The polyene antifungal candicidin is selectively packaged into membrane vesicles in Streptomyces S4.

In recent years, much attention has been focused on the biogenesis, engineering and utilisation of outer membrane vesicles (OMVs) in Gram-negative bacteria in a range of environments and niches. While the precise mechanism of biogenesis is unknown, it is focused on the modification of the Gram-negative cell wall to facilitate blebbing at sites of weakness in and around the characteristically thin peptidoglycan layer within the periplasm. Here, we investigate the biogenesis of membrane vesicles (MVs) in the Gram-positive organism Streptomyces albus S4 (Seipke et al. J Bacteriol 193:4270-4271, 2011 and Fazal et al. Antonie Van Leeuwenhoek 113:511-520, 2020). The S. albus S4 strain is an antifungal (candicidin and antimycin) producing organism that was isolated from attine ants (Barke et al. BMC Biol 8:109, 2010). The biogenesis and characterisation of S. albus S4 MVs is demonstrated using the wild-type (WT) and mutant strains ΔantC (no antimycin production) ΔfscC (no candicidin production) and ΔantC ΔfscC (produces neither antimycin nor candicidin). Here, we have shown that the S. albus S4 strain produces MVs and that these are comprised of both specific protein profiles and secondary metabolites, with a clear demonstration of the ability to selectively package one antifungal (candicidin) but not the other (antimycin).

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.

Quest for the Molecular Basis of Improved Selective Toxicity of All-Trans Isomers of Aromatic Heptaene Macrolide Antifungal Antibiotics.

Three aromatic heptaene macrolide antifungal antibiotics, Candicidin D, Partricin A (Gedamycin) and Partricin B (Vacidin) were subjected to controlled cis-trans→ all trans photochemical isomerization. The obtained all-trans isomers demonstrated substantially improved in vitro selective toxicity in the Candida albicans cells: human erythrocytes model. This effect was mainly due to the diminished hemotoxicity. The molecular modeling studies on interactions between original antibiotics and their photoisomers with ergosterol and cholesterol revealed some difference in free energy profiles of formation of binary antibiotic/sterol complexes in respective membrane environments. Moreover, different geometries of heptaene: sterol complexes and variations in polyene macrolide molecule alignment in cholesterol-and ergosterol-containing membranes were found. None of these effects are of the crucial importance for the observed improvement of selective toxicity of aromatic heptaene antifungals but each seems to provide a partial contribution.

A Hierarchical Network of Four Regulatory Genes Controlling Production of the Polyene Antibiotic Candicidin in Streptomyces sp. Strain FR-008.

The four regulatory genes fscR1 to fscR4 in Streptomyces sp. strain FR-008 form a genetic arrangement that is widely distributed in macrolide-producing bacteria. Our previous work has demonstrated that fscR1 and fscR4 are critical for production of the polyene antibiotic candicidin. In this study, we further characterized the roles of the other two regulatory genes, fscR2 and fscR3, focusing on the relationship between these four regulatory genes. Disruption of a single or multiple regulatory genes did not affect bacterial growth, but transcription of genes in the candicidin biosynthetic gene cluster decreased, and candicidin production was abolished, indicating a critical role for each of the four regulatory genes, including fscR2 and fscR3, in candicidin biosynthesis. We found that fscR1 to fscR4, although differentially expressed throughout the growth phase, displayed similar temporal expression patterns, with an abrupt increase in the early exponential phase, coincident with initial detection of antibiotic production in the same phase. Our data suggest that the four regulatory genes fscR1 to fscR4 have various degrees of control over structural genes in the biosynthetic cluster under the conditions examined. Extensive transcriptional analysis indicated that complex regulation exists between these four regulatory genes, forming a regulatory network, with fscR1 and fscR4 functioning at a lower level. Comprehensive cross-complementation analysis indicates that functional complementation is restricted among the four regulators and unidirectional, with fscR1 complementing the loss of fscR3 or -4 and fscR4 complementing loss of fscR2 Our study provides more insights into the roles of, and the regulatory network formed by, these four regulatory genes controlling production of an important pharmaceutical compound.IMPORTANCE The regulation of antibiotic biosynthesis by Streptomyces species is complex, especially for biosynthetic gene clusters with multiple regulatory genes. The biosynthetic gene cluster for the polyene antibiotic candicidin contains four consecutive regulatory genes, which encode regulatory proteins from different families and which form a subcluster within the larger biosynthetic gene cluster in Streptomyces sp. FR-008. Syntenic arrangements of these regulatory genes are widely distributed in polyene gene clusters, such as the amphotericin and nystatin gene clusters, suggesting a conserved regulatory mechanism controlling production of these clinically important medicines. However, the relationships between these multiple regulatory genes are unknown. In this study, we determined that each of these four regulatory genes is critical for candicidin production. Additionally, using transcriptional analyses, bioassays, high-performance liquid chromatography (HPLC) analysis, and genetic cross-complementation, we showed that FscR1 to FscR4 comprise a hierarchical regulatory network that controls candicidin production and is likely representative of how expression of other polyene biosynthetic gene clusters is controlled.

Dynamic changes of metabolomics and expression of candicidin biosynthesis gene cluster caused by the presence of a pleiotropic regulator AdpA in Streptomyces ZYJ-6.

Candicidin is one of the frequent antibiotics for its high antifungal activity, but the productivity is still extremely low. Introduction of adpA into Streptomyces ZYJ-6 could improve candicidin productivity significantly and achieved 9338 µg/mL, which was the highest value ever reported in the literature. Combined analyses of transcriptional levels, metabolic flux and metabolomics indicate that para-aminobenzoic acid and the first step of shikimic acid metabolism were not the bottleneck for the candicidin production in the control. However, methylmalonyl-CoA played a central role in the candicidin production and the gene methB responsible for the biosynthesis of methylmalonyl-CoA might be the candidate gene target for further improving the production of candicidin.

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.

Enhancing candicidin biosynthesis by medium optimization and pH stepwise control strategy with process metabolomics analysis of Streptomyces ZYJ-6.

Candicidin is one of the most effective antimonilial agents. In order to enhance candicidin productivity, medium optimization and pH stepwise control strategy in process optimization were conducted by Streptomyces ZYJ-6. With the aid of Design Expert software and N/C/P-sources regulation, chemically defined medium fit for cell growth and candicidin biosynthesis was developed. Moreover, pH effects on cell growth and metabolism were investigated. The results indicated that the optimal pH for cell growth and candicidin biosynthesis were 6.8 and 7.8, respectively. The metabolomics analysis revealed that the pH stepwise control strategy (pH 6.8-7.8) combined the advantages of pH 6.8 and pH 7.8 and avoided precursor limitation in pH 6.8 and 7.8. Consequently, the pH stepwise control strategy played positive performance on cell growth and candicidin biosynthesis with the maximum titer of 5161 µg/mL. The titer of 5161 µg/mL was the highest level ever reported for candicidin production, which laid a solid foundation for industrial application. Additionally, pH stepwise control strategy was important reference for process optimization.

Elimination of indigenous linear plasmids in Streptomyces hygroscopicus var. jinggangensis and Streptomyces sp. FR008 to increase validamycin A and candicidin productivities.

Giant linear plasmids, which replicate independently of the chromosomes, widely exist in actinobacteria. Previous studies mostly focused on the replication and evolution of the linear plasmids or the secondary metabolite gene clusters and the resistance gene clusters therein. However, the relationships of the linear plasmids to the productivities of secondary metabolites have not been studied. In this work, we developed a method to eliminate the indigenous linear plasmid pSHJG1 in Streptomyces hygroscopicus var. jinggangensis, and validamycin A titer increased by 12.5% (from 19.16 ± 1.93 to 21.56 ± 2.25 g/L) in the high-yielding strain TL01 and 43.7% (from 4.67 ± 0.05 to 6.71 ± 0.21 g/L) in the wild-type strain 5008, whereas the cellular growth of the plasmid-cured mutant was reduced. Subsequently, the plasmid-cured mutant was complemented with three structure genes involved in cellular growth in pSHJG1 under the control of a strong PvalA promoter. Among them, the complementation of genes pSHJG1.069 and pSHJG1.072, encoding a putative hydrolase and putative P-loop ATPase, respectively, resulted in the restoration of cellular growth and validamycin A titer. Furthermore, the elimination of indigenous linear plasmid pHZ228 in the candicidin producer Streptomyces sp. FR008 also led to enhanced candicidin production and reduced cellular growth. Because of the wide distribution of indigenous linear plasmids in actinobacteria, the engineering strategy described here could be implemented in a variety of strains for the overproduction of various natural products.

Production of the antibiotic FR-008/candicidin in Streptomyces sp. FR-008 is co-regulated by two regulators, FscRI and FscRIV, from different transcription factor families.

In Streptomyces sp. FR-008, the biosynthetic gene cluster of the polyene antibiotic FR-008, also known as candicidin, consists of 21 genes, including four regulatory genes, fscRI-fscRIV. Our bioinformatics analyses indicate that FscRI has an N-terminal PAS domain, whereas the other three regulators have N-terminal AAA domains and are members of the LAL (large ATP-binding regulators of the LuxR type) family. Deletion of fscRI abolished the production of FR-008, with production restored in the complemented strain, supporting a critical role for FscRI in FR-008 biosynthesis. Consistent with these findings, transcription of genes involved in the biosynthesis and efflux of FR-008 was greatly downregulated in a ΔfscRI mutant. Interestingly, the regulatory gene fscRIV was also downregulated in the ΔfscRI mutant. Production of FR-008 was reduced, but not abrogated, in an fscRIV deletion mutant, and although structural genes were downregulated in ΔfscRIV, the changes were much less dramatic than in ΔfscRI, suggesting a stronger regulatory role for FscRI. Remarkably, transcription of fscRI was also decreased in ΔfscRIV. Expression of fscRI restored antibiotic production in a ΔfscRIV mutant, but not vice versa. Putative binding sequences for FscRI were identified upstream of fscRIV and the three structural genes fscA, fscB and fscD, which encode large modular polyketide synthases. Our findings suggest that fscRI and fscRIV are interregulatory, whereas expression of fscRII and fscRIII appears to be independent of fscRI and fscRIV. This study demonstrates that the regulation of polyene antibiotic synthesis can involve mutually regulated transcriptional activators that belong to different families.

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.

[Function of transporter genes fscTI and fscTII in the biosynthetic cluster of candicidin/FR-008].

UNLABELLED: OBJECTIVE To investigate function of transporter genes fscTI and fscTII in the biosynthetic gene cluster of candicidin/FR-008. METHODS: We constructed a plasmid pJTU4137 for disruption of transporter genes fscTI and fscTII by conjugation and homologous recombinant. The transporter genes were also PCR amplified and cloned into the high-copy plasmid pJTU1278 for overexpression in strain ZYJ-6 derived from Streptomyces sp. FR-008. RESULTS: The disruption mutant LX10 was unable to produce candicidin and its analogues. Overexpression of FscTI and FscTII in ZYJ-6 caused a 1.5-fold increase in FR-008-III production compared with the control. CONCLUSION: We confirmed that fscTI and fscTII are function as ATP dependent ATP binding cassetle (ABC) transporters in the biosynthetic gene cluster of FR-008. Furthermore, a positive example was provided for improving antibiotic production in other polyene producing strains based on the results that overexpression of fscTI and fscTI increased candicidin production.

A mixed community of actinomycetes produce multiple antibiotics for the fungus farming ant Acromyrmex octospinosus.

BACKGROUND: Attine ants live in an intensely studied tripartite mutualism with the fungus Leucoagaricus gongylophorus, which provides food to the ants, and with antibiotic-producing actinomycete bacteria. One hypothesis suggests that bacteria from the genus Pseudonocardia are the sole, co-evolved mutualists of attine ants and are transmitted vertically by the queens. A recent study identified a Pseudonocardia-produced antifungal, named dentigerumycin, associated with the lower attine Apterostigma dentigerum consistent with the idea that co-evolved Pseudonocardia make novel antibiotics. An alternative possibility is that attine ants sample actinomycete bacteria from the soil, selecting and maintaining those species that make useful antibiotics. Consistent with this idea, a Streptomyces species associated with the higher attine Acromyrmex octospinosus was recently shown to produce the well-known antifungal candicidin. Candicidin production is widespread in environmental isolates of Streptomyces, so this could either be an environmental contaminant or evidence of recruitment of useful actinomycetes from the environment. It should be noted that the two possibilities for actinomycete acquisition are not necessarily mutually exclusive. RESULTS: In order to test these possibilities we isolated bacteria from a geographically distinct population of A. octospinosus and identified a candicidin-producing Streptomyces species, which suggests that they are common mutualists of attine ants, most probably recruited from the environment. We also identified a Pseudonocardia species in the same ant colony that produces an unusual polyene antifungal, providing evidence for co-evolution of Pseudonocardia with A. octospinosus. CONCLUSIONS: Our results show that a combination of co-evolution and environmental sampling results in the diversity of actinomycete symbionts and antibiotics associated with attine ants.

Optimisation of synthetic medium composition for levorin biosynthesis by Streptomyces levoris 99/23 and investigation of its accumulation dynamics using mathematical modelling methods.

The composition of a synthetic culture medium for levorin biosynthesis by Streptomyces levoris 99/23 was optimised using mathematical modelling methods. The optimal concentrations of the medium components were established by means of an optimum composition design at three factor variation levels. An adequate regression model was obtained. Levorin biosynthesis by Streptomyces levoris 99/23 in the optimised synthetic medium was over 38% higher than in the initial medium. The antibiotic biosynthesis dynamics in the optimised culture medium was studied by means of a non-linear differential equation system. The resultant model was valid.

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).

Tailoring the P450 monooxygenase gene for FR-008/candicidin biosynthesis.

Tailoring steps are often important for the activity of mature antibiotics. Here, we report that novel decarboxylated FR-008/candicidin derivatives were obtained from the P450 monooxygenase gene fscP mutant of Streptomyces sp. strain FR-008. The toxicity of decarboxylated FR-008/candicidin derivatives has been shown to be greatly reduced compared to that of wild-type FR-008/candicidin.

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.

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.

Selective removal of aberrant extender units by a type II thioesterase for efficient FR-008/candicidin biosynthesis in Streptomyces sp. strain FR-008.

Gene fscTE, encoding a putative type II thioesterase (TEII), was associated with the FR-008/candicidin gene cluster. Deletion of fscTE reduced approximately 90% of the FR-008/candicidin production, while the production level was well restored when fscTE was added back to the mutant in trans. FscTE was unable to compensate for the release of the maturely elongated polyketide as site-directed inactivation of the type I thioesterase (TEI) totally abolished FR-008/candicidin production. Direct biochemical analysis of FscTE in parallel with its homologue TylO from the tylosin biosynthetic pathway demonstrated their remarkable preferences for acyl-thioesters (i.e., propionyl-S-N-acetylcysteamine [SNAC] over methylmalonyl-SNAC and acetyl-SNAC over malonyl-SNAC) and thus concluded that TEII could maintain effective polyketide biosynthesis by selectively removing the nonelongatable residues bound to acyl carrier proteins. Overexpression of FscTE under the strong constitutive ermE*p promoter in the wild-type strain did not suppress FR-008/candicidin formation, which confirmed its substrate specificity in vivo. Furthermore, successful complementation of the fscTE mutant was obtained with fscTE and tylO, whereas no complementation was detected with nonribosomal peptide synthetase (NRPS) TEII tycF and srfAD, reflecting substrate specificities of TEIIs distinctive from those of either polyketide synthases or NRPSs.