Streptomyces griseus KJ623766: A Natural Producer of Two Anthracycline Cytotoxic Metabolites ß- and γ-Rhodomycinone.

BACKGROUND: This study aimed to produce, purify, structurally elucidate, and explore the biological activities of metabolites produced by Streptomyces (S.) griseus isolate KJ623766, a recovered soil bacterium previously screened in our lab that showed promising cytotoxic activities against various cancer cell lines. METHODS: Production of cytotoxic metabolites from S. griseus isolate KJ623766 was carried out in a 14L laboratory fermenter under specified optimum conditions. Using a 3-(4,5-dimethylthazol-2-yl)-2,5-diphenyl tetrazolium-bromide assay, the cytotoxic activity of the ethyl acetate extract against Caco2 and Hela cancer cell lines was determined. Bioassay-guided fractionation of the ethyl acetate extract using different chromatographic techniques was used for cytotoxic metabolite purification. Chemical structures of the purified metabolites were identified using mass, 1D, and 2D NMR spectroscopic analysis. RESULTS: Bioassay-guided fractionation of the ethyl acetate extract led to the purification of two cytotoxic metabolites, R1 and R2, of reproducible amounts of 5 and 1.5 mg/L, respectively. The structures of R1 and R2 metabolites were identified as ß- and γ-rhodomycinone with CD50 of 6.3, 9.45, 64.8 and 9.11, 9.35, 67.3 µg/mL against Caco2, Hela and Vero cell lines, respectively. Values were comparable to those of the positive control doxorubicin. CONCLUSIONS: This is the first report about the production of ß- and γ-rhodomycinone, two important scaffolds for synthesis of anticancer drugs, from S. griseus.

Zealpeptaibolin, an 11-mer cytotoxic peptaibol group with 3 Aib-Pro motifs isolated from Trichoderma sp. RK10-F026.

Six new 11-mer peptaibols designed as zealpeptaibolins, A - F were isolated from the soil fungus, Trichoderma sp. RK10-F026, based on the principal component analysis of the MS data from five different culture compositions. Previously, 20-mer peptaibols from the same fungal strain were identified; 11-mer peptaibols in contrast were discovered from a different culture condition, signifying peptaibol production was culture condition-dependent. These peptaibols contained three Aib-Pro motifs in the sequence. The structures were established by NMR and HR-MS experiments including detailed MS/MS fragmentations. The absolute configurations were determined by Marfey's analysis. Zealpeptaibolin F exhibited the strongest cytotoxicity toward K562 leukemia cells with an IC50 value of 0.91 µM.

Heterologous reconstitution of the biosynthesis pathway for 4-demethyl-premithramycinone, the aglycon of antitumor polyketide mithramycin.

BACKGROUND: Mithramycin is an anti-tumor compound of the aureolic acid family produced by Streptomyces argillaceus. Its biosynthesis gene cluster has been cloned and characterized, and several new analogs with improved pharmacological properties have been generated through combinatorial biosynthesis. To further study these compounds as potential new anticancer drugs requires their production yields to be improved significantly. The biosynthesis of mithramycin proceeds through the formation of the key intermediate 4-demethyl-premithramycinone. Extensive studies have characterized the biosynthesis pathway from this intermediate to mithramycin. However, the biosynthesis pathway for 4-demethyl-premithramycinone remains unclear. RESULTS: Expression of cosmid cosAR7, containing a set of mithramycin biosynthesis genes, in Streptomyces albus resulted in the production of 4-demethyl-premithramycinone, delimiting genes required for its biosynthesis. Inactivation of mtmL, encoding an ATP-dependent acyl-CoA ligase, led to the accumulation of the tricyclic intermediate 2-hydroxy-nogalonic acid, proving its essential role in the formation of the fourth ring of 4-demethyl-premithramycinone. Expression of different sets of mithramycin biosynthesis genes as cassettes in S. albus and analysis of the resulting metabolites, allowed the reconstitution of the biosynthesis pathway for 4-demethyl-premithramycinone, assigning gene functions and establishing the order of biosynthetic steps. CONCLUSIONS: We established the biosynthesis pathway for 4-demethyl-premithramycinone, and identified the minimal set of genes required for its assembly. We propose that the biosynthesis starts with the formation of a linear decaketide by the minimal polyketide synthase MtmPKS. Then, the cyclase/aromatase MtmQ catalyzes the cyclization of the first ring (C7-C12), followed by formation of the second and third rings (C5-C14; C3-C16) catalyzed by the cyclase MtmY. Formation of the fourth ring (C1-C18) requires MtmL and MtmX. Finally, further oxygenation and reduction is catalyzed by MtmOII and MtmTI/MtmTII respectively, to generate the final stable tetracyclic intermediate 4-demethyl-premithramycinone. Understanding the biosynthesis of this compound affords enhanced possibilities to generate new mithramycin analogs and improve their production titers for bioactivity investigation.

Sesquiterpenoids from Cultures of the Basidiomycetes Irpex lacteus.

Eight previously undescribed sesquiterpenoids, tremutins A-H (1-8), together with three known ones (9-11), were isolated from cultures of the basidiomycetes Irpex lacteus. Structures of the new compounds together with absolute configurations were elucidated on the basis of extensive spectroscopic methods, as well as single-crystal X-ray diffractions and equivalent circulating density calculations. Compounds 1 and 2 possess an unusual 6/7-fused ring system that might be derived from a tremulane framework. Compounds 3-7 and 9-11 are tremulane sesquiterpenoids of which 4 and 5 are the first tremulane examples with a 1,2-epoxy moiety to be reported. Compounds 6, 7, 10, and 11 possess weak activities to several human cancer cell lines. Compound 8 shows a weak inhibitory effect on NO production with a half maximal inhibitory concentration (IC50) value of 22.7 µM. Compound 1 inhibits the lipopolysaccharide (LPS)-induced proliferation of B lymphocyte cells with an IC50 value of 22.4 µM, while 2 inhibits concanavalin A (Con A)-induced T cell proliferation and LPS-induced B lymphocyte cell proliferation with IC50 values of 16.7 and 13.6 µM, respectively.

Biosynthesis of depsipeptides with a 3-hydroxybenzoate moiety and selective anticancer activities involves a chorismatase.

Neoantimycins are anticancer compounds of 15-membered ring antimycin-type depsipeptides. They are biosynthesized by a hybrid multimodular protein complex of nonribosomal peptide synthetase (NRPS) and polyketide synthase (PKS), typically from the starting precursor 3-formamidosalicylate. Examining fermentation extracts of Streptomyces conglobatus, here we discovered four new neoantimycin analogs, unantimycins B-E, in which 3-formamidosalicylates are replaced by an unusual 3-hydroxybenzoate (3-HBA) moiety. Unantimycins B-E exhibited levels of anticancer activities similar to those of the chemotherapeutic drug cisplatin in human lung cancer, colorectal cancer, and melanoma cells. Notably, they mostly displayed no significant toxicity toward noncancerous cells, unlike the serious toxicities generally reported for antimycin-type natural products. Using site-directed mutagenesis and heterologous expression, we found that unantimycin productions are correlated with the activity of a chorismatase homolog, the nat-hyg5 gene, from a type I PKS gene cluster. Biochemical analysis confirmed that the catalytic activity of Nat-hyg5 generates 3-HBA from chorismate. Finally, we achieved selective production of unantimycins B and C by engineering a chassis host. On the basis of these findings, we propose that unantimycin biosynthesis is directed by the neoantimycin-producing NRPS-PKS complex and initiated with the starter unit of 3-HBA. The elucidation of the biosynthetic unantimycin pathway reported here paves the way to improve the yield of these compounds for evaluation in oncotherapeutic applications.

Directed Biosynthesis of Iso-aclacinomycins with Improved Anticancer Activity.

A four-enzyme catalyzed hydroxy regioisomerization of anthracycline was integrated into the biosynthetic pathway of aclacinomycin A (ALM-A), to generate a series of iso-ALMs via directed combinatorial biosynthesis combined with precursor-directed mutasynthesis. Most of the newly acquired iso-ALMs exhibit obviously (1-5-fold) improved antitumor activity. Therefore, we not only developed iso-ALMs with potential as clinical drugs but also demonstrated the utility of this tailoring tool for modification of anthracycline antibiotics in drug discovery and development.

Engineering Fluorine into Verticillins (Epipolythiodioxopiperazine Alkaloids) via Precursor-Directed Biosynthesis.

Precursor-directed biosynthesis was used to generate a series of fluorinated verticillins. The biosynthesis of these epipolythiodioxopiperazine alkaloids was monitored in situ via the droplet liquid microjunction surface sampling probe (droplet probe), and a suite of NMR and mass spectrometry data were used for their characterization. All analogues demonstrated nanomolar IC50 values vs a panel of cancer cell lines. This approach yielded new compounds that would be difficult to generate via synthesis.

Negative regulation of bleomycins biosynthesis by ArsR/SmtB family repressor BlmR in Streptomyces verticillus.

Bleomycin, a broad-spectrum antibiotic, has been widely used for various tumor treatments. However, its poor fermentation yield is not satisfactory for industrial production. Here, the ArsR/SmtB family regulator BlmR was characterized as a repressor of bleomycin production. As an autoregulator, BlmR was found to bind to a 12-2-12 imperfect palindrome sequence in its own promoter, and deletion of blmR led to a 34% increase of bleomycin B2 production compared with the wild-type strain. Using reverse transcription and quantitative PCR (RT-qPCR), blmT, which encoded a putative transporter, was identified as the target gene regulated by BlmR. Therefore, high-production strain was constructed by blmT overexpression in a blmR deletion strain, and the bleomycin B2 titer reached to 80 mg/L, which was 1.9-fold higher than the wild-type strain. Moreover, electrophoretic mobility shift assay (EMSA) showed neither metal-binding motifs nor redox switches in BlmR. In order to elucidate the regulatory mechanism, a model of BlmR was constructed by homology modeling and protein-protein docking. The BlmR-DNA complex was generated by protein-DNA docking with the assistance of site-directed mutagenesis and molecular dynamic (MD) simulation, which directly revealed several key amino acid residues needed for the maintenance and stabilization of the interface between BlmR and target DNA. The interface information could provide the configuration reference and seek the potential effectors that could interact with BlmR, thereby extending the regulation role of ArsR/SmtB family members on the improvement of antibiotic production.

The secondary metabolite pactamycin with potential for pharmaceutical applications: biosynthesis and regulation.

The antitumor antibiotic pactamycin is a highly substituted aminocyclopentitol-derived secondary metabolite produced by the soil bacterium Streptomyces pactum. It has exhibited potent antibacterial, antitumor, antiviral, and antiprotozoal activities. Despite its outstanding biological activities, the complex chemical structure and broad-spectrum toxicity have hampered its development as a therapeutic, limiting its contribution to biomedical science to a role as a molecular probe for ribosomal function. However, a detailed understanding of its biosynthesis and how the biosynthesis is regulated has made it possible to tactically design and produce new pactamycin analogues, some of which have shown improved pharmacological properties. This mini-review describes the biosynthesis, regulation, engineered production, and biological activities of pactamycin and its congeners. It also highlights the suitability of biosynthetic methods as a feasible approach to generate new analogues of complex natural products and underscores the importance of utilizing biosynthetic enzymes as tools for chemoenzymatic production of structurally diverse bioactive compounds.

The antitumor antibiotic rebeccamycin-challenges and advanced approaches in production processes.

Rebeccamycin is an antibiotic and antitumor substance isolated from the filamentous bacterium Lentzea aerocolonigenes. After its discovery, investigations of rebeccamycin focused on elucidating its structure, biological activity, and biosynthetic pathway. For potential medical application, a sufficient drug supply has to be ensured, meaning that the production process of rebeccamycin plays a major role. In addition to the natural production of rebeccamycin in L. aerocolonigenes, where the complex cell morphology is an important factor for a sufficient production, rebeccamycin can also be heterologously produced or chemically synthesized. Each of these production processes has its own challenges, and first approaches to production often lead to low final product concentrations, which is why process optimizations are performed. This review provides an overview of the production of rebeccamycin and the different approaches used for rebeccamycin formation including process optimizations.

Enzymology of Anthraquinone-γ-Pyrone Ring Formation in Complex Aromatic Polyketide Biosynthesis.

Aromatic-fused γ-pyrones are structural features of many bioactive natural products and valid scaffolds for medicinal chemistry. However, the enzymology of their formation has not been completely established. Now it is demonstrated that TxnO9, a CalC-like protein belonging to a START family, functions as an unexpected anthraquinone-γ-pyrone synthase involved in the biosynthesis of antitumor antibiotic trioxacarcin A (TXN-A). Structural analysis by NMR identified a likely substrate/product-binding mode and putative key active sites of TxnO9, which allowed an enzymatic mechanism to be proposed. Moreover, a subset of uncharacterized homologous proteins bearing an unexamined Lys-Thr dyad exhibit the same function. Therefore, the functional assignment and mechanistic investigation of this γ-pyrone synthase elucidated an undescribed step in TXN-A biosynthesis, and the discovery of this new branch of polyketide heterocyclases expands the functions of the START superfamily.

P450-Catalyzed Tailoring Steps in Leinamycin Biosynthesis Featuring Regio- and Stereoselective Hydroxylations and Substrate Promiscuities.

Leinamycin (LNM) is a potent antitumor antibiotic produced by Streptomyces atroolivaceus S-140. Both in vivo and in vitro characterization of the LNM biosynthetic machinery have established the formation of the 18-membered macrolactam backbone and the C-3 alkyl branch; the nascent product, LNM E1, of the hybrid nonribosomal peptide synthetase (NRPS)-acyltransferase (AT)-less type I polyketide synthase (PKS); and the generation of the thiol moiety at C-3 of LNM E1. However, the tailoring steps converting LNM E1 to LNM are still unknown. Based on gene inactivation and chemical investigation of three mutant strains, we investigated the tailoring steps catalyzed by two cytochromes P450 (P450s), LnmA and LnmZ, in LNM biosynthesis. Our studies revealed that (i) LnmA and LnmZ regio- and stereoselectively hydroxylate the C-8 and C-4' positions, respectively, on the scaffold of LNM; (ii) both LnmA and LnmZ exhibit substrate promiscuity, resulting in multiple LNM analogs from several shunt pathways; and (iii) the C-8 and C-4' hydroxyl groups play important roles in the cytotoxicity of LNM analogs against different cancer cell lines, shedding light on the structure-activity relationships of the LNM scaffold and the LNM-type natural products in general. These studies set the stage for future biosynthetic pathway engineering and combinatorial biosynthesis of the LNM family of natural products for structure diversity and drug discovery.

A radical S-adenosyl-L-methionine enzyme and a methyltransferase catalyze cyclopropane formation in natural product biosynthesis.

Cyclopropanation of unactivated olefinic bonds via addition of a reactive one-carbon species is well developed in synthetic chemistry, whereas natural cyclopropane biosynthesis employing this strategy is very limited. Here, we identify a two-component cyclopropanase system, composed of a HemN-like radical S-adenosyl-L-methionine (SAM) enzyme C10P and a methyltransferase C10Q, catalyzes chemically challenging cyclopropanation in the antitumor antibiotic CC-1065 biosynthesis. C10P uses its [4Fe-4S] cluster for reductive cleavage of the first SAM to yield a highly reactive 5'-deoxyadenosyl radical, which abstracts a hydrogen from the second SAM to produce a SAM methylene radical that adds to an sp2-hybridized carbon of substrate to form a SAM-substrate adduct. C10Q converts this adduct to CC-1065 via an intramolecular SN2 cyclization mechanism with elimination of S-adenosylhomocysteine. This cyclopropanation strategy not only expands the enzymatic reactions catalyzed by the radical SAM enzymes and methyltransferases, but also sheds light on previously unnoticed aspects of the versatile SAM-based biochemistry.

Implication of orphan histidine kinase (OhkAsp) in biosynthesis of doxorubicin and daunorubicin in Streptomyces peucetius ATCC 27952.

The orphan histidine kinase (HK) from Streptomyces peucetius ATCC 27952 (ohkAsp) was found to be implicated in the regulation of doxorubicin (DOX)/daunorubicin (DNR) biosynthesis, self-defense and developmental attributes. OhkAsp is a homolog of OhkA from Streptomyces coelicolor and Streptomyces avermitilis (with 73 and 75% identity). As in its homologs, S. peucetius mutant with deletion of ohkAsp was found to enhance metabolite biosynthesis and impaired the morphological differentiation. But, unlike its homologs from Streptomyces coelicolor and Streptomyces avermitilis, differential enhancement in level of secondary metabolite production was found in overexpression mutants apart from deletion mutant. The deflection in characteristics of OhkA in its homologue from S. peucetius ATCC 27952, and its imminent implications was monitered by making various mutants with differential expression level of ohkAsp. The variations were observed in the morphology of mutants, transcriptional level of effectors and regulators of DOX/DNR biosynthesis pathway, DOX/DNR precursor pool and biomass accumulation. Based on comparisons of domain arrangements among its homologs, Low Complexity Region (LCR) present on the OhkAsp was the only domain that stood out. Further, the LCR on OhkAsp was found to be overlapping with a putative receiver domain responsible for interaction with response regulator. The imminent implications of differential expression level of ohkAsp on: regulation and biosynthesis of DOX/DNR, morphological differentiation, DOX/DNR precursor pool and biomass accumulation were explored in this study.

Enhanced doxorubicin production by Streptomyces peucetius using a combination of classical strain mutation and medium optimization.

Doxorubicin (DXR), which is produced by Streptomyces peucetius, is an important anthracycline-type antibiotic used for the treatment of various cancers. However, due to the low DXR productivity of wild-type S. peucetius, it is difficult to produce DXR by one-step fermentation. In this study, a DXR-resistance screening method was developed to screen for DXR high-producing mutants. Then, S. peucetius SIPI-11 was treated several times with UV and ARTP (atmospheric and room temperature plasma) to induce mutations. Treated strains were screened by spreading on a DXR-containing plate, isolating a mutant (S. peucetius 33-24) with enhanced DXR yield (570 mg/L vs. 119 mg/L for the original strain). The components of the fermentation medium, including the carbon and nitrogen sources, were optimized to further enhance DXR yield (to 850 mg/L). The pH of the fermentation medium and culture temperature were also optimized for effective DXR production. Finally, DXR production by S. peucetius 33-24 was investigated in flask culture and a fermenter. The yield of DXR was as high as 1100 mg/L in a 5-L fermenter, which is the highest DXR productivity reported thus far, suggesting that S. peucetius 33-24 has the potential to produce DXR by direct fermentation.

Hamuramicins A and B, 22-membered macrolides, produced by an endophytic actinomycete Allostreptomyces sp. K12-0794.

Two new compounds, designated as hamuramicins A (1) and B (2), were isolated from the cultured broth of an endophytic actinomycete Allostreptomyces sp. K12-0794 by silica gel column chromatography and HPLC. The structures of 1 and 2 were elucidated as 22-membered macrolide containing triene and trienone with an alkyl side chain by spectroscopic analyses including NMR experiments. Both compounds showed growth inhibition activity against Kocuria rhizophia and Xanthomonas oryzae pv. oryzae as well as human cell line toxicity.

Prodigiosin R2, a new prodigiosin from the roseophilin producer Streptomyces griseoviridis 2464-S5.

Roseophilin (2) is a unique prodigiosin-related compound produced by Streptomyces griseoviridis 2464-S5, and is characterized by a central furan ring and a bicyclic alkyl chain. During a search for biosynthetic intermediates of 2, a new metabolite designated prodigiosin R2 (1) was isolated from the culture of the roseophilin producer. The molecular formula of 1 was established as C27H35N3O by high-resolution FAB-MS. The structure of 1 was determined by NMR spectroscopic analyses as a prodigiosin derivative with the same bicyclic alkyl chain as 2. Prodigiosin R2 (1) showed potent cytotoxicity against HeLa human cervical carcinoma cells and HT1080 human fibrosarcoma cells with IC50s of 0.41 and 0.82 µM, respectively.

Strain improvement by combined UV mutagenesis and ribosome engineering and subsequent fermentation optimization for enhanced 6'-deoxy-bleomycin Z production.

The bleomycins (BLMs) are important clinical drugs extensively used in combination chemotherapy for the treatment of various cancers. Dose-dependent lung toxicity and the development of drug resistance have restricted their wide applications. 6'-Deoxy-BLM Z, a recently engineered BLM analogue with improved antitumor activity, has the potential to be developed into the next-generation BLM anticancer drug. However, its low titer in the recombinant strain Streptomyces flavoviridis SB9026 has hampered current efforts, which require sufficient compound, to pursue preclinical studies and subsequent clinical development. Here, we report the strain improvement by combined UV mutagenesis and ribosome engineering, as well as the fermentation optimization, for enhanced 6'-deoxy-BLM production. A high producer, named S. flavoviridis G-4F12, was successfully isolated, producing 6'-deoxy-BLM at above 70 mg/L under the optimized fermentation conditions, representing a sevenfold increase in comparison with that of the original producer. These findings demonstrated the effectiveness of combined empirical breeding methods in strain improvement and set the stage for sustainable production of 6'-deoxy-BLM via pilot-scale microbial fermentation.

Catalysis of Extracellular Deamination by a FAD-Linked Oxidoreductase after Prodrug Maturation in the Biosynthesis of Saframycin†A.

The biosynthesis of antibiotics in bacteria is usually believed to be an intracellular process, at the end of which the matured compounds are exported outside the cells. The biosynthesis of saframycin A (SFM-A), an antitumor antibiotic, requires a cryptic fatty acyl chain to guide the construction of a pentacyclic tetrahydroisoquinoline scaffold; however, the follow-up deacylation and deamination steps remain unknown. Herein we demonstrate that SfmE, a membrane-bound peptidase, hydrolyzes the fatty acyl chain to release the amino group; and SfmCy2, a secreted oxidoreductase covalently associated with FAD, subsequently performs an oxidative deamination extracellularly. These results not only fill in the missing steps of SFM-A biosynthesis, but also reveal that a FAD-binding oxidoreductase catalyzes an unexpected deamination reaction through an unconventional extracellular pathway in Streptmyces bacteria.