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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Interrogating the Tailoring Steps of Pactamycin Biosynthesis and Accessing New Pactamycin Analogues.

Pactamycin is a bacteria-derived aminocyclitol antibiotic with a wide-range of biological activity. Its chemical structure and potent biological activities have made it an interesting lead compound for drug discovery and development. Despite its unusual chemical structure, many aspects of its formation in nature remain elusive. Using a combination of genetic inactivation and metabolic analysis, we investigated the tailoring processes of pactamycin biosynthesis in Streptomyces pactum. The results provide insights into the sequence of events during the tailoring steps of pactamycin biosynthesis and explain the unusual production of various pactamycin analogues by S. pactum mutants. We also identified two new pactamycin analogues that have better selectivity indexes than pactamycin against malarial parasites.

Characterization of representative rpoB gene mutations leading to a significant change in toyocamycin production of Streptomyces diastatochromogenes 1628.

Modification of enzymes involved in transcription- or translation-processes is an interesting way to increase secondary metabolite production in Streptomycetes. However, application of such methods has not been widely described for strains which produce nucleoside antibiotics. The nucleoside antibiotic toyocamycin (TM) is produced by Streptomyces diastatochromogenes 1628. For improving TM production in S. diastatochromogenes 1628, the strain was spread on rifamycin-resistant (Rif(r)) medium. Several spontaneous mutants were obtained with mutations in the rpoB gene which encodes a RNA polymerase ß-subunit. The mutants which showed increased TM production were detected at a frequency of 7.5 % among the total Rif(r) mutants. Mutant 1628-T15 harboring amino acid substitution His437Arg was the best TM producer with a 4.5-fold increase in comparison to that of the wild-type strain. The worst producer was mutant 1628-T62 which also showed a poor sporulation behavior. RT-PCR was performed to study the transcription levels of the TM biosynthetic gene toyG in the parental strain as well as in mutants 1628-T15 and 1628-T62. The transcriptional level of toyG was higher in mutant 1628-T15 than that in parental strain 1628, while much lower in mutant 1628-T62. In mutant strain 1628-T62 the expression of adpA sd gene, which is required for morphological differentiation, was also much lower. Our studies also indicate that the introduction of mutations into rpoB is an effective strategy to improve the production of TM which is an important nucleoside antibiotic.

[THE ROLE OF (p)ppGpp MOLECULES IN FORMATION OF "STRICT RESPONSE" IN BACTERIA AND BIOSYNTHESIS OF ANTIBIOTICS AND MORPHOLOGICAL DIFFERENTIATION IN ACTINOMYCETES].

Strict response is a pleiotropic physiological response of cells caused by lack of aminoacetylated tRNAs. Experimentally, this response occurs due to the lack of amino acids in the environment and the limitation of tRNA aminoacylation even in the presence of the corresponding amino acids in the cell. Many features of this response indicate its dependence on the accumulation of ppGpp molecules. There is a correlation between the growth rate of actinomycetes and biosynthesis of their secondary metabolites. Introduction of additional relA gene copies of ppGpp synthetase can affect the production of antibiotics in streptomycetes. The article presents the authors' own experimental data, dedicated to the influence of heterologous relA gene expression in Streptomyces nogalater cells.

[THE ROLE OF STREPTOMYCES NOGALATER Lv65 snoaM, snoaL and snoaE GENES IN NOGALAMYCIN BIOSYNTHESIS].

The results of phylogenetic analysis indicate high similarity of SnoaM, SnoaL SnoaE to the cyclases involved in the biosynthesis of various antibiotics. Genes snoaM, snoaL and snoaE disruption in S. nogalater chromosome was carried on and S. nogalater MI, LI and EI strains were generated. The gene replacement events in M1, L1 and E1 were verified by Southern hybridization. Recombinant strains were characterised by lack of nogalamycin biosynthesis. Originally, M1, L1 and E1 were complemented with plasmids expressing putative cyclase genes from S. nogalater leading to restoration of nogalamycine production. The complementation results clearly indicate that obtained strains are cyclase deficient mutants. Furthermore, complementation of M1, L1 and E1 with a cyclase genes from wild-type strain is consistent with the suggested function of these enzymes.

Comparative characterization of the lactimidomycin and iso-migrastatin biosynthetic machineries revealing unusual features for acyltransferase-less type I polyketide synthases and providing an opportunity to engineer new analogues.

Lactimidomycin (LTM, 1) and iso-migrastatin (iso-MGS, 2) belong to the glutarimide-containing polyketide family of natural products. We previously cloned and characterized the mgs biosynthetic gene cluster from Streptomyces platensis NRRL 18993. The iso-MGS biosynthetic machinery featured an acyltransferase (AT)-less type I polyketide synthase (PKS) and three tailoring enzymes (MgsIJK). We now report cloning and characterization of the ltm biosynthetic gene cluster from Streptomyces amphibiosporus ATCC 53964, which consists of nine genes that encode an AT-less type I PKS (LtmBCDEFGHL) and one tailoring enzyme (LtmK). Inactivation of ltmE or ltmH afforded the mutant strain SB15001 or SB15002, respectively, that abolished the production of 1, as well as the three cometabolites 8,9-dihydro-LTM (14), 8,9-dihydro-8S-hydroxy-LTM (15), and 8,9-dihydro-9R-hydroxy-LTM (13). Inactivation of ltmK yielded the mutant strain SB15003 that abolished the production of 1, 13, and 15 but led to the accumulation of 14. Complementation of the ΔltmK mutation in SB15003 by expressing ltmK in trans restored the production of 1, as well as that of 13 and 15. These results support the model for 1 biosynthesis, featuring an AT-less type I PKS that synthesizes 14 as the nascent polyketide intermediate and a cytochrome P450 desaturase that converts 14 to 1, with 13 and 15 as minor cometabolites. Comparative analysis of the LTM and iso-MGS AT-less type I PKSs revealed several unusual features that deviate from those of the collinear type I PKS model. Exploitation of the tailoring enzymes for 1 and 2 biosynthesis afforded two analogues, 8,9-dihydro-8R-hydroxy-LTM (16) and 8,9-dihydro-8R-methoxy-LTM (17), that provided new insights into the structure-activity relationship of 1 and 2. While 12-membered macrolides, featuring a combination of a hydroxyl group at C-17 and a double bond at C-8 and C-9 as found in 1, exhibit the most potent activity, analogues with a single hydroxyl or methoxy group at C-8 or C-9 retain most of the activity whereas analogues with double substitutions at C-8 and C-9 lose significant activity.

Biosynthetic modularity rules in the bisintercalator family of antitumor compounds.

Diverse actinomycetes produce a family of structurally and biosynthetically related non-ribosomal peptide compounds which belong to the chromodepsipeptide family. These compounds act as bisintercalators into the DNA helix. They give rise to antitumor, antiparasitic, antibacterial and antiviral bioactivities. These compounds show a high degree of conserved modularity (chromophores, number and type of amino acids). This modularity and their high sequence similarities at the genetic level imply a common biosynthetic origin for these pathways. Here, we describe insights about rules governing this modular biosynthesis, taking advantage of the fact that nowadays five of these gene clusters have been made public (thiocoraline, triostin, SW-163 and echinomycin/quinomycin). This modularity has potential application for designing and producing novel genetic engineered derivatives, as well as for developing new chemical synthesis strategies. These would facilitate their clinical development.