Di-Isatropolone C, a Spontaneous Isatropolone C Dimer Derivative with Autophagy Activity.

Isatropolone C from Streptomyces sp. CPCC 204095 features a fused cyclopentadienone-tropolone-oxacyclohexadiene tricyclic moiety in its structure. Herein, we report an isatropolone C dimer derivative, di-isatropolone C, formed spontaneously from isatropolone C in methanol. Notably, the structure of di-isatropolone C resolved by NMR reveals a newly formed cyclopentane ring to associate the two isatropolone C monomers. The configurations of four chiral carbons, including a ketal one, in the cyclopentane ring are assigned using quantum NMR calculations and DP4+ probability. The plausible molecular mechanism for di-isatropolone C formation is proposed, in which complex dehydrogenative C-C bond coupling may have happened to connect the two isatropolone C monomers. Like isatropolone C, di-isatropolone C shows the biological activity of inducing autophagy in HepG2 cells.

Engineering Streptomyces sp. CPCC 204095 for the targeted high-level production of isatropolone A by elucidating its pathway-specific regulatory mechanism.

BACKGROUND: Isatropolone A and C, produced by Streptomyces sp. CPCC 204095, belong to an unusual class of non-benzenoid aromatic compounds and contain a rare seven-membered ring structure. Isatropolone A exhibits potent activity against Leishmania donovani, comparable to the only oral drug miltefosine. However, its variably low productivity represents a limitation for this lead compound in the future development of new anti-leishmaniasis drugs to meet unmet clinical needs. RESULTS: Here we first elucidated the regulatory cascade of biosynthesis of isatropolones, which consists of two SARP family regulators, IsaF and IsaJ. Through a series of in vivo and in vitro experiments, IsaF was identified as a pathway-specific activator that orchestrates the transcription of the gene cluster essential for isatropolone biosynthesis. Interestingly, IsaJ was found to only upregulate the expression of the cytochrome P450 monooxygenase IsaS, which is crucial for the yield and proportion of isatropolone A and C. Through targeted gene deletions of isaJ or isaS, we effectively impeded the conversion of isatropolone A to C. Concurrently, the facilitation of isaF overexpression governed by selected promoters, prompted the comprehensive activation of the production of isatropolone A. Furthermore, meticulous optimization of the fermentation parameters was conducted. These strategies culminated in the attainment of an unprecedented maximum yield-980.8 mg/L of isatropolone A-achieved in small-scale solid-state fermentation utilizing the genetically modified strains, thereby establishing the highest reported titer to date. CONCLUSION: In Streptomyces sp. CPCC 204095, the production of isatropolone A and C is modulated by the SARP regulators IsaF and IsaJ. IsaF serves as a master pathway-specific regulator for the production of isatropolones. IsaJ, on the other hand, only dictates the transcription of IsaS, the enzyme responsible for the conversion of isatropolone A and C. By engineering the expression of these pivotal genes, we have devised a strategy for genetic modification aimed at the selective and high-yield biosynthesis of isatropolone A. This study not only unveils the unique regulatory mechanisms governing isatropolone biosynthesis for the first time, but also establishes an essential engineering framework for the targeted high-level production of isatropolone A.

Iterative Methylation Leads to 3-Methylchuangxinmycin Production in Actinoplanes tsinanensis CPCC 200056.

A new congener of chuangxinmycin (CM) was identified from Actinoplanes tsinanensis CPCC 200056. Its structure was determined as 3-methylchuangxinmycin (MCM) by 1D and 2D NMR. MCM could be generated in vivo from CM by heterologous expression of the vitamin B12-dependent radical SAM enzyme CxnA/A1 responsible for methylation of 3-demethylchuangxinmycin (DCM) in CM biosynthesis, indicating that CxnA/A1 could perform iterative methylation for MCM production. In vitro assays revealed significant activities of CM, DCM, and MCM against Mycobacterium tuberculosis H37Rv and clinically isolated isoniazid/rifampin-resistant M. tuberculosis, suggesting that CM and its derivatives may have potential for antituberculosis drug development.

Geninthiocins E and F, two new cyclic thiopeptides with antiviral activities from soil-derived Streptomyces sp. CPCC 200267 using OSMAC strategy.

On the basis of the one strain-many compounds (OSMAC) strategy, two new cyclic thiopeptides, geninthiocins E and F, together with four known geninthiocin derivatives, geninthiocins A, B, C, and val-geninthiocin were isolated from Streptomyces sp. CPCC 200267. Their structures and absolute configurations were elucidated by extensive spectroscopic analyses and Marfey's method. Geninthiocin E (1), val-geninthiocin (3), geninthiocin A (4), and geninthiocin B (5) exhibited significant anti-influenza A virus activities with the IC50 values of 28.7, 15.3, 7.3, and 18.3 µM, respectively. Compounds 3 and 4 showed moderate antibacterial activities against Staphylococcus aureus.

Isatropolone/isarubrolone Cm from Streptomyces with biological activity of inducing incomplete autophagy.

Isatropolones/isarubrolones are Streptomyces secondary metabolites featuring a tropolone ring in the pentacyclic scaffolds of these molecules. They are able to induce complete autophagy in human HepG2 cells. Here, methyl isatropolones (1-2) and isarubrolone (3) are identified from Streptomyces CPCC 204095. They all have a methyl tropolone ring in the pentacyclic scaffold of these molecules resolved by MS and NMR spectra. Biological activity assay indicates that isatropolone Cm (1) and isarubrolone Cm (3) induce incomplete autophagy in human HepG2 cells.

Production of chain-extended cinnamoyl compounds by overexpressing two adjacent cluster-situated LuxR regulators in Streptomyces globisporus C-1027.

Natural products from microorganisms are important sources for drug discovery. With the development of high-throughput sequencing technology and bioinformatics, a large amount of uncharacterized biosynthetic gene clusters (BGCs) in microorganisms have been found, which show the potential for novel natural product production. Nine BGCs containing PKS and/or NRPS in Streptomyces globisporus C-1027 were transcriptionally low/silent under the experimental fermentation conditions, and the products of these clusters are unknown. Thus, we tried to activate these BGCs to explore cryptic products of this strain. We constructed the cluster-situated regulator overexpressing strains which contained regulator gene(s) under the control of the constitutive promoter ermE*p in S. globisporus C-1027. Overexpression of regulators in cluster 26 resulted in significant transcriptional upregulation of biosynthetic genes. With the separation and identification of products from the overexpressing strain OELuxR1R2, three ortho-methyl phenyl alkenoic acids (compounds 1-3) were obtained. Gene disruption showed that compounds 1 and 2 were completely abolished in the mutant GlaEKO, but were hardly affected by deletion of the genes orf3 or echA in cluster 26. The type II PKS biosynthetic pathway of chain-extended cinnamoyl compounds was deduced by bioinformatics analysis. This study showed that overexpression of the two adjacent cluster-situated LuxR regulator(s) is an effective strategy to connect the orphan BGC to its products.

Dihydroisatropolone C from Streptomyces and Its Implication in Tropolone-Ring Construction for Isatropolone Biosynthesis.

Isatropolones/isarubrolones are actinomycete secondary metabolites featuring a tropolone-ring in their structures. From the isatropolone/isarubrolone producer Streptomyces sp. CPCC 204095, 7,12-dihydroisatropolone C (H2ITC) is discovered and identified as a mixture of two interchangeable diastereomers differing in the C-6 configuration. As a major metabolite in the mycelial growth period of Streptomyces sp. CPCC 204095, H2ITC can be oxidized spontaneously to isatropolone C (ITC), suggesting H2ITC is the physiological precursor of ITC. Characterization of H2ITC makes us propose dihydrotropolone-ring construction in the biosynthesis of isatropolones.

Mintaimycins, a Group of Novel Peptide Metabolites from Micromonospora sp. C-3509.

A group of peptide metabolites (1-4), designated as mintaimycins, were isolated from Micromonospora sp. C-3509. The planar structures of mintaimycins were determined by combination of mass spectrometry, 1D and 2D NMR spectroscopy, and the stereochemistry of mintaimycins were partially resolved by Marfey's or Mosher's method. Mintaimycins featured a central ß-methylphenylalanine or phenylalanine linked at its amino group with 5-methyl-2-hexenoic acid, and at its carboxyl group with 5-hydroxy-norleucine or leucine that combined a derivative of hexanoic acid or 4-methylpentanoic acid. Mintaimycin A1 (1), the principal component, was found to exhibit the biological activity of inducing pre-adipocyte differentiation of 3T3-L1 fibroblast cells at 10.0 µmol/L.

Cytochrome P450 Monooxygenase for Catalyzing C-42 Hydroxylation of the Glycine-Derived Fragment in Hangtaimycin Biosynthesis.

A hybrid trans-AT PKS/NRPS gene cluster htm was identified with defined boundaries for hangtaimycin biosynthesis in Streptomyces spectabilis CPCC200148. Deoxyhangtaimycin, a new derivative of hangtaimycin, was identified from the htm11 gene knockout mutant. In vitro biochemical assays demonstrated that the cytochrome P450 monooxygenase Htm11 was responsible for the stereoselective hydroxylation of deoxyhangtaimycin to hangtaimycin. More importantly, deoxyhangtaimycin showed activity against influenza A virus at the micromolar level, highlighting its potential as an antiviral lead compound.

Isarubrolone C Promotes Autophagic Degradation of Virus Proteins via Activating ATG10S in HepG2 Cells.

Isarubrolone C is a bioactive polycyclic tropoloalkaloid from Streptomyces. Our previous study showed that isarubrolone C could trigger autophagy. Here, we report isarubrolone C potential in broad-spectrum antiviral effect and its antiviral mechanism in vitro. Our results show that isarubrolone C activated autophagy and reduced levels of viral proteins in the cells harboring HCV-CORE/NS5B, HBx, ZIKV-NS5, and HIV-RT, respectively. The role of isarubrolone C in suppression of the viral proteins was via an autophagic degradation pathway rather than a proteasome pathway. Co-immunoprecipitation assays revealed that isarubrolone C promoted both autophagy flux opening and the viral proteins being enwrapped in autolysosomes. PCR assays showed that isarubrolone C elevated the transcription levels of ATG10/ATG10S and IL28A. Further, ATG10S high expression could efficiently enhance IL28A expression and the ability of isarubrolone C to degrade the viral proteins by promoting the colocalization of viral proteins with autolysosomes. Additionally, knockdown of endogenous IL28A caused both losses of the isarubrolone C antiviral effect and autolysosome formation. These results indicate that the role of isarubrolone C antiviruses is achieved by triggering the autophagic mechanism, which is mediated by endogenous ATG10S and IL28A activation. This is the first report about isarubrolone C potential of in vitro broad-spectrum antiviruses.

The Cytochrome P450 Catalyzing C-S Bond Formation in S-Heterocyclization of Chuangxinmycin Biosynthesis.

Microbial sulfur-containing secondary metabolites show various biological activities, but the C-S bond-forming in their biosynthetic metabolism has not been thoroughly understood. Here, we present genetic, biochemical and structural characterization of a cytochrome P450 monooxygenase CxnD exhibiting C-S bond forming activity in S-heterocyclization of chuangxinmycin biosynthesis. In vivo and in vitro analyses demonstrated that CxnD generated an indole-fused dihydrothiopyran skeleton from a L-Trp-derived thiol intermediate. Furthermore, X-ray crystal structure of CxnD in complex with a substrate analogue and structure-based mutagenesis revealed intimate details of the substrate binding mode. A radical mechanism initiated by abstraction of the imino hydrogen atom or an electron from indole group of the substrate was proposed for CxnD, which provided valuable insights into the molecular basis for the intra-molecular C(sp2 )-H thiolation by the P450 in chuangxinmycin biosynthesis.

Cervinomycins C1-4 with cytotoxic and antibacterial activity from Streptomyces sp. CPCC 204980.

Polycyclic xanthones are secondary metabolites from actinomycetes and cervinomycin A and B are bioactive 26-membered polycyclic xanthones from Streptomyces sp. CPCC 204980. Herein, we report cervinomycins C1-4 (1-4) from the same strain. The structures of 1-4 were determined by 1D- and 2D-NMR, or single-crystal X-ray diffraction. Compounds 1-4 feature the open or loss of A (oxazolidine) ring in their angular polycyclic framework compared with cervinomycin B. Compounds 1-4 showed potent cytotoxicity against human cancer cell lines HCT116 and BxPC-3, with IC50 at 0.9-801.0 nM and strong anti-Gram-positive bacterial activity.

1-hydroxy-7-oxolavanducyanin and Δ7″,8″-6″-hydroxynaphthomevalin from Streptomyces sp. CPCC 203577.

Lavanducyanin is a bioactive phenazine-containing secondary metabolite, and naphthomevalin is an antibacterial polyketide secondary metabolite. Herein, new analogues of lavanducyanin (2) and of naphthomevalin (4), together with lavanducyanin (1) and naphthomevalin (3), were identified from Streptomyces sp. CPCC 203577, an actinomycete soil isolate. The structures of 2 and 4 were elucidated as 1-hydroxy-7-oxolavanducyanin and Δ7″,8″-6″-hydroxynaphthomevalin, respectively, by 1D and 2D NMR. Antibacterial assays revealed that 2 had significant but reduced anti-Gram-positive bacterial activity compared with 1, and 4 was devoid of anti-Gram-positive bacterial activity. This indicated that the phenazinone nucleus in lavanducyanin and the monoterpene side chain in naphthomevalin might be important for their anti-Gram-positive bacterial activity. Compounds 1-4 were all inactive against Gram-negative bacteria.

Cytotoxic and Antibacterial Cervinomycins B1-4 from a Streptomyces Species.

AntiSMASH analysis of genome DNA of Streptomyces CPCC 204980, a soil isolate with potent antibacterial activity, revealed a gene cluster for polycyclic xanthones. A subsequent chemical study confirmed that the microorganism produced polycyclic xanthone cervinomycin A2 (1) and the new congeners cervinomycins B1-4 (2-5). The structures of 1-5 were determined by comprehensive analyses of MS and NMR data, which indicated that 2-5 featured a common dihydro-D ring in the polycyclic xanthone core moiety of their molecules. 2-5 are toxic to human cancer cells and active against Gram-positive bacteria.

Genome-Guided Discovery of Pretilactam from Actinosynnema pretiosum ATCC 31565.

Actinosynnema is a small but well-known genus of actinomycetes for production of ansamitocin, the payload component of antibody-drug conjugates against cancers. However, the secondary metabolite production profile of Actinosynnema pretiosum ATCC 31565, the most famous producer of ansamitocin, has never been fully explored. Our antiSMASH analysis of the genomic DNA of Actinosynnema pretiosum ATCC 31565 revealed a NRPS-PKS gene cluster for polyene macrolactam. The gene cluster is very similar to gene clusters for mirilactam and salinilactam, two 26-membered polyene macrolactams from Actinosynnema mirum and Salinispora tropica, respectively. Guided by this bioinformatics prediction, we characterized a novel 26-membered polyene macrolactam from Actinosynnema pretiosum ATCC 31565 and designated it pretilactam. The structure of pretilactam was elucidated by a comprehensive analysis of HRMS, 1D and 2D-NMR, with absolute configuration of chiral carbons predicted bioinformatically. Pretilactam features a dihydroxy tetrahydropyran moiety, and has a hexaene unit and a diene unit as its polyene system. A preliminary antibacterial assay indicated that pretilactam is inactive against Bacillus subtilis and Candida albicans.

Isarubrolones Containing a Pyridooxazinium Unit from Streptomyces as Autophagy Activators.

Isarubrolones are bioactive polycyclic tropoloalkaloids from Streptomyces. Three new isarubrolones (2-4), together with the known isarubrolone C (1) and isatropolones A (5) and C (6, 3( R)-hydroxyisatropolone A), were identified from Streptomyces sp. CPCC 204095. The structures of these compounds were determined using a combination of mass spectrometry, 1D and 2D NMR spectroscopy, and ECD. Compounds 3 and 4 feature a pyridooxazinium unit, which is rarely seen in natural products. Compound 6 could conjugate with amino acids or amines to expand the structural diversity of isarubrolones with a pentacyclic or hexacyclic core. Importantly, 1 and 3-6 were found to induce complete autophagy.

Geninthiocins C and D from Streptomyces as 35-membered macrocyclic thiopeptides with modified tail moiety.

Geninthiocin is a thiopeptide with 35-membered macrocyclic core moiety. It has potent anti-Gram-positive (G+) bacteria activity. Herein, we reported two new congeners (2-3) of geninthiocin (geninthiocin A, 1) from Streptomyces sp. CPCC 200267, and designated them as geninthiocins C and D, whose structures were determined by NMR. Geninthiocins A, C and D had the same 35-membered macrocyclic core moiety, but possessed a -Dha-Dha-NH2, -Dha-Ala-NH2, and -NH2 tail, respectively. Besides, the Ala residue in geninthiocin C was determined as L- configuration by C3 Marfey's method. In vitro assays indicated that geninthiocins C-D showed no antibacterial activity, in contrast to the potent anti-G+ bacteria activity displayed by geninthiocin A. Therefore, the -Dha-Dha-NH2 tail of geninthiocin A played an important role in its potent activity against G+ bacteria.

Quinohemanine, a quinoxalinone-bohemamine hybrid compound from Streptomyces sp. CPCC 200497.

A quinoxalinone-bohemamine hybrid compound quinohemanine (1), together with 1-methyl-2(H)-quinoxalin-2-one (2), was isolated from Streptomyces sp. CPCC 200497, a producer of quinomycins and bohemamines. Compounds 1 and 2 were purified using standard chromatographic methods, and their structures were defined through interpretation of HRMS, 1D, and 2D NMR data. Both 1 and 2 displayed moderate cytotoxicity against cancer cell line HepG2.

Biosynthesis of antibiotic chuangxinmycin from Actinoplanes tsinanensis.

Chuangxinmycin is an antibiotic isolated from Actinoplanes tsinanensis CPCC 200056 in the 1970s with a novel indole-dihydrothiopyran heterocyclic skeleton. Chuangxinmycin showed in vitro antibacterial activity and in vivo efficacy in mouse infection models as well as preliminary clinical trials. But the biosynthetic pathway of chuangxinmycin has been obscure since its discovery. Herein, we report the identification of a stretch of DNA from the genome of A. tsinanensis CPCC 200056 that encodes genes for biosynthesis of chuangxinmycin by bioinformatics analysis. The designated cxn cluster was then confirmed to be responsible for chuangxinmycin biosynthesis by direct cloning and heterologous expressing in Streptomyces coelicolor M1146. The cytochrome P450 CxnD was verified to be involved in the dihydrothiopyran ring closure reaction by the identification of seco-chuangxinmycin in S. coelicolor M1146 harboring the cxn gene cluster with an inactivated cxnD. Based on these results, a plausible biosynthetic pathway for chuangxinmycin biosynthesis was proposed, by hijacking the primary sulfur transfer system for sulfur incorporation. The identification of the biosynthetic gene cluster of chuangxinmycin paves the way for elucidating the detail biochemical machinery for chuangxinmycin biosynthesis, and provides the basis for the generation of novel chuangxinmycin derivatives by means of combinatorial biosynthesis and synthetic biology.

Cytotoxic Dibohemamines D-F from a Streptomyces Species.

Three dimeric analogues of bohemamines, dibohemamines D-F (1-3), together with dibohemamine A (4), were isolated from Streptomyces sp. CPCC 200497. Their structures were solved using a combination of mass spectrometry, 1D and 2D NMR spectroscopy, and CD. Dibohemamines D and E were new dimeric analogues of bohemamines, and dibohemamine F was a known compound obtained previously by semisynthesis. Dibohemamine F displayed potent cytotoxicity against cancer cell lines A549 and HepG2 with IC50 values of 1.1 and 0.3 µM, respectively. Dibohemamines D and E showed moderate cytotoxicity against cancer cell lines A549 and HepG2.