An overview of the two-component system GarR/GarS role on antibiotic production in Streptomyces coelicolor.

The Streptomyces genus comprises Gram-positive bacteria known to produce over two-thirds of the antibiotics used in medical practice. The biosynthesis of these secondary metabolites is highly regulated and influenced by a range of nutrients present in the growth medium. In Streptomyces coelicolor, glucose inhibits the production of actinorhodin (ACT) and undecylprodigiosin (RED) by a process known as carbon catabolite repression (CCR). However, the mechanism mediated by this carbon source still needs to be understood. It has been observed that glucose alters the transcriptomic profile of this actinobacteria, modifying different transcriptional regulators, including some of the one- and two-component systems (TCSs). Under glucose repression, the expression of one of these TCSs SCO6162/SCO6163 was negatively affected. We aimed to study the role of this TCS on secondary metabolite formation to define its influence in this general regulatory process and likely establish its relationship with other transcriptional regulators affecting antibiotic biosynthesis in the Streptomyces genus. In this work, in silico predictions suggested that this TCS can regulate the production of the secondary metabolites ACT and RED by transcriptional regulation and protein-protein interactions of the transcriptional factors (TFs) with other TCSs. These predictions were supported by experimental procedures such as deletion and complementation of the TFs and qPCR experiments. Our results suggest that in the presence of glucose, the TCS SCO6162/SCO6163, named GarR/GarS, is an important negative regulator of the ACT and RED production in S. coelicolor. KEY POINTS: • GarR/GarS is a TCS with domains for signal transduction and response regulation • GarR/GarS is an essential negative regulator of the ACT and RED production • GarR/GarS putatively interacts with and regulates activators of ACT and RED.

SCO6992, a Protein with ß-Glucuronidase Activity, Complements a Mutation at the absR Locus and Promotes Antibiotic Biosynthesis in Streptomyces coelicolor.

Streptomyces coelicolor is a filamentous soil bacterium producing several kinds of antibiotics. S. coelicolor abs8752 is an abs (antibiotic synthesis deficient)-type mutation at the absR locus; it is characterized by an incapacity to produce any of the four antibiotics synthesized by its parental strain J1501. A chromosomal DNA fragment from S. coelicolor J1501, capable of complementing the abs- phenotype of the abs8752 mutant, was cloned and analyzed. DNA sequencing revealed that two complete ORFs (SCO6992 and SCO6993) were present in opposite directions in the clone. Introduction of SCO6992 in the mutant strain resulted in a remarkable increase in the production of two pigmented antibiotics, actinorhodin and undecylprodigiosin, in S. coelicolor J1501 and abs8752. However, introduction of SCO6993 did not show any significant difference compared to the control, suggesting that SCO6992 is primarily involved in stimulating the biosynthesis of antibiotics in S. coelicolor. In silico analysis of SCO6992 (359 aa, 39.5 kDa) revealed that sequences homologous to SCO6992 were all annotated as hypothetical proteins. Although a metalloprotease domain with a conserved metal-binding motif was found in SCO6992, the recombinant rSCO6992 did not show any protease activity. Instead, it showed very strong ß-glucuronidase activity in an API ZYM assay and toward two artificial substrates, p-nitrophenyl-ß-D-glucuronide and AS-BI-ß-D-glucuronide. The binding between rSCO6992 and Zn2+ was confirmed by circular dichroism spectroscopy. We report for the first time that SCO6992 is a novel protein with ß-glucuronidase activity, that has a distinct primary structure and physiological role from those of previously reported ß-glucuronidases.

Butylcycloheptylprodigiosin and undecylprodigiosin are potential photosensitizer candidates for photodynamic cancer therapy.

BACKGROUND: Prodiginines are bacterial red polypyrrole pigments and multifaceted secondary metabolites. These agents have anti-proliferative, immunosuppressive, antimicrobial, and anticancer effects. Recent analysis revealed that prodigiosin hypersensitizes Serratia marcescens to gamma radiation. In the present study, we report the cytotoxicity and genotoxicity properties of undecylprodigiosin and butylcycloheptylprodigiosin in the presence and absence of radiation through the MTT and alkaline comet experiments. METHODS AND RESULTS: Findings demonstrated that undecylprodigiosin was at least a fivefold more cytotoxic at low radiation doses (1 and 3 Gy) on both MCF7 and HDF lines rather than in the absence or high radiation doses (5 Gy) (P value < 0.05). Although butylcycloheptylprodigiosin toxicity on MCF7 and HDF was dose-dependent, it was not influenced by any radiation doses (P value > 0.05). Comet findings confirmed that these compounds' genotoxicity is only dose-dependent. Radiation had no significant effects on DNA damage on any of the cells (P value > 0.05). CONCLUSIONS: In general, it can be concluded that the prodiginines are cytotoxic agents that act as a double-edged sword, radiosensitizers and radio-protective, respectively at low and high radiation doses in cancer treatment process. As the results they could be used in antitumor therapies very soon.

Total Synthesis and Antimalarial Activity of 2-(p-Hydroxybenzyl)-prodigiosins, Isoheptylprodigiosin, and Geometric Isomers of Tambjamine MYP1 Isolated from Marine Bacteria.

Highly efficient and straightforward synthetic routes toward the first total synthesis of 2-(p-hydroxybenzyl)-prodigiosins (2-5), isoheptylprodigiosin (6), and geometric isomers of tambjamine MYP1 ((E/Z)-7) have been developed. The crucial steps involved in these synthetic routes are the construction of methoxy-bipyrrole-carboxaldehydes (MBCs) and a 20-membered macrocyclic core and a regioselective demethylation of MBC analogues. These new synthetic routes enabled us to generate several natural prodiginines 24-27 in larger quantity. All of the synthesized natural products exhibited potent asexual blood-stage antiplasmodial activity at low nanomolar concentrations against a panel of Plasmodium falciparum parasites, with a great therapeutic index. Notably, prodiginines 6 and 24-27 provided curative in vivo efficacy against erythrocytic Plasmodium yoelii at 25 mg/kg × 4 days via oral route in a murine model. No overt clinical toxicity or behavioral change was observed in any mice treated with prodiginines and tambjamines.

Antibacterial Performance of a Streptomyces spectabilis Strain Producing Metacycloprodigiosin.

After separation of bacterial colonies on solid plates, purification, and screening through the agar cup-plate method, an antibiotic-resistant bacterial isolate was obtained, and named strain L20190601, the 16S rRNA gene sequence data of strain L20190601 to GenBank, NCBI have provided GenBank accession number MW931615. 16S rRNA gene sequencing revealed that this isolate was highly similar to a number of Streptomyces species. Among them, the homology with S. spectabilis was the highest, reaching 99.9, together with curved hyphal morphology and biochemical tests, allowed us to identify strain L20190601 as S. spectabilis. The red pigment produced by S. spectabilis strain L20190601 was structurally identified. An acid-base color reaction assay showed that when this pigment was dissolved in a solution at pH 3.0 and 9.0, the color of the solution was red and yellow, respectively. In addition, the analysis of absorption spectra revealed that at pH 8.0 and 3.0, the maximum absorption peaks were at 466 and 531 nm, respectively. These results are consistent with the spectral absorption characteristics of metacycloprodigiosin reported in the literature. Moreover, the retention time of purified pigments was identical to those of standard metacycloprodigiosin solutions. Mass spectrometry analysis revealed that the molecular weight of the red compound was 392.2 [M + H]+. Finally, metacycloprodigiosin was found to be effective against eight clinically common pathogens: Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Streptococcus pyogenes, Pseudomonas aeruginosa, Bacillus typhi, Candida albicans, and Trichophyton rubrum. In summary, metacycloprodigiosin exhibited strong antibacterial activity and a broad antibacterial spectrum, and thus is a promising compound for the development of a new type of antibacterial drug.

Differential regulation of undecylprodigiosin biosynthesis in the yeast-scavenging Streptomyces strain MBK6.

Streptomyces are efficient chemists with a capacity to generate diverse and potent chemical scaffolds. The secondary metabolism of these soil-dwelling prokaryotes is stimulated upon interaction with other microbes in their complex ecosystem. We observed such an interaction when a Streptomyces isolate was cultivated in a media supplemented with dead yeast cells. Whole-genome analysis revealed that Streptomyces sp. MBK6 harbors the red cluster that is cryptic under normal environmental conditions. An interactive culture of MBK6 with dead yeast triggered the production of the red pigments metacycloprodigiosin and undecylprodigiosin. Streptomyces sp. MBK6 scavenges dead-yeast cells and preferentially grows in aggregates of sequestered yeasts within its mycelial network. We identified that the activation depends on the cluster-situated regulator, mbkZ, which may act as a cross-regulator. Cloning of this master regulator mbkZ in S. coelicolor with a constitutive promoter and promoter-deprived conditions generated different production levels of the red pigments. These surprising results were further validated by DNA-protein binding assays. The presence of the red cluster in Streptomyces sp. MBK6 provides a vivid example of horizontal gene transfer of an entire metabolic pathway followed by differential adaptation to a new environment through mutations in the receiver domain of the key regulatory protein MbkZ.

Stringent Starvation Protein Regulates Prodiginine Biosynthesis via Affecting Siderophore Production in Pseudoalteromonas sp. Strain R3.

Prodiginines are a family of red-pigmented secondary metabolites with multiple biological activities. The biosynthesis of prodiginines is affected by various physiological and environmental factors. Thus, prodiginine biosynthesis regulation is highly complex and multifaceted. Although the regulatory mechanism for prodiginine biosynthesis has been extensively studied in Serratia and Streptomyces species, little is known about that in the marine betaproteobacterium Pseudoalteromonas In this study, we report that stringent starvation protein A (SspA), an RNA polymerase-associated regulatory protein, is required for the biosynthesis of prodiginine in Pseudoalteromonas sp. strain R3. The strain lacking sspA (ΔsspA) fails to produce prodiginine, which resulted from the downregulation of the prodiginine biosynthetic gene (pig) cluster. The effect of SspA on prodiginine biosynthesis is independent of histone-like nucleoid structuring protein (H-NS) and RpoS (σS). Further analysis demonstrates that the ΔsspA strain has a significant decrease in the transcription of the siderophore biosynthesis gene (pvd) cluster, leading to the inhibition of siderophore production and iron uptake. The ΔsspA strain regains the ability to synthesize prodiginine by cocultivation with siderophore producers or the addition of iron. Therefore, we conclude that SspA-regulated prodiginine biosynthesis is due to decreased siderophore levels and iron deficiency. We further show that the iron homeostasis master regulator Fur is also essential for pig transcription and prodiginine biosynthesis. Overall, our results suggest that SspA indirectly regulates the biosynthesis of prodiginine, which is mediated by the siderophore-dependent iron uptake pathway.IMPORTANCE The red-pigmented prodiginines are attracting increasing interest due to their broad biological activities. As with many secondary metabolites, the biosynthesis of prodiginines is regulated by both environmental and physiological factors. At present, studies on the regulation of prodiginine biosynthesis are mainly restricted to Serratia and Streptomyces species. This work focused on the regulatory mechanism of prodiginine biosynthesis in Pseudoalteromonas sp. R3. We found that stringent starvation protein A (SspA) positively regulates prodiginine biosynthesis via affecting the siderophore-dependent iron uptake pathway. The connections among SspA, iron homeostasis, and prodiginine biosynthesis were investigated. These findings uncover a novel regulatory mechanism for prodigiosin biosynthesis.

Enhancement of undecylprodigiosin production from marine endophytic recombinant strain Streptomyces sp. ALAA-R20 through low-cost induction strategy.

Genetic manipulation of the undecylprodigiosin-producing strains and engineered culture medium approaches were applied as the most economical induction strategy for improving production. The hyper-producing recombinant strain ALAA-R20 was obtained after applying protoplast fusion strategy between the potent producer marine endophytic strains Streptomyces sp. ESRAA-10 (P1) and Streptomyces sp. ESRAA-31 (P2) of Dendronephthya hemprichi. Recombinant strain ALAA-R20 produced undecylprodigiosin yield higher than its parental strains ESRAA-10 and ESRAA-31 by 82.45% and 105.52% under submerged fermentation using modified R2YE medium. In order to reduce the costs of producing undecylprodigiosin, a solid-state fermentation (SSF) was applied. Scaled-up of optimized SSF parameters consisting of groundnut oil cake (GOC) sized to 3 mm, initial moisture content 80% with a mixture of dairy mill and fruit processing wastewaters (1:1), pH 7.0, inoculum size equal to 3 × 105 spores/g dry substrate (gds), incubation temperature 30 °C, and 7-day incubation period yielded the highest yield of 181.78 mg/gds of undecylprodigiosin by the recombinant strain Streptomyces sp. ALAA-R20. Extraction and purification of the pigment using the chromatographic techniques as well as mass spectral analysis exhibited maximum absorbance at 539 nm which is physiological property of the undecylprodigiosin. Undecylprodigiosin was stable over a wide temperature ranged from - 20 to 35 °C even after storage for 6 months. The maximum yield and stability of pigment was obtained at the acidic pH (acidified methanol, pH 4.0). Undecylprodigiosin obtained from the recombinant strain Streptomyces sp. ALAA-R20 demonstrated strong antimicrobial activity against all multidrug-resistant bacterial and fungal strains tested with minimum inhibitory, minimum bactericidal, and minimum fungicidal concentrations ranged between 0.5 and 4.0, 0.5 to 4.0, and 1.0 to 8.0 µg/mL, respectively. It also showed complete inhibition of cancer cells; HCT-116, HepG-2, MCF-7 and A-549 at 5, 8, 4, and 7 µM with IC50 equal to 2.0, 4.7, 1.2, and 2.8 µM, respectively.

Prodigiosin Modulates the Immune Response and Could Promote a Stable Atherosclerotic Lession in C57bl/6 Ldlr-/- Mice.

Atherosclerosis is a chronic inflammatory disease, whose progression and stability are modulated, among other factors, by an innate and adaptive immune response. Prodiginines are bacterial secondary metabolites with antiproliferative and immunomodulatory activities; however, their effect on the progression or vulnerability of atheromatous plaque has not been evaluated. This study assessed the therapeutic potential of prodigiosin and undecylprodigiosin on inflammatory marker expression and atherosclerosis. An in vitro and in vivo study was carried out. Migration, low-density lipoprotein (LDL) uptake and angiogenesis assays were performed on cell types involved in the pathophysiology of atherosclerosis. In addition, male LDL receptor null (Ldlr-/-) C57BL/6J mice were treated with prodigiosin or undecylprodigiosin for 28 days. Morphometric analysis of atherosclerotic plaques, gene expression of atherogenic factors in the aortic sinus and serum cytokine quantification were performed. The treatments applied had slight effects on the in vitro tests performed, highlighting the inhibitory effect on the migration of SMCs (smooth muscle cells). On the other hand, although no significant difference in atherosclerotic plaque progression was observed, gene expression of IL-4 and chemokine (C-C motif) ligand 2 (Ccl2) was downregulated. In addition, 50 µg/Kg/day of both treatments was sufficient to inhibit circulating tumor necrosis factor alpha (TNF-α), interleukin-2 (IL-2) and interferon-gamma (IFN-γ) in serum. These results suggested that prodigiosin and undecylprodigiosin modulated inflammatory markers and could have an impact in reducing atherosclerotic plaque vulnerability.

The different anion transport capability of prodiginine- and tambjamine-like molecules.

Prodiginines and tambjamines are anion-selective ionophores capable of facilitating the transport of anions across the plasma membrane in mammalian cells. One of the potential applications of these anionophores is the possibility of employing them as a substitutive therapy for pathologies involving anion channels, as in cystic fibrosis. We have studied the interaction of a large anion as gluconate with three prodiginine- and two tambjamine-like compounds. Apparent dissociation constants for the chloride, iodide and gluconate complexes were estimated from iodide influx experiments in mammalian cells exposed to different extracellular anion combinations. Our experiments indicate that gluconate is not transported by the prodiginines, leaving the anionophores free to transport chloride and iodide. Conversely, gluconate would be transported to some extent by the tambjamines, competing with halides for the anionophores, and consequently reducing their flux. This might be related to the different structural features of both families of compounds. These data have important implications for the selection of impermeable anions in the analysis of the anionophore mechanism.

Survivin modulation in the antimelanoma activity of prodiginines.

Melanoma is a type of skin cancer with an elevated incidence of metastasis and chemoresistance. Such features hamper treatment success of these neoplasms, demanding the search for new therapeutic options. Using a two-step resin-based approach, we recently demonstrated that cytotoxic prodiginines bind to the inhibitor of apoptosis protein, survivin. Herein, we explore the role of survivin in melanoma and whether its modulation is related to the antimelanoma properties of three cytotoxic prodiginines (prodigiosin, cyclononylprodigiosin, and nonylprodigiosin) isolated from marine bacteria. In melanoma patients and cell lines, survivin is overexpressed, and higher levels negatively impact survival. All three prodiginines caused a decrease in cell growth with reduced cytotoxicity after 24 h compared to 72 h treatment, suggesting that low concentrations promote cytostatic effects in SK-Mel-19 (BRAF mutant) and SK-Mel-28 (BRAF mutant), but not in SK-Mel-147 (NRAS mutant). An increase in G1 population was observed after 24 h treatment with prodigiosin and cyclononylprodigiosin in SK-Mel-19. Further studies indicate that prodigiosin induced apoptosis and DNA damage, as detected by increased caspase-3 cleavage and histone H2AX phosphorylation, further arguing for the downregulation of survivin. Computer simulations suggest that prodigiosin and cyclononylprodigiosin bind to the BIR domain of survivin. Moreover, knockdown of survivin increased long-term toxicity of prodigiosin, as observed by reduced clonogenic capacity, but did not alter short-term cytotoxicity. In summary, prodiginine treatment provoked cytostatic rather than cytotoxic effects, cell cycle arrest at G0/G1 phase, induction of apoptosis and DNA damage, downregulation of survivin, and decreased clonogenic capacity in survivin knockdown cells.

Marine Actinomycetes-Derived Secondary Metabolites Overcome TRAIL-Resistance via the Intrinsic Pathway through Downregulation of Survivin and XIAP.

Resistance of cancer cells to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis represents the major hurdle to the clinical use of TRAIL or its derivatives. The discovery and development of lead compounds able to sensitize tumor cells to TRAIL-induced cell death is thus likely to overcome this limitation. We recently reported that marine actinomycetes' crude extracts could restore TRAIL sensitivity of the MDA-MB-231 resistant triple negative breast cancer cell line. We demonstrate in this study, that purified secondary metabolites originating from distinct marine actinomycetes (sharkquinone (1), resistomycin (2), undecylprodigiosin (3), butylcyclopentylprodigiosin (4), elloxizanone A (5) and B (6), carboxyexfoliazone (7), and exfoliazone (8)), alone, and in a concentration-dependent manner, induce killing in both MDA-MB-231 and HCT116 cell lines. Combined with TRAIL, these compounds displayed additive to synergistic apoptotic activity in the Jurkat, HCT116 and MDA-MB-231 cell lines. Mechanistically, these secondary metabolites induced and enhanced procaspase-10, -8, -9 and -3 activation leading to an increase in PARP and lamin A/C cleavage. Apoptosis induced by these compounds was blocked by the pan-caspase inhibitor QvD, but not by a deficiency in caspase-8, FADD or TRAIL agonist receptors. Activation of the intrinsic pathway, on the other hand, is likely to explain both their ability to trigger cell death and to restore sensitivity to TRAIL, as it was evidenced that these compounds could induce the downregulation of XIAP and survivin. Our data further highlight that compounds derived from marine sources may lead to novel anti-cancer drug discovery.

Antimicrobial and Antioxidant Effects of a Forest Actinobacterium V002 as New Producer of Spectinabilin, Undecylprodigiosin and Metacycloprodigiosin.

The aim of the study is the research and identification of a Streptomyces strain as a new producer of spectinabilin, undecylprodigiosin and metacycloprodigiosin. Among 54 actinomycete isolates isolated from El-Ogbane forest soils in Algeria, only one isolate, designated V002, was selected for its ability to produce prodigiosins. The selected strain was analysed for its ability to produce three different secondary metabolites as well as their biological activities. V002 belongs to the Streptomyces genus and has significant antimicrobial and antioxidant activities. The taxonomic position of V002 by 16S rRNA sequence analysis showed a similarity of 99.93% with Streptomyces lasiicapitis DSM 103124T and 98.96% with Streptomyces spectabilis DSM 40512T. Fractionation of crude secondary metabolites produced by the strain using HPLC-MS revealed the presence of spectinabilin, undecylprodigiosin and metacycloprodigiosin, which demonstrated significant activity. Strain V002 is considered a new producer of spectinabilin, undecylprodigiosin and metacycloprodigiosin with significant antimicrobial and antioxidant activity.

The production and bioactivity of prodigiosin: quo vadis?

Prodigiosin (PG), a red tripyrrole pigment, belongs to a member of the prodiginine family and is normally secreted by various sources including Serratia marcescens and other Gram-negative bacteria. The studies of PG have received innovative devotion as a result of reported antimicrobial, larvicidal and anti-nematoid immunomodulation and antitumor properties, owing to its antibiotic and cytotoxic activities. This review provides a comprehensive summary of research undertaken toward the isolation and structural elucidation of the prodiginine family of natural products. Additionally, the current evidence-based understanding of the biological activities and medicinal potential of PG is employed to determine the efficacy, with some reports of information related to pharmacokinetics, pharmacodynamics and toxicology.

Screening of new cell cycle suppressive compounds from marine-derived microorganisms in Chinese hamster ovary cells.

Monoclonal antibodies (mAbs) are active pharmaceutical ingredients in antibody drugs, produced mainly using recombinant Chinese hamster ovary (CHO) cells. The regulation of recombinant CHO cell proliferation can improve the productivity of heterologous proteins. Chemical compound approaches for cell cycle regulation have the advantages of simplicity and ease of use in industrial processes. However, CHO cells have genetic and phenotypic diversity, and the effects of such compounds might depend on cell line and culture conditions. Increasing the variety of cell cycle inhibitors is a promising strategy to overcome the dependency. Marine microorganisms are a vast and largely undeveloped source of secondary metabolites with physiological activity. In this study, we focused on secondary metabolites of marine microorganisms and evaluated their effectiveness as cell cycle inhibitory compounds. Of 720 extracts from microorganisms (400 actinomycetes and 320 filamentous fungi) collected from the Okinawan Sea, we identified nine extracts that decreased the specific growth rate and increased the specific production rate without reducing cell viability. After fractionating the extracts, the components of active fractions were estimated using time-of-flight mass spectrometry analysis. Then, four compounds, including staurosporine and undecylprodigiosin were deduced to be active compounds. These compounds have been reported to exert a cell cycle inhibitory effect on mammalian cells. These compounds might serve as additives to improve mAb production in CHO cells. This study indicates that secondary metabolites of marine microorganisms are a useful source for new cell cycle inhibitory compounds that can increase mAb production in CHO cells.

Ion Mobility Mass Spectrometry as an Efficient Tool for Identification of Streptorubin B in Streptomyces coelicolor M145.

Ion mobility spectrometry was utilized to corroborate the identity of streptorubin B (2) as the natural product produced by Streptomyces coelicolor. Natural product 2 was initially assigned as butylcycloheptylprodigiosin (3), and only relatively recently was this assignment clarified. We present additional evidence of this assignment by comparing collisional cross sections (Ω) of synthetic standards of 2, 3, and metacycloprodigiosin (4) to the cyclic prodiginine produced by S. coelicolor. Calculated theoretical Ω values demonstrate that cyclic prodiginines could be identified without standards. This work highlights ion mobility as an efficient tool for the dereplication of natural products.

RedH and PigC Catalyze the Biosynthesis of Hybrubins via Phosphorylation of 4'-Methoxy-2,2'-Bipyrrole-5'-Carbaldehyde.

Hybrubins are "unnatural" alkaloids with the same 4'-methoxy-2,2'-bipyrrole-5'-methine moiety found in prodiginines and a different ring derived from tetramic acids. Here, we demonstrated that RedH, a homologue of prodigiosin synthetase PigC, was responsible for the biosynthesis of hybrubins A and B in Streptomyces lividansIn vitro reactions indicated that RedH and PigC catalyzed the intermolecular condensation between 4'-methoxy-2,2'-bipyrrole-5'-carbaldehyde (MBC) and (Z)-5-ethylidenetetramic acid (ETA) to produce hybrubin B. Moreover, we demonstrated that RedH and PigC activated MBC via phosphorylation of the aldehyde group to form an intermediate Pi-MBC and that the subsequent condensation between Pi-MBC and (Z)-5-ethylidenetetramic acid occurs in a nonenzymatic way.IMPORTANCE Hybrubins are an emerging class of prodiginines possessing a new C ring derived from 5'-substituted tetramic acids and the methylene bridge connecting the C ring at a different position. We have supposed that condensation between 4'-methoxy-2,2'-bipyrrole-5'-carbaldehyde (MBC) and 5-ethylidenetetramic acid (ETA) yields the hybrid natural products hybrubins, which was proposed to be catalyzed by the undecylprodigiosin synthetase RedH. However, it is doubted whether RedH is able to catalyze another type of condensation between MBC and tetramic acids. In this study, we have demonstrated that the MBC-ETA condensation proceeds through RedH/PigC-catalyzed enzymatic activation of MBC via phosphorylation and a nonenzymatic condensation of Pi-MBC with ETA. Since MBC analogues have been shown to be accepted by PigC, more hybrubin analogues might be produced by using combinations of MBC analogues and other tetramic acids in future studies.

Purification and Kinetic Characterization of the Essential Condensation Enzymes Involved in Prodiginine and Tambjamine Biosynthesis.

Prodiginines and tambjamines are related families of bioactive alkaloid natural products with pharmaceutical potential. Both compound families result from a convergent biosynthetic pathway ending in the condensation of a conserved bipyrrole core with a variable partner. This reaction is performed by unique condensation enzymes, and has the potential to be manipulated to produce new pyrrolic compounds. We have purified and reconstituted the in vitro activity of the condensation enzymes PigC and TamQ from Pseudoalteromonas sp., which are involved, respectively, in the prodiginine and tambjamine biosynthetic pathways. Kinetic analysis confirmed a Uni Uni Bi Uni ping-pong reaction sequence with competitive and uncompetitive substrate inhibition for PigC and TamQ respectively. The kinetic parameters of each enzyme provide insight into their differing substrate scope, and suggest that TamQ may have evolved a wide substrate tolerance that can be used for the production of novel prodiginines and tambjamines.

The complete genomic sequence of Streptomyces spectabilis NRRL-2792 and identification of secondary metabolite biosynthetic gene clusters.

This is the first report of a fully annotated genomic sequence of Streptomyces spectabilis NRRL-2792, isolated and identified by The Upjohn Company in 1961. The genome was assembled into a single scaffold for annotation and analysis. The chromosome is linear, 9.5 Mb in size which is one of the largest Streptomyces genomes yet described, has a G+C content of 72%, and encodes for approximately 7943 genes. Antibiotic Secondary Metabolite Analysis Shell (antiSMASH) and Basic Local Alignment Search Tool (BLAST) bioinformatics analyses identified six complete secondary metabolite biosynthetic gene clusters for ectoine, melanin, albaflavenone, spectinomycin, 2-methylisoborneol and coelichelin. Additionally, biosynthetic clusters were identified that shared ≥ 90% gene content with complestatin, hopene, neoaureothin, or undecylprodigiosin. Thirty-one other likely secondary metabolite gene clusters were identified by antiSMASH. BLAST identified two subsets of undecylprodigiosin biosynthetic genes at polar opposites of the chromosome; their duplication was subsequently confirmed by primer walking.

Overexpression of the diguanylate cyclase CdgD blocks developmental transitions and antibiotic biosynthesis in Streptomyces coelicolor.

Cyclic dimeric GMP (c-di-GMP) has emerged as the nucleotide second messenger regulating both development and antibiotic production in high-GC, Gram-positive streptomycetes. Here, a diguanylate cyclase (DGC), CdgD, encoded by SCO5345 from the model strain Streptomyces coelicolor, was functionally identified and characterized to be involved in c-di-GMP synthesis through genetic and biochemical analysis. cdgD overexpression resulted in significantly reduced production of actinorhodin and undecylprodigiosin, as well as completely blocked sporulation or aerial mycelium formation on two different solid media. In the cdgD-overexpression strain, intracellular c-di-GMP levels were 13-27-fold higher than those in the wild-type strain. In vitro enzymatic assay demonstrated that CdgD acts as a DGC, which could efficiently catalyze the synthesis of c-di-GMP from two GTP molecules. Heterologous overproduction of cdgD in two industrial Streptomyces strains could similarly impair developmental transitions as well as antibiotic biosynthesis. Collectively, our results combined with previously reported data clearly demonstrated that c-di-GMP-mediated signalling pathway plays a central and universal role in the life cycle as well as secondary metabolism in streptomycetes.