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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Binding of a biosynthetic intermediate to AtrA modulates the production of lidamycin by Streptomyces globisporus.

The control of secondary production in streptomycetes involves the funneling of environmental and physiological signals to the cluster-situated (transcriptional) regulators (CSRs) of the biosynthetic genes. For some systems, the binding of biosynthetic products to the CSR has been shown to provide negative feedback. Here we show for the production of lidamycin (C-1027), a clinically relevant antitumor agent, by Streptomyces globisporus that negative feedback can extend to a point higher in the regulatory cascade. We show that the DNA-binding activity of the S. globisporus orthologue of AtrA, which was initially described as a transcriptional activator of actinorhodin biosynthesis in S. coelicolor, is inhibited by the binding of heptaene, a biosynthetic intermediate of lidamycin. Additional experiments described here show that S. globisporus AtrA binds in vivo as well as in vitro to the promoter region of the gene encoding SgcR1, one of the CSRs of lidamycin production. The feedback to the pleiotropic regulator AtrA is likely to provide a mechanism for coordinating the production of lidamycin with that of other secondary metabolites. The activity of AtrA is also regulated by actinorhodin. As AtrA is evolutionarily conserved, negative feedback of the type described here may be widespread within the streptomycetes.

[Identification of 3-demethylchuangxinmycin from Actinoplanes tsinanensis CPCC 200056].

Chuangxinmycin (CM) from Actinoplanes tsinanensis was an antibiotic discovered by Chinese scientists about 40 years ago. It contains a new heterocyclic system of indole fused with dihydrothiopyran, whose biosynthetic mechanism remains unclear. CM is used as an oral medicine in the treatment of bacterial infections in China. The simple structure makes CM as an attractive candidate of structure modification for improvement of antibacterial activity. Recently, we analyzed the secondary metabolites of Actinoplanes tsinanensis CPCC 200056, a CM producing strain, as a natural CM analogue. We discovered the first natural CM analogue 3-demethylchuangxinmycin (DCM) as a new natural product. Compared to CM, DCM exhibited a much weaker activity in the inhibition of the bacterial strains tested. The finding provides valuable information for the structure-activity relationship in the biosynthesis of CM.

NRPS substrate promiscuity leads to more potent antitubercular sansanmycin analogues.

Sansanmycins, members of the uridyl peptide antibiotics, are assembled by nonribosomal peptide synthetases (NRPSs), the substrate promiscuity of which results in the diversity of products. Further exploration of the NRPSs' substrate promiscuity by reinvestigating sansanmycin producer strain led to the isolation and structural elucidation of eight new uridyl peptides, sansanmycins H-O (1-8). Among them, sansanmycin L, containing a 6-OH-bicyclic residue and Phe3 first found at the position AA3, exhibited activity against M. tuberculosis H37Rv with an MIC value of 2 µg/mL, 8-fold more potent than that of the major compound, sansanmycin A (MIC = 16 µg/mL).

6-Deoxy-13-hydroxy-8,11-dione-dihydrogranaticin B, an intermediate in granaticin biosynthesis, from Streptomyces sp. CPCC 200532.

A new granaticin analogue and its hydrolysis product were isolated from Streptomyces sp. CPCC 200532. Their structures were determined to be 6-deoxy-13-hydroxy-8,11-dione-dihydrogranaticins B (1) and A (2), respectively, by detailed analysis of spectroscopic data. Compound 1 was regarded as an intermediate in granaticin biosynthesis, as it was bioconvertable to granaticin B. Compared to granaticin B, 1 showed similar cytotoxicity against cancer cell line HCT116, but decreased cytotoxicity against cancer cell lines A549, HeLa, and HepG2. Compound 2 displayed lower cytotoxicity than 1 against all four cancer cell lines tested.

19-[(1'S,4'R)-4'-Hydroxy-1'-methoxy-2'-oxopentyl]geldanamycin, a natural geldanamycin analogue from Streptomyces hygroscopicus 17997.

A novel natural geldanamycin analogue was discovered in Streptomyces hygroscopicus 17997. Its 4,5-dihydro form was also identified in the gdmP gene disruption mutant of Streptomyces hygroscopicus 17997. The structures of the two compounds were determined to be 19-[(1'S,4'R)-4'-hydroxy-1'-methoxy-2'-oxopentyl]geldanamycin (1) and 19-[(1'S,4'R)-4'-hydroxy-1'-methoxy-2'-oxopentyl]-4,5-dihydrogeldanamycin (2), respectively, by extensive spectroscopic data analysis, including 2D NMR, modified Mosher's method, and electronic circular dichroism. Compared to geldanamycin, 1 and 2 showed increased water solubility and decreased cytotoxicity against HepG2 cells.

[Synthetic biology toward microbial secondary metabolites and pharmaceuticals].

Microbial secondary metabolites are one of the major sources of anti-bacterial, anti-fungal, antitumor, anti-virus and immunosuppressive agents for clinical use. Present challenges in microbial pharmaceutical development are the discovery of novel secondary metabolites with significant biological activities, improving the fermentation titers of industrial microbial strains, and production of natural product drugs by re-establishing their biosynthetic pathways in suitable microbial hosts. Synthetic biology, which is developed from systematic biology and metabolic engineering, provides a significant driving force for microbial pharmaceutical development. The review describes the major applications of synthetic biology in novel microbial secondary metabolite discovery, improved production of known secondary metabolites and the production of some natural drugs in genetically modified or reconstructed model microorganisms.

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.

Identification of 4,5-dihydro-4-hydroxygeldanamycins as shunt products of geldanamycin biosynthesis.

Two new geldanamycin (GDM) analogues, (4S)-4,5-dihydro-4-hydroxygeldanamycin (1) and (4R)-4,5-dihydro-4-hydroxygeldanamycin (2), were identified from Streptomyces hygroscopicus 17997. Compounds 1 and 2 were not normal intermediates of GDM biosynthesis but shunt products of C-4,5 oxidation catalyzed by GdmP, a cytochrome P450 oxidase acting as a desaturase in GDM biosynthesis. Preliminary assays implied that, compared with GDM, 1 and 2 exhibited decreased cytotoxicity.

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.