug development. To elucidate the biosynthetic machinery associated with production of these two unique antibiotics, the gene cluster responsible for both GV and VG production was identified within the Streptomyces griseoviridis genome and characterized, and its function in GV and VG biosynthesis was confirmed by inactivation of 30 genes and complementation experiments. This sgv gene cluster is localized to a 105 kb DNA region that consists of 36 open reading frames (ORFs), including four nonribosomal peptide synthetases (NRPSs) for VG biosynthesis and a set of hybrid polyketide synthases (PKS)‐NRPSs with a discrete acyltransferase (AT), SgvQ, to assemble the GV backbone. The enzyme encoding genes for VG versus GV biosynthesis are separated into distinct “halves” of the cluster. A series of four genes: sgvA, sgvB, sgvC, and sgvK, were found downstream of the PKS‐NRPS; these likely code for construction of a γ‐butyrolactone (GBL)‐like molecule. GBLs and the corresponding GBL receptor systems are the highest ranked regulators that are able to coordinate the two streptomyces antibiotic regulatory protein (SARP) family positive regulators SgvR2 and SgvR3; both are key biosynthetic activators. Models of GV, VG, and GBL biosynthesis were proposed by using functional gene assignments, determined on the basis of bioinformatics analysis and further supported by in vivo gene inactivation experiments. Overall, this work provides new insights into the biosyntheses of the GV and VG streptogramins that are potentially applicable to a host of combinatorial biosynthetic scenarios.     

Biosynthetic Gene Cluster for Surugamide A Encompasses an Unrelated Decapeptide,Surugamide F

Genome mining is a powerful method for finding novel secondary metabolites. In our study on the biosynthetic gene cluster for the cyclic octapeptides surugamides A–E (inhibitors of cathepsin B), we found a putative gene cluster consisting of four successive non‐ribosomal peptide synthetase (NRPS) genes, surA, surB, surC, and surD. Prediction of amino acid sequence based on the NRPSs and gene inactivation revealed that surugamides A–E are produced by two NRPS genes, surA and surD, which were separated by two NRPS genes, surB and surC. The latter genes are responsible for the biosynthesis of an unrelated peptide, surugamide F. The pattern of intercalation observed in the sur genes is unprecedented. The structure of surugamide F, a linear decapeptide containing one 3‐amino‐2‐methylpropionic acid (AMPA) residue, was determined by spectroscopic methods and was confirmed by solid‐phase peptide synthesis.     

New Aminocoumarins from the Rare Actinomycete Catenulispora acidiphila DSM 44928: Identification,Structure Elucidation,and Heterologous Production

Genome mining led to the discovery of a novel aminocoumarin gene cluster in the rare actinomycete Catenulispora acidiphila DSM 44928. Sequence analysis revealed the presence of genes putatively involved in export/resistance, regulation, and biosynthesis of the aminocoumarin moiety and its halogenation, as well as several genes with so far unknown function. Two new aminocoumarins, cacibiocin A and B, were identified in the culture broth of C. acidiphila. Heterologous expression of the putative gene cluster in Streptomyces coelicolor M1152 confirmed that this cluster is responsible for cacibiocin biosynthesis. Furthermore, total production levels of cacibiocins could be increased by heterologous expression and screening of different culture media from an initial yield of 4.9 mg L ^?1 in C. acidiphila to 60 mg L ^?1 in S. coelicolor M1152. By HR‐MS and NMR analysis, cacibiocin A was found to contain a 3‐amino‐4,7‐dihydroxycoumarin moiety linked by an amide bond to a pyrrole‐2,5‐dicarboxylic acid. The latter structural motif has not been identified previously in any natural compound. Additionally, cacibiocin B contains two chlorine atoms at positions 6′ and 8′ of the aminocoumarin moiety.     

New Insights into the Glycosylation Steps in the Biosynthesis of Sch47554 and Sch47555

Sch47554 and Sch47555 are antifungal compounds from Streptomyces sp. SCC‐2136. The availability of the biosynthetic gene cluster made it possible to track genes that encode biosynthetic enzymes responsible for the structural features of these two angucyclines. Sugar moieties play important roles in the biological activities of many natural products. An investigation into glycosyltransferases (GTs) might potentially help to diversify pharmaceutically significant drugs through combinatorial biosynthesis. Sequence analysis indicates that SchS7 is a putative C‐GT, whereas SchS9 and SchS10 are proposed to be O‐GTs. In this study, the roles of these three GTs in the biosynthesis of Sch47554 and Sch47555 are characterized. Coexpression of the aglycone and sugar biosynthetic genes with schS7 in Streptomyces lividans K4 resulted in the production of C‐glycosylated rabelomycin, which revealed that SchS7 attached a d ‐amicetose moiety to the aglycone core structure at the C‐9 position. Gene inactivation studies revealed that subsequent glycosylation steps took place in a sequential manner, in which SchS9 first attached either an l ‐aculose or l ‐amicetose moiety to 4′‐OH of the C‐glycosylated aglycone, then SchS10 transferred an l ‐aculose moiety to 3‐OH of the angucycline core.     

A Single Gene Cluster for Chalcomycins and Aldgamycins: Genetic Basis for Bifurcation of Their Biosynthesis

Aldgamycins are 16‐membered macrolide antibiotics with a rare branched‐chain sugar d ‐aldgarose or decarboxylated d ‐aldgarose at C‐5. In our efforts to clone the gene cluster for aldgamycins from a marine‐derived Streptomyces sp. HK‐2006‐1 capable of producing both aldgamycins and chalcomycins, we found that both are biosynthesized from a single gene cluster. Whole‐genome sequencing combined with gene disruption established the entire gene cluster of aldgamycins: nine new genes are incorporated with the previously identified chalcomycin gene cluster. Functional analysis of these genes revealed that almDI/almDII, (encoding α/β subunits of pyruvate dehydrogenase) triggers the biosynthesis of aldgamycins, whereas almCI (encoding an oxidoreductase) initiates chalcomycins biosynthesis. This is the first report that aldgamycins and chalcomycins are derived from a single gene cluster and of the genetic basis for bifurcation in their biosynthesis.     

Biosynthetic Gene Cluster of a d‐Tryptophan‐Containing Lasso Peptide,MS‐271

MS‐271, produced by Streptomyces sp. M‐271, is a lasso peptide natural product comprising 21 amino acid residues with a d ‐tryptophan at its C terminus. Because lasso peptides are ribosomal peptides, the biosynthesis of MS‐271, especially the mechanism of d ‐Trp introduction, is of great interest. The MS‐271 biosynthetic gene cluster was identified by draft genome sequencing of the MS‐271 producer, and it was revealed that the precursor peptide contains all 21 amino acid residues including the C‐terminal tryptophan. This suggested that the d ‐Trp residue is introduced by epimerization. Genes for modification enzymes such as a macrolactam synthetase (mslC), precursor peptide recognition element (mslB1), cysteine protease (mslB2), disulfide oxidoreductases (mslE, mslF), and a protein of unknown function (mslH) were found in the flanking region of the precursor peptide gene. Although obvious epimerase genes were absent in the cluster, heterologous expression of the putative MS‐271 cluster in Streptomyces lividans showed that it contains all the necessary genes for MS‐271 production including a gene for a new peptide epimerase. Furthermore, a gene‐deletion experiment indicated that MslB1, ‐B2, ‐C and ‐H were indispensable for MS‐271 production and that some interactions of the biosynthetic enzymes were essential for the biosynthesis of MS‐271.     

Cloning and Characterization of the Biosynthetic Gene Cluster of 16‐Membered Macrolide Antibiotic FD‐891: Involvement of a Dual Functional Cytochrome P450 Monooxygenase Catalyzing Epoxidation and Hydroxylation

FD‐891 is a 16‐membered cytotoxic antibiotic macrolide that is especially active against human leukemia such as HL‐60 and Jurkat cells. We identified the FD‐891 biosynthetic (gfs) gene cluster from the producer Streptomyces graminofaciens A‐8890 by using typical modular type I polyketide synthase (PKS) genes as probes. The gfs gene cluster contained five typical modular type I PKS genes (gfsA, B, C, D, and E), a cytochrome P450 gene (gfsF), a methyltransferase gene (gfsG), and a regulator gene (gfsR). The gene organization of PKSs agreed well with the basic polyketide skeleton of FD‐891 including the oxidation states and α‐alkyl substituent determined by the substrate specificities of the acyltransferase (AT) domains. To clarify the involvement of the gfs genes in the FD‐891 biosynthesis, the P450 gfsF gene was inactivated; this resulted in the loss of FD‐891 production. Instead, the gfsF gene‐disrupted mutant accumulated a novel FD‐891 analogue 25‐O‐methyl‐FD‐892, which lacked the epoxide and the hydroxyl group of FD‐891. Furthermore, the recombinant GfsF enzyme coexpressed with putidaredoxin and putidaredoxin reductase converted 25‐O‐methyl‐FD‐892 into FD‐891. In the course of the GfsF reaction, 10‐deoxy‐FD‐891 was isolated as an enzymatic reaction intermediate, which was also converted into FD‐891 by GfsF. Therefore, it was clearly found that the cytochrome P450 GfsF catalyzes epoxidation and hydroxylation in a stepwise manner in the FD‐891 biosynthesis. These results clearly confirmed that the identified gfs genes are responsible for the biosynthesis of FD‐891 in S. graminofaciens.     

A Tryptophan 6‐Halogenase and an Amidotransferase Are Involved in Thienodolin Biosynthesis

The biosynthetic gene cluster for the plant growth‐regulating compound thienodolin was identified in and cloned from the producer organism Streptomyces albogriseolus MJ286‐76F7. Sequence analysis of a 27 kb DNA region revealed the presence of 21 ORFs, 14 of which are involved in thienodolin biosynthesis. Three insertional inactivation mutants were generated in the sequenced region to analyze their involvement in thienodolin biosynthesis and to functionally characterize specific genes. The gene inactivation experiments together with enzyme assays with enzymes obtained by heterologous expression and feeding studies showed that the first step in thienodolin biosynthesis is catalyzed by a tryptophan 6‐halogenase and that the last step is the formation of a carboxylic amide group catalyzed by an amidotransferase. The results led to a hypothetical model for thienodolin biosynthesis.     

Deciphering Pactamycin Biosynthesis and Engineered Production of New Pactamycin Analogues

Pactamycin is an aminocyclopentitol‐derived natural product that has potent antibacterial and antitumor activities. Sequence analysis of an 86 kb continuous region of the chromosome from Streptomyces pactum ATCC 27456 revealed a gene cluster involved in the biosynthesis of pactamycin. Gene inactivation of the Fe‐S radical SAM oxidoreductase (ptmC) and the glycosyltransferase (ptmJ), individually abrogated pactamycin biosynthesis; this confirmed the involvement of the ptm gene cluster in pactamycin biosynthesis. The polyketide synthase gene (ptmQ) was found to support 6‐methylsalicylic acid (6‐MSA) synthesis in a heterologous host, S. lividans T7. In vivo inactivation of ptmQ in S. pactum impaired pactamycin and pactamycate production but led to production of two new pactamycin analogues, de‐6‐MSA‐pactamycin and de‐6‐MSA‐pactamycate. The new compounds showed equivalent cytotoxic and antibacterial activities with the corresponding parent molecules and shed more light on the structure–activity relationship of pactamycin.     

Functional Characterization of Different ORFs Including Luciferase‐Like Monooxygenase Genes from the Mensacarcin Gene Cluster

The biologically active compound mensacarcin is produced by Streptomyces bottropensis. The cosmid cos2 contains a large part of the mensacarcin biosynthesis gene cluster. Heterologous expression of this cosmid in Streptomyces albus J1074 led to the production of the intermediate didesmethylmensacarcin (DDMM). In order to gain more insights into the biosynthesis, gene inactivation experiments were carried out by λ‐Red/ET‐mediated recombination, and the deletion mutants were introduced into the host S. albus. In total, 23 genes were inactivated. Analysis of the metabolic profiles of the mutant strains showed the complete collapse of DDMM biosynthesis, but upon overexpression of the SARP regulatory gene msnR1 in each mutant new intermediates were detected. The compounds were isolated, and their structures were elucidated. Based on the results the specific functions of several enzymes were determined, and a pathway for mensacarcin biosynthesis is proposed.     

Insights into the Bioactivity of Mensacarcin and Epoxide Formation by MsnO8

Mensacarcin, a potential antitumour drug, is produced by Streptomyces bottropensis. The structure consists of a three‐membered ring system with many oxygen atoms. Of vital importance in this context is an epoxy moiety in the side chain of mensacarcin. Our studies with different mensacarcin derivatives have demonstrated that this epoxy group is primarily responsible for the cytotoxic effect of mensacarcin. In order to obtain further information about this epoxy moiety, inactivation experiments in the gene cluster were carried out to identify the epoxy‐forming enzyme. Therefore the cosmid cos2, which covers almost the complete type II polyketide synthase (PKS) gene cluster, was heterologously expressed in Streptomyces albus. This led to production of didesmethylmensacarcin, due to the fact that methyltransferase genes are missing in the cosmid. Further gene inactivation experiments on this cosmid showed that MsnO8, a luciferase‐like monooxygenase, introduces the epoxy group at the end of the biosynthesis of mensacarcin. In addition, the protein MsnO8 was purified, and its crystal structure was determined to a resolution of 1.80 Å.     

Identifying the Minimal Enzymes for Unusual Carbon–Sulfur Bond Formation in Thienodolin Biosynthesis

Thienodolin (THN) features a tricyclic indole‐S‐hetero scaffold that encompasses two unique carbon–sulfur bonds. Although its biosynthetic gene cluster has been recently identified in Streptomyces albogriseolus, the essential enzymes for the formation of C?S bonds have been relatively unexplored. Here, we isolated and characterized a new biosynthetic gene cluster from Streptomyces sp. FXJ1.172. Heterologous expression, systematic gene inactivation, and in vitro biochemical characterization enable us to determine the minimum set of genes for THN synthesis, and an aminotransferase (ThnJ) for catalyzing the downstream conversion of tryptophan chlorination. In addition, we evaluated (and mainly excluded) a previously assumed pivotal intermediate by feeding experiments. With these results, we narrowed down four enzymes (ThnC–F) that are responsible for the two unprecedented C?S bond formations. Our study provides a solid basis for further unraveling of the unique C?S mechanisms.     

Diversity of Polyketide Chains Achieved by Deleting the Tailoring Genes in the Biosynthesis of Ansatrienins

The ast gene cluster (GenBank accession numbers KF813023.1 and KP284551) was characterized to be responsible for the biosynthesis of ansatrienins in Streptomyces sp. XZQH13, which contains astC, astF1, and astF2 genes involved in the assembly of the N‐cyclohexanoyl d ‐alanyl side chain and the hydroxylation of C‐19, respectively. Further to investigating the biosynthetic mechanism of ansatrienins, herein we constructed the mutant strains XZQH13OEΔastF2 and XZQH13OEΔastCΔastF2. Three new ansatrienin analogues, namely, ansatrienols I–K ( 1 – 3 ), along with trienomycinol ( 4 ) and 3‐O‐demethyltrienomycinol ( 5 ), were isolated from the XZQH13OEΔastCΔastF2 strain, and trienomycin A ( 6 ) and trienomycin G ( 7 ) were isolated from the XZQH13OEΔastF2 strain. Their structures were determined by a combination of high‐resolution MS (ESI) and 1D and 2D NMR spectroscopy. Accordingly, a pathway for the biosynthesis of these new ansatrienins was proposed.     

Biosynthetic Code for Divergolide Assembly in a Bacterial Mangrove Endophyte

Divergolides are structurally diverse ansamycins produced by a bacterial endophyte (Streptomyces sp.) of the mangrove tree Bruguiera gymnorrhiza. By genomic analyses a gene locus coding for the divergolide pathway was detected. The div gene cluster encodes genes for the biosynthesis of 3‐amino‐5‐hydroxybenzoate and the rare extender units ethylmalonyl‐CoA and isobutylmalonyl‐CoA, polyketide assembly by a modular type I polyketide synthase (PKS), and enzymes involved in tailoring reactions, such as a Baeyer–Villiger oxygenase. A detailed PKS domain analysis confirmed the stereochemical integrity of the divergolides and provided valuable new insights into the formation of the diverse aromatic chromophores. The bioinformatic analyses and the isolation and full structural elucidation of four new divergolide congeners led to a revised biosynthetic model that illustrates the formation of four different types of ansamycin chromophores from a single polyketide precursor.     

Cloning and Characterization of the Ravidomycin and Chrysomycin Biosynthetic Gene Clusters

The gene clusters responsible for the biosynthesis of two antitumor antibiotics, ravidomycin and chrysomycin, have been cloned from Streptomyces ravidus and Streptomyces albaduncus, respectively. Sequencing of the 33.28 kb DNA region of the cosmid cosRav32 and the 34.65 kb DNA region of cosChry1‐1 and cosChryF2 revealed 36 and 35 open reading frames (ORFs), respectively, harboring tandem sets of type II polyketide synthase (PKS) genes, D ‐ravidosamine and D ‐virenose biosynthetic genes, post‐PKS tailoring genes, regulatory genes, and genes of unknown function. The isolated ravidomycin gene cluster was confirmed to be involved in ravidomycin biosynthesis through the production of a new analogue of ravidomycin along with anticipated pathway intermediates and biosynthetic shunt products upon heterologous expression of the cosmid, cosRav32, in Streptomyces lividans TK24. The identity of the cluster was further verified through cross complementation of gilvocarcin V (GV) mutants. Similarly, the chrysomycin gene cluster was demonstrated to be indirectly involved in chrysomycin biosynthesis through cross‐complementation of gilvocarcin mutants deficient in the oxygenases GilOII, GilOIII, and GilOIV with the respective chrysomycin monooxygenase homologues. The ravidomycin glycosyltransferase (RavGT) appears to be able to transfer both amino‐ and neutral sugars, exemplified through the structurally distinct 6‐membered D ‐ravidosamine and 5‐membered D ‐fucofuranose, to the coumarin‐based polyketide derived backbone. These results expand the library of biosynthetic genes involved in the biosyntheses of gilvocarcin class compounds that can be used to generate novel analogues through combinatorial biosynthesis.     

Identification of Genes Essential for Fluorination and Sulfamylation within the Nucleocidin Gene Clusters of Streptomyces calvus and Streptomyces virens

The gene cluster in Streptomyces calvus associated with the biosynthesis of the fluoro- and sulfamyl-metabolite nucleocidin was interrogated by systematic gene knockouts. Out of the 26 gene deletions, most did not affect fluorometabolite production, nine abolished sulfamylation but not fluorination, and three precluded fluorination, but had no effect on sulfamylation. In addition to nucI, nucG, nucJ, nucK, nucL, nucN, nucO, nucQ and nucP, we identified two genes (nucW_, nucA), belonging to a phosphoadenosine phosphosulfate (PAPS) gene cluster, as required for sulfamyl assembly. Three genes (orf(−3), orf2 and orf3) were found to be essential for fluorination, although the activities of their protein products are unknown. These genes as well as nucK, nucN, nucO and nucPNP, whose knockouts produced results differing from those described in a recent report, were also deleted in Streptomyces virens – with confirmatory outcomes. This genetic profile should inform biochemistry aimed at uncovering the enzymology behind nucleocidin biosynthesis.     

Definition of the common and divergent steps in carbapenem β-lactam antibiotic biosynthesis

Approximately 50 naturally occurring carbapenem β-lactam antibiotics are known. All but one of these have been isolated from Streptomyces species and are disubstituted structural variants of a simple core that is synthesized by Pectobacterium carotovorum (Erwinia carotovora), a phylogenetically distant plant pathogen. While the biosynthesis of the simple carbapenem, (5R)-carbapen-2-em-3-carboxylic acid, is impressively efficient requiring only three enzymes, CarA, CarB and CarC, the formation of thienamycin, one of the former group of metabolites from Streptomyces, is markedly more complex. Despite their phylogenetic separation, bioinformatic analysis of the encoding gene clusters suggests that the two pathways could be related. Here we demonstrate with gene swapping, stereochemical and kinetics experiments that CarB and CarA and their S. cattleya orthologues, ThnE and ThnM, respectively, are functionally and stereochemically equivalent, although their catalytic efficiencies differ. The biosynthetic pathways, therefore, to thienamycin, and likely to the other disubstituted carbapenems, and to the simplest carbapenem, (5R)-carbapen-2-em-3-carboxylic acid, are initiated in the same manner, but share only two common steps before diverging.     

Cloning and Heterologous Expression of the Aurachin RE Biosynthesis Gene Cluster Afford a New Cytochrome P450 for Quinoline N‐Hydroxylation

Aurachin RE is a prenylated quinoline antibiotic that was first isolated from the genus Rhodococcus. It shows potent antibacterial activity against a variety of Gram‐positive bacteria. Here we have identified a minimal biosynthesis gene cluster for aurachin RE in Rhodococcus erythropolis JCM 6824 by using random transposon mutagenesis and heterologous production. The Rhodococcus aurachin (rau) gene cluster consists of genes encoding cytochrome P450 (rauA), prenyltransferase, polyketide synthase, and farnesyl pyrophosphate synthase, as well as others including genes involved in regulation and transport. Markerless gene disruption of rauA resulted in the complete loss of aurachin RE production and in the accumulation of a new aurachin derivative lacking the N‐hydroxy group. When the recombinant RauA was expressed in Escherichia coli, it catalyzed N‐hydroxylation of the derivative to form aurachin RE. This study establishes the biosynthetic pathway of aurachin RE and provides experimental evidence for the role of P450 RauA in catalyzing N‐hydroxylation of the quinoline ring, which is indispensable for the antibacterial activity of aurachin RE.     

Identification and Analysis of the Biosynthetic Gene Cluster for the Indolizidine Alkaloid Iminimycin in Streptomyces griseus

Indolizidine alkaloids, which have versatile bioactivities, are produced by various organisms. Although the biosynthesis of some indolizidine alkaloids has been studied, the enzymatic machinery for their biosynthesis in Streptomyces remains elusive. Here, we report the identification and analysis of the biosynthetic gene cluster for iminimycin, an indolizidine alkaloid with a 6-5-3 tricyclic system containing an iminium cation from Streptomyces griseus. The gene cluster has 22 genes, including four genes encoding polyketide synthases (PKSs), which consist of eight modules in total. In vitro analysis of the first module revealed that its acyltransferase domain selects malonyl-CoA, although predicted to select methylmalonyl-CoA. Inactivation of seven tailoring enzyme-encoding genes and structural elucidation of four compounds accumulated in mutants provided important insights into iminimycin biosynthesis, although some of these compounds appeared to be shunt products. This study expands our knowledge of the biosynthetic machinery of indolizidine alkaloids and the enzymatic chemistry of PKS.     

Investigations into the Biosynthesis,Regulation, and Self‐Resistance of Toxoflavin in Pseudomonas protegens Pf‐5

Pseudomonas spp. are prolific producers of natural products from many structural classes. Here we show that the soil bacterium Pseudomonas protegens Pf‐5 is capable of producing trace levels of the triazine natural product toxoflavin ( 1 ) under microaerobic conditions. We evaluated toxoflavin production by derivatives of Pf‐5 with deletions in specific biosynthesis genes, which led us to propose a revised biosynthetic pathway for toxoflavin that shares the first two steps with riboflavin biosynthesis. We also report that toxM, which is not present in the well‐characterized cluster of Burkholderia glumae, encodes a monooxygenase that degrades toxoflavin. The toxoflavin degradation product of ToxM is identical to that of TflA, the toxoflavin lyase from Paenibacillus polymyxa. Toxoflavin production by P. protegens causes inhibition of several plant‐pathogenic bacteria, and introduction of toxM into the toxoflavin‐sensitive strain Pseudomonas syringae DC3000 results in resistance to toxoflavin.