Rifamycin W Analogues from Amycolatopsis mediterranei S699 Δrif-orf5 Strain.

Rifamycin W, the most predominant intermediate in the biosynthesis of rifamycin, needs to undergo polyketide backbone rearrangement to produce rifamycin B via an oxidative cleavage of the C-12/C-29 double bond. However, the mechanism of this putative oxidative cleavage has not been characterized yet. Rif-Orf5 (a putative cytochrome P450 monooxygenase) was proposed to be involved in the cleavage of this olefinic moiety of rifamycin W. In this study, the mutant strain Amycolatopsis mediterranei S699 Δrif-orf5 was constructed by in-frame deleting the rif-orf5 gene to afford thirteen rifamycin W congeners (1-13) including seven new ones (1-7). Their structures were elucidated by extensive analysis of 1D and 2D NMR spectroscopic data and high-resolution ESI mass spectra. Presumably, compounds 1-4 were derivatized from rifamycin W via C-5/C-11 retro-Claisen cleavage, and compounds 1-3, 9 and 10 featured a hemiacetal. Compounds 5-7 and 11 showed oxygenations at various sites of the ansa chain. In addition, compounds 1-3 exhibited antibacterial activity against Staphylococcus aureus with minimal inhibitory concentration (MIC) values of 5, 40 and 0.5 µg/mL, respectively. Compounds 1 and 3 showed modest antiproliferative activity against HeLa and Caco-2 cells with half maximal inhibitory concentration (IC50) values of about 50 µM.

8-Deoxy-Rifamycin Derivatives from Amycolatopsis mediterranei S699 ΔrifT Strain.

Proansamycin X, a hypothetical earliest macrocyclic precursor in the biosynthesis of rifamycin, had never been isolated and identified. According to bioinformatics analysis, it was proposed that RifT (a putative NADH-dependent dehydrogenase) may be a candidate target responsible for the dehydrogenation of proansamycin X. In this study, the mutant strain Amycolatopsis mediterranei S699 ΔrifT was constructed by deleting the rifT gene. From this strain, eleven 8-deoxy-rifamycin derivatives (1-11) and seven known analogues (12-18) were isolated. Their structures were elucidated by extensive analysis of 1D and 2D NMR spectroscopic data and high-resolution ESI mass spectra. Compound 1 is a novel amide N-glycoside of seco-rifamycin. Compounds 2 and 3 feature conserved 11,12-seco-rifamycin W skeleton. The diverse post-modifications in the polyketide chain led to the production of 4-11. Compounds 2, 3, 5, 6, 13 and 15 exhibited antibacterial activity against Staphylococcus aureus (MIC (minimal inhibitory concentration) values of 10, 20, 20, 20, 40 and 20 µg/mL, respectively). Compounds 14, 15, 16, 17 and 18 showed potent antiproliferative activity against KG1 cells with IC50 (half maximal inhibitory concentration) values of 14.91, 44.78, 2.16, 18.67 and 8.07 µM, respectively.

Michael additions in polyketide biosynthesis.

Covering: up to July 2018 Polyketides constitute a large family of natural products exhibiting various biological activities. Polyketide biosynthetic systems employ several strategies for the production of structurally diverse polyketides. Among the polyketide biosynthetic enzymes, a growing number of enzymes that catalyze a Michael-type addition have been identified. These enzymes are responsible for constructing unique polyketide backbone structures, forming heterocycles, and incorporating heteroatoms into the polyketide backbone, all of which contribute to the diversification of the polyketide structure. This review summarizes the current understanding of the function of enzymes catalyzing a Michael-type addition in polyketide biosynthesis, with a particular focus on mechanistic studies.

Discovery of 16-Demethylrifamycins by Removing the Predominant Polyketide Biosynthesis Pathway in Micromonospora sp. Strain TP-A0468.

A number of strategies have been developed to mine novel natural products based on biosynthetic gene clusters and there have been dozens of successful cases facilitated by the development of genomic sequencing. During our study on biosynthesis of the antitumor polyketide kosinostatin (KST), we found that the genome of Micromonospora sp. strain TP-A0468, the producer of KST, contains other potential polyketide gene clusters, with no encoded products detected. Deletion of kst cluster led to abolishment of KST and the enrichment of several new compounds, which were isolated and characterized as 16-demethylrifamycins (referred to here as compounds 3 to 6). Transcriptional analysis demonstrated that the expression of the essential genes related to the biosynthesis of compounds 3 to 6 was comparable to the level in the wild-type and in the kst cluster deletion strain. This indicates that the accumulation of these compounds was due to the redirection of metabolic flux rather than transcriptional activation. Genetic disruption, chemical complementation, and bioinformatic analysis revealed that the production of compounds 3 to 6 was accomplished by cross talk between the two distantly placed polyketide gene clusters pks3 and M-rif This finding not only enriches the analogue pool and the biosynthetic diversity of rifamycins but also provides an auxiliary strategy for natural product discovery through genome mining in polyketide-producing microorganisms.IMPORTANCE Natural products are essential in the development of novel clinically used drugs. Discovering new natural products and modifying known compounds are still the two main ways to generate new candidates. Here, we have discovered several rifamycins with varied skeleton structures by redirecting the metabolic flux from the predominant polyketide biosynthetic pathway to the rifamycin pathway in the marine actinomycetes species Micromonospora sp. strain TP-A0468. Rifamycins are indispensable chemotherapeutics in the treatment of various diseases such as tuberculosis, leprosy, and AIDS-related mycobacterial infections. This study exemplifies a useful method for the discovery of cryptic natural products in genome-sequenced microbes. Moreover, the 16-demethylrifamycins and their genetically manipulable producer provide a new opportunity in the construction of novel rifamycin derivates to aid in the defense against the ever-growing drug resistance of Mycobacterium tuberculosis.

GlnR positive transcriptional regulation of the phosphate-specific transport system pstSCAB in Amycolatopsis mediterranei U32.

Amycolatopsis mediterranei U32 is an important industrial strain for the production of rifamycin SV. Rifampicin, a derivative of rifamycin SV, is commonly used to treat mycobacterial infections. Although phosphate has long been known to affect rifamycin biosynthesis, phosphate transport, metabolism, and regulation are poorly understood in A. mediterranei. In this study, the functional phosphate transport system pstSCAB was isolated by RNA sequencing and inactivated by insertion mutation in A. mediterranei U32. The mycelium morphology changed from a filamentous shape in the wild-type and pstS1+ strains to irregular swollen shape at the end of filamentous in the ΔpstS1 strain. RT-PCR assay revealed that pstSCAB genes are co-transcribed as a polycistronic messenger. The pstSCAB transcription was significantly activated by nitrate supplementation and positively regulated by GlnR which is a global regulator of nitrogen metabolism in actinomycetes. At the same time, the yield of rifamycin SV decreased after mutation (ΔpstS1) compared with wild-type U32, which indicated a strong connection among phosphate metabolism, nitrogen metabolism, and rifamycin production in actinomycetes.

Deciphering the late steps of rifamycin biosynthesis.

Rifamycin-derived drugs, including rifampin, rifabutin, rifapentine, and rifaximin, have long been used as first-line therapies for the treatment of tuberculosis and other deadly infections. However, the late steps leading to the biosynthesis of the industrially important rifamycin SV and B remain largely unknown. Here, we characterize a network of reactions underlying the biosynthesis of rifamycin SV, S, L, O, and B. The two-subunit transketolase Rif15 and the cytochrome P450 enzyme Rif16 are found to mediate, respectively, a unique C-O bond formation in rifamycin L and an atypical P450 ester-to-ether transformation from rifamycin L to B. Both reactions showcase interesting chemistries for these two widespread and well-studied enzyme families.

A feedback regulatory model for RifQ-mediated repression of rifamycin export in Amycolatopsis mediterranei.

BACKGROUND: Due to the important role of rifamycin in curing tuberculosis infection, the study on rifamycin has never been stopped. Although RifZ, which locates within the rifamycin biosynthetic cluster, has recently been characterized as a pathway-specific regulator for rifamycin biosynthesis, little is known about the regulation of rifamycin export. RESULTS: In this work, we proved that the expression of the rifamycin efflux pump (RifP) was regulated by RifQ, a TetR-family transcriptional regulator. Deletion of rifQ had little impact on bacterial growth, but resulted in improved rifamycin production, which was consistent with the reverse transcription PCR results that RifQ negatively regulated rifP's transcription. With electrophoretic mobility shift assay and DNase I Footprinting assay, RifQ was found to directly bind to the promoter region of rifP, and a typical inverted repeat was identified within the RifQ-protected sequences. The transcription initiation site of rifP was further characterized and found to be upstream of the RifQ binding sites, well explaining the RifQ-mediated repression of rifP's transcription in vivo. Moreover, rifamycin B (the end product of rifamycin biosynthesis) remarkably decreased the DNA binding affinity of RifQ, which led to derepression of rifamycin export, reducing the intracellular concentration of rifamycin B as well as its toxicity against the host. CONCLUSIONS: Here, we proved that the export of rifamycin B was repressed by RifQ in Amycolatopsis mediterranei, and the RifQ-mediated repression could be specifically relieved by rifamycin B, the end product of rifamycin biosynthesis, based on which a feedback model was proposed for regulation of rifamycin export. With the findings here, one could improve the antibiotic yield by simply inactivating the negative regulator of the antibiotic transporter.

RifZ (AMED_0655) Is a Pathway-Specific Regulator for Rifamycin Biosynthesis in Amycolatopsis mediterranei.

Rifamycin and its derivatives are particularly effective against the pathogenic mycobacteria Mycobacterium tuberculosis and Mycobacterium leprae Although the biosynthetic pathway of rifamycin has been extensively studied in Amycolatopsis mediterranei, little is known about the regulation in rifamycin biosynthesis. Here, an in vivo transposon system was employed to identify genes involved in the regulation of rifamycin production in A. mediterranei U32. In total, nine rifamycin-deficient mutants were isolated, among which three mutants had the transposon inserted in AMED_0655 (rifZ, encoding a LuxR family regulator). The rifZ gene was further knocked out via homologous recombination, and the transcription of genes in the rifamycin biosynthetic gene cluster (rif cluster) was remarkably reduced in the rifZ null mutant. Based on the cotranscription assay results, genes within the rif cluster were grouped into 10 operons, sharing six promoter regions. By use of electrophoretic mobility shift assay and DNase I footprinting assay, RifZ was proved to specially bind to all six promoter regions, which was consistent with the fact that RifZ regulated the transcription of the whole rif cluster. The binding consensus sequence was further characterized through alignment using the RifZ-protected DNA sequences. By use of bionformatic analysis, another five promoters containing the RifZ box (CTACC-N8-GGATG) were identified, among which the binding of RifZ to the promoter regions of both rifK and orf18 (AMED_0645) was further verified. As RifZ directly regulates the transcription of all operons within the rif cluster, we propose that RifZ is a pathway-specific regulator for the rif cluster.IMPORTANCE To this day, rifamycin and its derivatives are still the first-line antituberculosis drugs. The biosynthesis of rifamycin has been extensively studied, and most biosynthetic processes have been characterized. However, little is known about the regulation of the transcription of the rifamycin biosynthetic gene cluster (rif cluster), and no regulator has been characterized. Through the employment of transposon screening, we here characterized a LuxR family regulator, RifZ, as a direct transcriptional activator for the rif cluster. As RifZ directly regulates the transcription of the entire rif cluster, it is considered a pathway-specific regulator for rifamycin biosynthesis. Therefore, as the first regulator characterized for direct regulation of rif cluster transcription, RifZ may provide a new clue for further engineering of high-yield industrial strains.

Aminorifamycins and Sporalactams Produced in Culture by a Micromonospora sp. Isolated from a Northeastern-Pacific Marine Sediment Are Potent Antibiotics.

The new ansa macrolide antibiotics 1 to 4 have been isolated from cultures of a Micromonospora sp. obtained from a marine sediment. Rifamycins 1 and 2 are the first natural ansa macrolides to have a 3-amino substituent. Sporalactams A (3) and B (4) are comprised of a heterocylic core 5 and a 14-membered ansa bridge that are both unprecedented. Sporalactam B (4) shows selective and potent inhibition of Mycobacterium tuberculosis.

Conjugation of ϕBT1-derived integrative plasmid pDZL802 in Amycolatopsis mediterranei U32.

The genus Amycolatopsis is well known for its ability to produce antibiotics, and an increasing number of valuable biotechnological applications, such as bioremediation, biodegradation, bioconversion, and potentially biofuel, that use this genus have been developed. Amycolatopsis mediterranei is an industrial-scale producer of the important antibiotic rifamycin, which plays a vital role in antimycobacterial therapy. Genetic studies of Amycolatopsis species have progressed slowly due to the lack of efficient transformation methods and stable plasmid vectors. In A. mediterranei U32, electroporation and replicable plasmid vectors have been developed. Here, we establish a simple and efficient conjugal system by transferring integrative plasmid pDZL802 from ET12567 (pUZ8002) to A. mediterranei U32, with an efficiency of 4 × 10-5 CFU per recipient cell. This integrative vector, based on the ϕBT1 int-attP locus, is a stable and versatile tool for A. mediterranei U32, and it may also be applicable to various other Amycolatopsis species for strain improvement, heterologous protein expression, and synthetic biology experiments.

Rifamycin Biosynthetic Congeners: Isolation and Total Synthesis of Rifsaliniketal and Total Synthesis of Salinisporamycin and Saliniketals A and B.

We describe the isolation, structure elucidation, and total synthesis of the novel marine natural product rifsaliniketal and the total synthesis of the structurally related variants salinisporamycin and saliniketals A and B. Rifsaliniketal was previously proposed, but not observed, as a diverted metabolite from a biosynthetic precursor to rifamycin S. Decarboxylation of rifamycin provides salinisporamycin, which upon truncation with loss of the naphthoquinone ring leads to saliniketals. Our synthetic strategy hinged upon a Pt(II)-catalyzed cycloisomerization of an alkynediol to set the dioxabicyclo[3.2.1]octane ring system and a fragmentation of an intermediate dihydropyranone to forge a stereochemically defined (E,Z)-dienamide unit. Multiple routes were explored to assemble fragments with high stereocontrol, an exercise that provided additional insights into acyclic stereocontrol during stereochemically complex fragment-assembly processes. The resulting 11-14 step synthesis of saliniketals then enabled us to explore strategies for the synthesis and coupling of highly substituted naphthoquinones or the corresponding naphthalene fragments. Whereas direct coupling with naphthoquinone fragments proved unsuccessful, both amidation and C-N bond formation tactics with the more electron-rich naphthalene congeners provided an efficient means to complete the first total synthesis of rifsaliniketal and salinisporamycin.

Bacterial Transcription as a Target for Antibacterial Drug Development.

Transcription, the first step of gene expression, is carried out by the enzyme RNA polymerase (RNAP) and is regulated through interaction with a series of protein transcription factors. RNAP and its associated transcription factors are highly conserved across the bacterial domain and represent excellent targets for broad-spectrum antibacterial agent discovery. Despite the numerous antibiotics on the market, there are only two series currently approved that target transcription. The determination of the three-dimensional structures of RNAP and transcription complexes at high resolution over the last 15 years has led to renewed interest in targeting this essential process for antibiotic development by utilizing rational structure-based approaches. In this review, we describe the inhibition of the bacterial transcription process with respect to structural studies of RNAP, highlight recent progress toward the discovery of novel transcription inhibitors, and suggest additional potential antibacterial targets for rational drug design.

["Nitrate stimulating effect" in Amycolatopsis mediterranei--from discovery to mechanistic studies].

Nitrate not only remarkably stimulates the rifamycinbiosynthesis in Amycolatopsis mediterranei, but also influences the primary metabolisms, including the inhibition of fatty acids biosynthesis in the bacterial. This phenomenon has been designated as "Nitrate Stimulating Effect" by the late Prof. J.S. Chiaosince its discovery in the 1970's, and has been found in many other antibiotics-producing actinomycetes subsequently. Based on the research in his laboratory, we have revealed that the nitrate stimulation effect mainly manifests in two aspects over the last two decades. First, nitrate promotes the supply of rifamycin precursors, e.g., UDP-glucose, AHBA, malonyl-CoA and methylmalonyl-CoA. Specifically, the biosynthesis of fatty acids is inhibited by nitrate consequently the acetyl-CoA is shunted into malonyl-CoA. Second, nitrate facilitates the expression of genes in the rifclulsterthat encodes rifamycin biosynthetic enzymes. Following our current understanding, the future research will focus on the signals, the signal transduction pathway and the molecular mechanisms that dictate nitrate-mediated transcriptional and post-translational regulations.

Evaluation of the Synthetic Potential of an AHBA Knockout Mutant of the Rifamycin Producer Amycolatopsis mediterranei.

Supplementing an AHBA(-) mutant strain of Amycolatopsis mediterranei, the rifamycin producer, with a series of benzoic acid derivatives yielded new tetraketides containing different phenyl groups. These mutasynthetic studies revealed unique reductive properties of A. mediterranei towards nitro- and azidoarenes, leading to the corresponding anilines. In selected cases, the yields of mutaproducts (fermentation products isolated after feeding bacteria with chemically prepared analogs of natural building blocks) obtained are in a range (up to 118 mg L(-1)) that renders them useful as chiral building blocks for further synthetic endeavors. The configuration of the stereogenic centers at C6 and C7 was determined to be 6R,7S for one representative tetraketide. Importantly, processing beyond the tetraketide stage is not always blocked when the formation of the bicyclic naphthalene precursor cannot occur. This was proven by formation of a bromo undecaketide, an observation that has implications regarding the evolutionary development of rifamycin biosynthesis.

Identification and Heterologous Expression of the Chaxamycin Biosynthesis Gene Cluster from Streptomyces leeuwenhoekii.

Streptomyces leeuwenhoekii, isolated from the hyperarid Atacama Desert, produces the new ansamycin-like compounds chaxamycins A to D, which possess potent antibacterial activity and moderate antiproliferative activity. We report the development of genetic tools to manipulate S. leeuwenhoekii and the identification and partial characterization of the 80.2-kb chaxamycin biosynthesis gene cluster, which was achieved by both mutational analysis in the natural producer and heterologous expression in Streptomyces coelicolor A3(2) strain M1152. Restoration of chaxamycin production in a nonproducing ΔcxmK mutant (cxmK encodes 3-amino-5-hydroxybenzoic acid [AHBA] synthase) was achieved by supplementing the growth medium with AHBA, suggesting that mutasynthesis may be a viable approach for the generation of novel chaxamycin derivatives.

Solid state fermentation and production of rifamycin SV using Amycolatopsis mediterranei.

UNLABELLED: Production of Rifamycin SV from cheaper agro-industrial by-products using mutant strain of Amycolatopsis mediterranei OVA5-E7 in solid state fermentation (SSF) was optimized. Among the agro-based substrates used, ragi bran was found suitable for maximizing the yield of Rifamycin SV (1310 mg 100 g(-1) ds). The yield can be further enhanced to 19·7 g Kg(-1) of dry substrate by supplementing the substrate with deoiled cotton cake (10% w/w) using optimized fermentation parameters such as maintaining 80% moisture, pH 7·0, 30°C incubation temperature, inoculum 25% v/w and carrying the solid state fermenting for 9 days. Manipulating these seven specifications, the end product yield achieved in our experimentation was 20 g of Rifamycin SV Kg(-1) ds. Eventually, an overall 5-fold improvement in Rifamycin SV production was achieved. SIGNIFICANCE AND IMPACT OF THE STUDY: Antibiotics such as rifamycin are broad-spectrum antimicrobial drugs used in large-scale worldwide as human medicine towards controlling diseases. Amycolatopsis mediterranei strain which produces this antibiotic was earlier used in submerged fermentation yielded lower amounts of rifamycin. By employing cheaper agro-industrial by-products, we produced upto 20 g rifamycin SV per Kg dry substrate used under optimized solid state fermentation conditions. Keeping in view, the role of rifamycin in meeting the medical demands of world's increasing population; we successfully used an improved strain on cheaper substrates with optimized fermentation parameters and achieved a 5-fold improvement in rifamycin SV production.

Effects of salinity on antibiotic production in sponge-derived Salinispora actinobacteria.

AIMS: To investigate the effects of growth conditions related to marine habitat on antibiotic production in sponge-derived Salinispora actinobacteria. METHODS AND RESULTS: Media with varying salt concentration were used to investigate the effects of salinity in relation to Salinispora growth and rifamycin production. The chemotypic profiles of the model strain Salinispora arenicola M413 was then assessed using metabolomic fingerprints from high-pressure liquid chromatography with diode array detection (HPLC-DAD) and multivariate data analysis, before extending this approach to two other strains of S. arenicola. Fingerprint data were generated from extracts of S. arenicola broth cultures grown in media of varying salt (NaCl) concentrations. These fingerprints were then compared using multivariate analysis methods such as principal components analysis (PCA) and orthogonal projection to latent structures discriminant analysis (OPLS-DA). From the analysis, a low-sodium growth condition (1% NaCl) was found to delay the onset of growth of the model S. arenicola M413 strain when compared to growth in media with either 3% artificial sea salt or 3% NaCl. However, low-sodium growth conditions also increased cell mass yield and contributed to at least a significant twofold increase in rifamycin yield when compared to growth in 3% artificial sea salt and 3% NaCl. CONCLUSIONS: The integration of HPLC-DAD and multivariate analysis proved to be an effective method of assessing chemotypic variations in Salinispora grown in different salt conditions, with clear differences between strain-related chemotypes apparent due to varying salt concentrations. SIGNIFICANCE AND IMPACT OF THE STUDY: The observed variation in S. arenicola chemotypic profiles further suggests diversity in secondary metabolites in this actinomycete in response to changes in the salinity of its environment.

Discovering the recondite secondary metabolome spectrum of Salinispora species: a study of inter-species diversity.

Patterns of inter-species secondary metabolite production by bacteria can provide valuable information relating to species ecology and evolution. The complex nature of this chemical diversity has previously been probed via directed analyses of a small number of compounds, identified through targeted assays rather than more comprehensive biochemical profiling approaches such as metabolomics. Insights into ecological and evolutionary relationships within bacterial genera can be derived through comparative analysis of broader secondary metabolite patterns, and this can also eventually assist biodiscovery search strategies for new natural products. Here, we investigated the species-level chemical diversity of the two marine actinobacterial species Salinispora arenicola and Salinispora pacifica, isolated from sponges distributed across the Great Barrier Reef (GBR), via their secondary metabolite profiles using LC-MS-based metabolomics. The chemical profiles of these two species were obtained by UHPLC-QToF-MS based metabolic profiling. The resultant data were interrogated using multivariate data analysis methods to compare their (bio)chemical profiles. We found a high level of inter-species diversity in strains from these two bacterial species. We also found rifamycins and saliniketals were produced exclusively by S. arenicola species, as the main secondary metabolites differentiating the two species. Furthermore, the discovery of 57 candidate compounds greatly increases the small number of secondary metabolites previously known to be produced by these species. In addition, we report the production of rifamycin O and W, a key group of ansamycin compounds, in S. arenicola for the first time. Species of the marine actinobacteria harbour a much wider spectrum of secondary metabolites than suspected, and this knowledge may prove a rich field for biodiscovery as well as a database for understanding relationships between speciation, evolution and chemical ecology.

Biosynthesis of 3,5-AHBA-derived natural products.

Covering: 1957 to 2011. 3-Amino-5-hydroxy benzoic acid (3,5-AHBA) is a precursor for a large group of natural products, including the family of naphthalenic and benzenic ansamycins, the unique saliniketals, and the family of mitomycins. This review covers the biosynthesis of AHBA-derived natural products from a molecular genetics, chemical, and biochemical perspectives, and 174 references are cited.

Two genes, rif15 and rif16, of the rifamycin biosynthetic gene cluster in Amycolatopsis mediterranei likely encode a transketolase and a P450 monooxygenase, respectively, both essential for the conversion of rifamycin SV into B.

Amycolatopsis mediterranei produces an important antibiotic rifamycin, the biosynthesis of which involves many unusual modifications. Previous work suggested a putative P450 enzyme encoded by rif16 within the rifamycin biosynthetic gene cluster (rif) was required for the conversion of the intermediate rifamycin SV into the end product rifamycin B. In this study, we genetically proved that a putative transketolase encoded by rif15 is another essential enzyme for this conversion. Expression of merely rif15 and rif16 in a rif cluster null mutant of A. mediterranei U32 was able to convert rifamycin SV into B. However, this Rif15- and Rif16-mediated conversion was only detected in intact cells of A. meidterranei, but not in Streptomyce coelicolor or Mycobacterium smegmatis, suggesting that yet-characterized gene(s) in A. mediterranei other than those encoded by the rif cluster should be involved in this process.