Candicidin Isomer Production Is Essential for Biocontrol of Cucumber Rhizoctonia Rot by Streptomyces albidoflavus W68.

Diseases caused by soilborne fungal pathogens result in significant crop yield losses and quality reduction. Streptomyces albidoflavus strain W68 is effective in controlling several soilborne fungal diseases. To identify antifungal substances critical for biocontrol activity of W68, the genome of W68 was sequenced and a linear chromosome of 6.80 Mb was assembled. A total of 21 secondary metabolite biosynthesis gene clusters (BGCs), accounting for 12.27% of the genome, were identified. Core gene deletion mutants for each of all 8 BGCs for nonribosomal peptide synthetases and polyketide synthases were created. Among them, only the mutant lacking ctg1-5755 (the gene was renamed as fscDW68) in BGC 19, which shares 100% sequence similarity with the BGC for candicidin synthesis, showed obvious reduction in antifungal activity. A pot experiment revealed that biocontrol effects of the ΔfscDW68 mutant in Rhizoctonia rot of cucumber were also significantly compromised relative to W68. Liquid chromatography-mass spectrometry (LC-MS) analysis revealed that W68 but not the ΔfscDW68 mutant can produce candicidin isomers, indicating that the production of candicidin isomers is key for antifungal activity and biocontrol activity of S. albidoflavus W68.IMPORTANCE This study reports that candicidin-like secondary metabolites produced by microbial cells in natural soil environments can effectively control soilborne fungal diseases, revealing a novel mechanism of microbial biocontrol agents. We demonstrated that the main antifungal activity and biocontrol activity of Streptomyces albidoflavus strain W68 are attributable to the production of candicidin isomers, suggesting that gene clusters for candicidin-like compound biosynthesis might be used as molecular markers to screen and breed microbial strains for biocontrol agent development.

The complete stereochemistry of the antibiotic candicidin A3 (syn. ascosin A3, levorin A3).

Herein, the stereostructure of the aromatic heptaene macrolide (AHM) antifungal antibiotic candicidin A3 (syn. ascosin A3, levorin A3) has been established upon the 2D NMR studies, consisting of DQF-COSY, TOCSY, ROESY, HSQC and HMBC experiments, as well as upon extensive molecular dynamics simulations. The geometry of the heptaenic chromophore was defined as: (22E, 24E, 26Z, 28Z, 30E, 32E, 34E). The previously unreported absolute configuration of the chiral centres of candicidin A3 was established as: (3R, 9R, 11S, 13S, 15R, 17S, 18R, 19S, 21R, 36S, 37R, 38S, 40S, 41S).

Effect of Chemically Transformed Macrocyclic Polyene Antibiotics on Tumor Cells.

Comparative analysis of the effects of chemically transformed polyene antibiotics pimaricin, nystatin, lucensomycin, amphotericin B, and levorin on biological objects in vivo and in vitro revealed the greatest biological activity of original amphotericin B and levorin with its derivatives. The study also examined the effects of alkyl derivatives of amphotericin B and levorin modified in certain parts of the lactone ring on the lipid and biological membranes. It is established that methylated levorin possesses larger biological activity than the original antibiotic. Examination of the effects of alkyl derivatives of levorin and amphotericin B on cell cultures C6 (rat glioma) and HeLa (human cervical carcinoma) in vitro revealed the antitumor action of methylated levorin and original amphotericin B.

Light-Induced Transformation of the Aromatic Heptaene Antifungal Antibiotic Candicidin D into Its All-Trans Isomer.

Illumination of the aromatic heptaene macrolide antifungal antibiotic candicicin D with UV light results in an isomerization of the molecule. The product formed after irradiation of the candicidin complex with UV light (λ = 365 nm), namely, iso-candicidin D, was isolated and subjected to 2D NMR studies, consisting of DQF-COSY, ROESY, TOCSY, HSQC, and HMBC experiments. The obtained spectral data unambiguously evidenced that iso-candicidin D was the all-trans isomer of the native antibiotic, and straightening of the heptaenic chromophore was the only light-induced structural change that occurred. Hence, iso-candicidin D was proclaimed to be a prototype of a novel class of polyene macrolide antifungal antibiotics: the all-trans aromatic heptaenes, containing a macrolide ring similar to that of amphotericin B.

Analytical studies on ascosin, candicidin and levorin multicomponent antifungal antibiotic complexes. The stereostructure of ascosin A2.

In the class of polyene macrolides, there is a subgroup of aromatic heptaenes, which exhibit the highest antifungal activity within this type of antibiotics. Yet, due to their complex nature, aromatic heptaenes were not extensively studied and their potential as drugs is currently underexploited. Moreover, there are many inconsistencies in the literature regarding the composition and the structures of the individual components of the aromatic heptaene complexes. Inspired by one of such cases, herein we conducted the analytical studies on ascosin, candicidin and levorin using HPLC-DAD-(ESI)Q-TOF techniques. The resulting chromatograms and the molecular masses of the individual components of these three complexes strongly indicated that the major components of ascosin, candicidin and levorin are structurally identical. In order to validate these results, the main component of previously structurally uncharacterized ascosin was derivatized, isolated and subjected to 2D NMR studies. The resulting structure of the ascosin's main component, herein named ascosin A2, was shown to be identical with the earlier reported structures of the main components of candicidin and levorin complexes: candicidin D and levorin A2. In the end, all the structural knowledge regarding these three antibiotic complexes was gathered, systematized and completed, and the new nomenclature was proposed.

The stereostructure of candicidin D.

The candicidin D stereostructure was established based on NMR studies including DQF-COSY, ROESY, HSQC and HMBC experiments. The relative configurations of the candicidin D stereogenic centers were assigned as the following: 9R*, 11S*, 13S*, 15R*, 17S*, 18R*, 19S*, 21R*, 36S*, 37R*, 38S*, 40S* and 41S*. The geometry of the heptaene chromophore was defined as 22E, 24E, 26Z, 28Z, 30E, 32E and 34E.

Isolating antifungals from fungus-growing ant symbionts using a genome-guided chemistry approach.

We describe methods used to isolate and identify antifungal compounds from actinomycete strains associated with the leaf-cutter ant Acromyrmex octospinosus. These ants use antibiotics produced by symbiotic actinomycete bacteria to protect themselves and their fungal cultivar against bacterial and fungal infections. The fungal cultivar serves as the sole food source for the ant colony, which can number up to tens of thousands of individuals. We describe how we isolate bacteria from leaf-cutter ants collected in Trinidad and analyze the antifungal compounds made by two of these strains (Pseudonocardia and Streptomyces spp.), using a combination of genome analysis, mutagenesis, and chemical isolation. These methods should be generalizable to a wide variety of insect-symbiont situations. Although more time consuming than traditional activity-guided fractionation methods, this approach provides a powerful technique for unlocking the complete biosynthetic potential of individual strains and for avoiding the problems of rediscovery of known compounds. We describe the discovery of a novel nystatin compound, named nystatin P1, and identification of the biosynthetic pathway for antimycins, compounds that were first described more than 60 years ago. We also report that disruption of two known antifungal pathways in a single Streptomyces strain has revealed a third, and likely novel, antifungal plus four more pathways with unknown products. This validates our approach, which clearly has the potential to identify numerous new compounds, even from well-characterized actinomycete strains.

Enzymology of the polyenes pimaricin and candicidin biosynthesis.

Pimaricin and candicidin are prototypical representatives of the "small" and the "aromatic" polyene macrolides, respectively. Pimaricin, produced by Streptomyces natalensis, is an important antifungal agent used in human therapy for the treatment of fungal keratitis, and in the food industry to prevent mould contamination. Five large polyketide synthase subunits are implicated in the formation of the pimaricin macrolactone ring, while P450 mono-oxygenases and a glycosyltransferase are responsible for ring "decoration." Two transcriptional regulators directly modulate transcription of certain genes in the cluster; an extracellular cholesterol oxidase also participates in such control. Two regulatory locus external to the pimaricin gene cluster, encoding the two-component PhoR-PhoP system for phosphate limitation response, and a gamma-butyrolactone receptor, contribute to the control of pimaricin production. A quorum-sensing inducer of pimaricin biosynthesis (PI-factor) has been identified recently. Candicidin (also named FR-008) contains an aromatic para-aminoacetophenone moiety derived from para-aminobenzoic acid (PABA), which acts as a starter unit in the biosynthesis. Two genes in the candicidin cluster, pabAB and pabC, are involved in the biosynthesis of PABA. Six polyketide synthase subunits encoded by fscA to fscF, containing 21 modules, are involved in the synthesis of the candicidin aglycone. At least three genes (fscO, fscP, and fscTE) encode aglycone modification enzymes. Three genes-fscM1, M2, and M3-are involved in mycosamine biosynthesis and its attachment to the aglycone. The candicidin cluster also includes two ABC transporter genes and four putative transcriptional regulators. Expression of the PABA synthase gene (pabAB) is drastically repressed by phosphate.

Candicidin biosynthesis gene cluster is widely distributed among Streptomyces spp. isolated from the sediments and the neuston layer of the Trondheim fjord, Norway.

A large number of Streptomyces bacteria with antifungal activity isolated from samples collected in the Trondheim fjord (Norway) were found to produce polyene compounds. Investigation of polyene-containing extracts revealed that most of the isolates produced the same compound, which had an atomic mass and UV spectrum corresponding to those of candicidin D. The morphological diversity of these isolates prompted us to speculate about the involvement of a mobile genetic element in dissemination of the candicidin biosynthesis gene cluster (can). Eight candicidin-producing isolates were analyzed by performing a 16S rRNA gene-based taxonomic analysis, pulsed-field gel electrophoresis, PCR, and Southern blot hybridization with can-specific probes. These analyses revealed that most of the isolates were related, although they were morphologically diverse, and that all of them contained can genes. The majority of the isolates studied contained large plasmids, and two can-specific probes hybridized to a 250-kb plasmid in one isolate. Incubation of the latter isolate at a high temperature resulted in loss of the can genes and candicidin production, while mating of the "cured" strain with a plasmid-containing donor restored candicidin production. The latter result suggested that the 250-kb plasmid contains the complete can gene cluster and could be responsible for conjugative transfer of this cluster to other streptomycetes.

Candicidin-producing Streptomyces support leaf-cutting ants to protect their fungus garden against the pathogenic fungus Escovopsis.

Leaf-cutting ants such as Acromyrmex octospinosus live in obligate symbiosis with fungi of the genus Leucoagaricus, which they grow with harvested leaf material. The symbiotic fungi, in turn, serve as a major food source for the ants. This mutualistic relation is disturbed by the specialized pathogenic fungus Escovopsis sp., which can overcome Leucoagaricus sp. and thus destroy the ant colony. Microbial symbionts of leaf-cutting ants have been suggested to protect the fungus garden against Escovopsis by producing antifungal compounds [Currie CR, Scott JA, Summerbell RC, Malloch D (1999) Fungus-growing ants use antibiotic-producing bacteria to control garden parasites. Nature 398:701-704.]. To date, however, the chemical nature of these compounds has remained elusive. We characterized 19 leaf-cutting ant-associated microorganisms (5 Pseudonocardia, 1 Dermacoccus, and 13 Streptomyces) from 3 Acromyrmex species, A. octospinosus, A. echinatior, and A. volcanus, using 16S-rDNA analysis. Because the strain Streptomyces sp. Ao10 proved highly active against the pathogen Escovopsis, we identified the molecular basis of its antifungal activity. Using bioassay-guided fractionation, high-resolution electrospray mass spectrometry (HR-ESI-MS), and UV spectroscopy, and comparing the results with an authentic standard, we were able identify candicidin macrolides. Candicidin macrolides are highly active against Escovopsis but do not significantly affect the growth of the symbiotic fungus. At least one of the microbial isolates from each of the 3 leaf-cutting ant species analyzed produced candicidin macrolides. This suggests that candicidins play an important role in protecting the fungus gardens of leaf-cutting ants against pathogenic fungi.

The pH shift and precursor feeding strategy in a low-toxicity FR-008/candicidin derivative CS103 fermentation bioprocess by a mutant of Streptomyces sp. FR-008.

CS103, the novel derivative of polyene macrolides antibiotic FR-008/candicidin with lower toxicity has been isolated from the culture mycelia of the mutant of Streptomyces sp. FR-008, with targeted deletions of the fscP cytochrome P450 gene from its chromosome. To enhance biosynthesis of CS103, pH shift and precursor feeding strategy for fermentation process by the mutant of Streptomyces sp. FR-008 in a stirred tank bioreactor was developed. According to the process parameters analysis, the effectiveness of the strategy was examined and confirmed by experiments. A maximal CS103 concentration of 139.98 microg/mL was obtained, 2.05-fold higher than that in the pH-uncontrolled fermentation. Compared to other three cases as pH-uncontrolled, pH-controlled, and two-stage pH-controlled batch cultures, the proposed "pH shift and precursor feeding strategy" effectively avoided the scarcity of the antibiotic precursor, increased the CS103 yield from biomass (Y (P/X)) and substrate (Y (P/S)) by 110.61% and 48.52%, respectively, and at the time the fermentation time was shortened from 120 to 96 h. The highest CS103 production rate (1.46 microg mL(-1) h(-1)) of the pH shift and precursor feeding strategy was 284.21%, 97.30%, and 58.70% higher than that of pH-uncontrolled, pH-controlled, and two-stage pH-controlled batch culture cases, respectively.

Incomplete beta-ketone processing as a mechanism for polyene structural variation in the FR-008/candicidin complex.

FR-008/candicidin is a heptaene macrolide with established antifungal activity, produced by Streptomyces sp. FR-008 as a complex mixture of compounds. Here, six components (FR-008-I to -VI) of the FR-008/candicidin complex were determined; III, V, and VI were confirmed as natural products, principally differing from each other at C-3 and C-9, while the other three were believed to originate from the respective conversions of the natural ones in vitro. Inactivation of KR21 and DH18, respectively, abolished production of V carrying a C-3 hydroxyl, and VI carrying a C-9 methylene. Combined inactivation created a mutant producing only III, with a C-3 ketone and a C-9 hydroxyl, and having antifungal activity superior to V and comparable to VI. Incomplete activities of KR21 and DH18 were, therefore, unambiguously identified as being involved in structural variations of FR-008 complex.

Organizational and mutational analysis of a complete FR-008/candicidin gene cluster encoding a structurally related polyene complex.

The complete gene cluster for biosynthesis of a polyene complex, FR-008, spans 137.2 kb of the genome of Streptomyces sp. FR-008 consisting of six genes for a modular PKS and 15 additional genes. The extensive similarity to the partially characterized candicidin gene cluster in Streptomyces griseus IMRU3570, especially for genes involved in mycosamine biosynthesis, prompted us to compare the compounds produced by Streptomyces sp. FR-008 and Streptomyces griseus IMRU3570, and we found that FR-008 and candicidin complex are identical. A model for biosynthesis of a set of four structurally related FR-008/candicidin compounds was proposed. Deletion of the putative regulatory genes abolished antibiotic production, while disruption of putative glycosyltransferase and GDP-ketosugar aminotransferase functionalities led to the productions of a set of nonmycosaminated aglycones and a novel polyene complex with attachment of altered sugar moiety, respectively.

Candicidin biosynthesis in Streptomyces griseus.

The biosynthesis of the aromatic polyene macrolide antibiotic candicidin, produced by Streptomyces griseus IMRU 3570, begins with a p-aminobenzoic acid (PABA) molecule which is activated to PABA-CoA and used as starter for the head-to-tail condensation of four propionate and 14 acetate units to produce a polyketide molecule to which the deoxysugar mycosamine is attached. Using the gene coding for the PABA synthase ( pabAB) from S. griseusIMRU 3570 as the probe, a 205-kb region of continuous DNA from the S. griseus chromosome was isolated and partially sequenced. Some of the genes possibly involved in the biosynthesis of candicidin were identified including part of the modular polyketide synthase (PKS), genes for thioesterase, deoxysugar biosynthesis, modification, transport, and regulatory proteins. The regulatory mechanisms involved in the production of candicidin, such as phosphate regulation, were studied using internal probes for some of the genes involved in the biosynthesis of the three moieties of candicidin (PKS, aromatic moiety and amino sugar). mRNAs specific for these genes were detected only in the production medium (SPG) but not in the SPG medium supplemented with phosphate or in the inoculum medium, indicating that phosphate represses the expression of genes involved in candicidin biosynthesis. The modular architecture of the candicidin PKS and the availability of the PKSs involved in the biosynthesis of three polyene antibiotics (pimaricin, nystatin, and amphotericin B) shall make possible the creation of new, less toxic and more active polyene antibiotics through combinatorial biosynthesis and targeted mutagenesis.

Solution NMR structure of five representative glycosylated polyene macrolide antibiotics with a sterol-dependent antifungal activity.

Glycosylated polyene macrolide antibiotics, as nystatins and amphotericins, are amphiphilic structures known to exert antifungal activity by disrupting the fungal cell membrane, leading to leakage of cellular materials, and cell death. This membrane disruption is strongly influenced by the presence and the exact nature of the membrane sterols. The solution structures of five representative glycosylated members, three tetraenes (pimaricin, nystatin A1 and rimocidin) and two heptaenes (candidin and vacidin A) have been calculated using geometric restraints derived from 1H-NMR data and random searches of their conformational space. Despite a different apparent structural order, the NMR solutions structure indicate that the hydroxyl groups all clustered on one side of the rod-shaped structures, and the glycosyl moieties are structurally conserved both in their conformation and their apparent order. The molecular structures afford an understanding of their selective interaction with the membrane sterols and the design of new polyene macrolides with improved activities.

[The cis-trans isomerization of aromatic heptaenes catalyzed by sulfur and phosphorus compounds]. / Tsis-trans-izomerizatsiia aromaticheskikh geptaenov, kataliziruemaia soedineniiami sery i fosfora.

It was shown that sulfur and phosphorus compounds (sodium thiosulfate, sodium tripolyphosphate, sodium hexametaphosphate, monosodium phosphate) catalyze cis-trans isomerization of aromatic heptaens. Preparative method of levorin isomerezation at the presence of sodium thiosulfate was elaborated. The isolated product was a fully trans-isomer.

[Ionol phosphorus organic analogs and stabilization of levorin]. / Ispol'zovaniephosphoroorganicheskikh analogov ionola dlia stabilizatsii levorina.

Stability of levorin isolated and purified with the use of ionole phosphorus organic analogs having in their structure phosphate, phosphonate and phosphonite groups was studied. The compound having in its structure (in addition to tertiary butyl substitutes) the phosphonite group with the P-H bond increasing the compound antioxidant property was shown to have the highest stabilizing effect. The results of the study made it possible to recommend the use of the space complicated phenols with the structure fragments of the P-H bond type as antioxidants in production of levorin.

[Stabilization of roseofungin by antioxidants]. / Stabilizatsiia rozeofungina antioksidantami.

Stabilization of roseofungin, a pentaene antibiotic, by antioxidants of different chemical structure was studied. The highest inhibitory effect was observed with the use of antioxidants X-1 (a partially hydrated oxyquinoline) and P-1 (a polyaromatic nitrogen containing compound). The results of the study correlated with the data on nystatin and levorin. The stabilizing effect markedly depended on the temperature: with increasing of the temperature the antioxidant effect decreased. The spectrophotometric study revealed that the decrease of the biological activity of the antibiotic in the presence of the antioxidants was more pronounced than the change of its structure.