Structural and bioactivity characterization of filipin derivatives from engineered streptomyces filipinensis strains reveals clues for reduced haemolytic action

The rise in the number of immunocompromised patients has led to an increased incidence of fungal infections, with high rates of morbidity and mortality. Furthermore, misuse of antifungals has boosted the number of resistant strains to these agents; thus, there is urgent need for new drugs against these infections. Here, the in vitro antifungal activity of filipin III metabolic intermediates has been characterized against a battery of opportunistic pathogenic fungi—Candida albicans, Candida glabrata, Candida krusei, Cryptococcus neoformans, Trichosporon cutaneum, Trichosporon asahii, Aspergillus nidulans, Aspergillus niger, and Aspergillus fumigatus—using the Clinical and Laboratory Standards Institute broth microdilution method. Structural characterization of these compounds was undertaken by mass spectrometry (MS) and nuclear magnetic resonance (NMR) following HPLC purification. Complete NMR assignments were obtained for the first time for filipins I and II. In vitro haemolytic assays revealed that the haemolytic action of these compounds relies largely on the presence of a hydroxyl function at C26, since derivatives lacking such moiety show remarkably reduced activity. Two of these derivatives, 1′-hydroxyfilipin I and filipin I, show decreased toxicity towards cholesterol-containing membranes while retaining potent antifungal activity, and could constitute excellent leads for the development of efficient pharmaceuticals, particularly against CryptococcosisThis research was funded by the Spanish Ministerio de Economía, Industria y Competitividad (grants BIO2013-42983-P and PCIN-2016-190 to J.F.A.), an FPU contract of the Ministerio de Educación, Cultura y Deporte (FPU13/01537 to A.P.), and contracts from the Junta de Castilla y León (to E.G.B.) and from the youth employment initiative (to E.J.), both co-financed by the European Social Fun

Activation of Secondary Metabolite Gene Clusters in Streptomyces clavuligerus by the PimM Regulator of Streptomyces natalensis

Expression of non-native transcriptional activators may be a powerful general method to activate secondary metabolites biosynthetic pathways. PAS-LuxR regulators, whose archetype is PimM, activate the biosynthesis of polyene macrolide antifungals and other antibiotics, and have been shown to be functionally preserved across multiple Streptomyces strains. In this work we show that constitutive expression of pimM in Streptomyces clavuligerus ATCC 27064 significantly affected its transcriptome and modifies secondary metabolism. Almost all genes in three secondary metabolite clusters were overexpressed, including the clusters responsible for the biosynthesis of the clinically important clavulanic acid and cephamycin C. In comparison to a control strain, this resulted in 10- and 7-fold higher production levels of these metabolites, respectively. Metabolomic and bioactivity studies of S. clavuligerus::pimM also revealed deep metabolic changes. Antifungal activity absent in the control strain was detected in S. clavuligerus::pimM, and determined to be the result of a fivefold increase in the production of the tunicamycin complex

Phosphate effect on filipin production and morphological differentiation in Streptomyces filipinensis and the role of the PhoP transcription factor.

The biosynthesis of the antifungal filipin in Streptomyces filipinensis is very sensitive to phosphate regulation. Concentrations as low as 2.5 mM block filipin production. This effect is, at least in part, produced by repression of the transcription of most filipin biosynthetic genes. The role of the two-component PhoRP system in this process was investigated. The phoRP system of S. filipinensis was cloned and transcriptionally characterised. PhoP binds to two PHO boxes present in one of its two promoters. Filipin production was greatly increased in ΔphoP and ΔphoRP mutants, in agreement with a higher transcription of the fil genes, and the effect of phosphate repression on the antibiotic production of these strains was significantly reduced. No PhoP binding was observed by electrophoretic mobility gel shift assays (EMSAs) with the promoter regions of the fil gene cluster thus suggesting an indirect effect of mutations. Binding assays with cell-free extracts from the wild-type and mutant strains on fil genes promoters revealed retardation bands in the parental strain that were absent in the mutants, thus suggesting that binding of the putative transcriptional regulator or regulators controlled by PhoP was PhoP dependent. Noteworthy, PhoP or PhoRP deletion also produced a dramatic decrease in sporulation ability, thus indicating a clear relationship between the phosphate starvation response mediated by PhoP and the sporulation process in S. filipinensis. This effect was overcome upon gene complementation, but also by phosphate addition, thus suggesting that alternative pathways take control in the absence of PhoRP

Biotechnological production and application of the antibiotic pimaricin: biosynthesis and its regulation

Pimaricin (natamycin) is a small polyene macrolide antibiotic used worldwide. This efficient antimycotic and antiprotozoal agent, produced by several soil bacterial species of the genus Streptomyces, has found application in human therapy, in the food and beverage industries and as pesticide. It displays a broad spectrum of activity, targeting ergosterol but bearing a particular mode of action different to other polyene macrolides. The biosynthesis of this only antifungal agent with a GRAS status has been thoroughly studied, which has permitted the manipulation of producers to engineer the biosynthetic gene clusters in order to generate several analogues. Regulation of its production has been largely unveiled, constituting a model for other polyenes and setting the leads for optimizing the production of these valuable compounds. This review describes and discusses the molecular genetics, uses, mode of action, analogue generation, regulation and strategies for increasing pimaricin production yields