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).

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.

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.

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.

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).

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.

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.

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.

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.

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.

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.

In vivo investigation of the substrate recognition capability and activity affecting amino acid residues of glycosyltransferase FscMI in the biosynthesis of candicidin.

Alteration of sugar moieties of natural products often leads to novel antibiotics with different chemical and physical properties. fscMI is a putative glycosyltransferase (GT) in a gene cluster for the production of candicidin, a polyene macrolide antibiotic, produced by Streptomyces sp. FR-008. In this report, we established an in vivo biochemical detection system by inactivating fscMI and the DH11 domain of polyketide synthase (PKS) through double homologous recombination to unveil the interaction between polyene GTs and their substrates. We found that homologous GT genes including amphDI, nysDI and pimK can catalyze the conversion of candicidin aglycone into candicidin/FR-008-III in fscMI mutant, suggesting that homologous polyene GTs show some tolerance toward aglycones and that it is possible to create new polyene analogues with altered aglycones through genetic engineering. Inactivation of the DH11 domain of PKS led to novel polyene derivatives with mycosamine added to the altered polyketide backbones, further confirming the loose substrate specificity of polyene GTs. Furthermore, mutation of Ser346, Ser361, His362 or Cys387 of FscMI by site-directed mutagenesis significantly reduced its catalytic activity. Further analysis suggested that Ser361 and Cys387 are likely the critical donor interacting residues that could affect the activity of GT FscMI. To our knowledge, this is the first report of the critical residues in a polyene GT.

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.

[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 single Streptomyces symbiont makes multiple antifungals to support the fungus farming ant Acromyrmex octospinosus.

Attine ants are dependent on a cultivated fungus for food and use antibiotics produced by symbiotic Actinobacteria as weedkillers in their fungus gardens. Actinobacterial species belonging to the genera Pseudonocardia, Streptomyces and Amycolatopsis have been isolated from attine ant nests and shown to confer protection against a range of microfungal weeds. In previous work on the higher attine Acromyrmex octospinosus we isolated a Streptomyces strain that produces candicidin, consistent with another report that attine ants use Streptomyces-produced candicidin in their fungiculture. Here we report the genome analysis of this Streptomyces strain and identify multiple antibiotic biosynthetic pathways. We demonstrate, using gene disruptions and mass spectrometry, that this single strain has the capacity to make candicidin and multiple antimycin compounds. Although antimycins have been known for >60 years we report the sequence of the biosynthetic gene cluster for the first time. Crucially, disrupting the candicidin and antimycin gene clusters in the same strain had no effect on bioactivity against a co-evolved nest pathogen called Escovopsis that has been identified in ∼30% of attine ant nests. Since the Streptomyces strain has strong bioactivity against Escovopsis we conclude that it must make additional antifungal(s) to inhibit Escovopsis. However, candicidin and antimycins likely offer protection against other microfungal weeds that infect the attine fungal gardens. Thus, we propose that the selection of this biosynthetically prolific strain from the natural environment provides A. octospinosus with broad spectrum activity against Escovopsis and other microfungal weeds.

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.