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.
The DNA region encoding the filipin gene cluster in Streptomyces avermitilis (pte) contains a PAS-LuxR regulatory gene, pteF, orthologue to pimM, the final pathway-specific positive regulatory ...protein of pimaricin biosynthesis in Streptomyces natalensis. Gene replacement of the gene from S. avermitilis chromosome resulted in a severe loss of filipin production and delayed spore formation in comparison to that of the wild-type strain, suggesting that it acts as a positive regulator of filipin biosynthesis and that it may also have a role in sporulation. Complementation of the mutant with a single copy of the gene integrated into the chromosome restored wild-type phenotypes. Heterologous complementation with the regulatory counterpart from S. natalensis also restored parental phenotypes. Gene expression analyses in S. avermitilis wild-type and the mutant by reverse transcription-quantitative polymerase chain reaction of the filipin gene cluster suggested the targets for the regulatory protein. Transcription start points of all the genes of the cluster were studied by 5′-rapid amplification of complementary DNA ends. Transcription start point analysis of the pteF gene revealed that the annotated sequence in the databases is incorrect. Confirmation of target promoters was performed by in silico search of binding sites among identified promoters and the binding of the orthologous regulator for pimaricin biosynthesis PimM to gene promoters by electrophoretic mobility shift assays. Precise binding regions were investigated by DNAse I protection studies. Our results indicate that PteF activates the transcription from two promoters of polyketide synthase genes directly, and indirectly of other genes of the cluster.
The biosynthesis of small-size polyene macrolides is ultimately controlled by a couple of transcriptional regulators that act in a hierarchical way. A
antibiotic regulatory protein-large ATP-binding ...regulator of the LuxR family (SARP-LAL) regulator binds the promoter of a PAS-LuxR regulator-encoding gene and activates its transcription, and in turn, the gene product of the latter activates transcription from various promoters of the polyene gene cluster directly. The primary operator of PimR, the archetype of SARP-LAL regulators, contains three heptameric direct repeats separated by four-nucleotide spacers, but the regulator can also bind a secondary operator with only two direct repeats separated by a 3-nucleotide spacer, both located in the promoter region of its unique target gene,
A similar arrangement of operators has been identified for PimR counterparts encoded by gene clusters for different antifungal secondary metabolites, including not only polyene macrolides but peptidyl nucleosides, phoslactomycins, or cycloheximide. Here, we used promoter engineering and quantitative transcriptional analyses to determine the contributions of the different heptameric repeats to transcriptional activation and final polyene production. Optimized promoters have thus been developed. Deletion studies and electrophoretic mobility assays were used for the definition of DNA-binding boxes formed by 22-nucleotide sequences comprising two conserved heptameric direct repeats separated by four-nucleotide less conserved spacers. The cooperative binding of PimR
appears to be the mechanism involved in the binding of regulator monomers to operators, and at least two protein monomers are required for efficient binding.
Here, we have shown that a modulation of the production of the antifungal pimaricin in
can be accomplished via promoter engineering of the PAS-LuxR transcriptional activator
The expression of this gene is controlled by the
antibiotic regulatory protein-large ATP-binding regulator of the LuxR family (SARP-LAL) regulator PimR, which binds a series of heptameric direct repeats in its promoter region. The structure and importance of such repeats in protein binding, transcriptional activation, and polyene production have been investigated. These findings should provide important clues to understand the regulatory machinery that modulates antibiotic biosynthesis in
and open new possibilities for the manipulation of metabolite production. The presence of PimR orthologues encoded by gene clusters for different secondary metabolites and the conservation of their operators suggest that the improvements observed in the activation of pimaricin biosynthesis by
could be extrapolated to the production of different compounds by other species.
PAS-LuxR regulators are highly conserved proteins devoted to the control of antifungal production by binding to operators located in given promoters of polyene biosynthetic genes. The canonical ...operator of PimM, archetype of this class of regulators, has been used here to search for putative targets of orthologous protein PteF in the genome of Streptomyces avermitilis, finding 97 putative operators outside the pentaene filipin gene cluster (pte). The processes putatively affected included genetic information processing; energy, carbohydrate, and lipid metabolism; DNA replication and repair; morphological differentiation; secondary metabolite biosynthesis; and transcriptional regulation, among others. Seventeen of these operators were selected, and their binding to PimM DNA-binding domain was assessed by electrophoretic mobility shift assays. Strikingly, the protein bound all predicted operators suggesting a direct control over targeted processes. As a proof of concept, we studied the biosynthesis of the ATP-synthase inhibitor oligomycin whose gene cluster included two operators. Regulator mutants showed a severe loss of oligomycin production, whereas gene complementation of the mutant restored phenotype, and gene duplication in the wild-type strain boosted oligomycin production. Comparative gene expression analyses in parental and mutant strains by reverse transcription-quantitative polymerase chain reaction of selected olm genes corroborated production results. These results demonstrate that PteF is able to cross-regulate the biosynthesis of two related secondary metabolites, filipin and oligomycin, but might be extended to all the processes indicated above. This study highlights the complexity of the network of interactions in which PAS-LuxR regulators are involved and opens new possibilities for the manipulation of metabolite production in Streptomycetes.
LAL regulators (Large ATP-binding regulators of the LuxR family) constitute a poorly studied family of transcriptional regulators. Several regulators of this class have been identified in antibiotic ...and other secondary metabolite gene clusters from actinomycetes, thus they have been considered pathway-specific regulators. In this study we have obtained two disruption mutants of LAL genes from S. coelicolor (Δ0877 and Δ7173). Both mutants were deficient in the production of the polyketide antibiotic actinorhodin, and antibiotic production was restored upon gene complementation of the mutants. The use of whole-genome DNA microarrays and quantitative PCRs enabled the analysis of the transcriptome of both mutants in comparison with the wild type. Our results indicate that the LAL regulators under study act globally affecting various cellular processes, and amongst them the phosphate starvation response and the biosynthesis of the blue-pigmented antibiotic actinorhodin. Both regulators act as negative modulators of the expression of the two-component phoRP system and as positive regulators of actinorhodin biosynthesis. To our knowledge this is the first characterization of LAL regulators with wide implications in Streptomyces metabolism.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Control of polyene macrolide production in
Streptomyces natalensis is mediated by the PAS–LuxR transcriptional activator PimM. Expression of target genes in this strain is positively regulated by ...binding of the regulator to 14-nucleotide sites showing dyad symmetry, and overlapping the -35 element of each promoter. These sequences have been found in the upstream regions of genes belonging to different polyene biosynthetic gene clusters. All the sequences in the amphotericin, nystatin, and filipin clusters were cloned and the binding of PimM to all of them has been shown by electrophoretic mobility shift assays. The precise binding regions were investigated by DNaseI protection studies. Results indicated that PAS–luxR regulators share the same regulatory pattern in different polyene-producing strains, these genes being responsible for polyketide chain construction, and when available, the genes for sugar dehydration and attachment, and the ABC transporters, the targets for regulation. Information content analysis of the 24 sequences protected in target promoters was used to refine the information-based model of the binding site. This site now spans 16 nucleotides and adjusts to the consensus CTVGGGAWWTCCCBAG. Gene complementation of
S. natalensis Δ
pimM with a single copy of heterologous regulators of the PAS/LuxR class integrated into the chromosome, such as
amphRIV,
nysRIV, or
pteF, restored antifungal production, thus proving the functional conservation of these regulators. Introduction of a single copy of
pimM into the amphotericin producing strain
Streptomyces nodosus, or into the filipin producing strain
S. avermitilis, boosted the production of both polyenes, thus indicating that the expression of the PAS–LuxR regulator constitutes a bottleneck in the biosynthesis of the antifungal, and also that these regulators are fully exchangeable. This work is the first report of a general mechanism regulating polyene production.
► Control of polyene production is mediated by PAS–LuxR transcriptional activators. ► These regulators share the same regulatory pattern in different strains. ► PAS–LuxR regulators bind the consensus sequence CTVGGGAWWTCCCBAG. ► Expression of these regulators constitutes a bottleneck in polyene biosynthesis.
Streptomyces filipinensis is the industrial producer of filipin, a pentaene macrolide, archetype of non-glycosylated polyenes, and widely used for the detection and the quantitation of cholesterol in ...biological membranes and as a tool for the diagnosis of Niemann-Pick type C disease. Genetic manipulations of polyene biosynthetic pathways have proven useful for the discovery of products with improved properties. Here, we describe the late biosynthetic steps for filipin III biosynthesis and strategies for the generation of bioactive filipin III derivatives at high yield.
A region of 13,778 base pairs of DNA from the S. filipinensis genome was isolated, sequenced, and characterized. Nine complete genes and two truncated ORFs were located. Disruption of genes proved that this genomic region is part of the biosynthetic cluster for the 28-membered ring of the polyene macrolide filipin. This set of genes includes two cytochrome P450 monooxygenase encoding genes, filC and filD, which are proposed to catalyse specific hydroxylations of the macrolide ring at C26 and C1' respectively. Gene deletion and complementation experiments provided evidence for their role during filipin III biosynthesis. Filipin III derivatives were accumulated by the recombinant mutants at high yield. These have been characterized by mass spectrometry and nuclear magnetic resonance following high-performance liquid chromatography purification thus revealing the post-polyketide steps during polyene biosynthesis. Two alternative routes lead to the formation of filipin III from the initial product of polyketide synthase chain assembly and cyclization filipin I, one trough filipin II, and the other one trough 1'-hydroxyfilipin I, all filipin III intermediates being biologically active. Moreover, minimal inhibitory concentration values against Candida utilis and Saccharomyces cerevisiae were obtained for all filipin derivatives, finding that 1'-hydroxyfilipin and especially filipin II show remarkably enhanced antifungal bioactivity. Complete nuclear magnetic resonance assignments have been obtained for the first time for 1'-hydroxyfilipin I.
This report reveals the existence of two alternative routes for filipin III formation and opens new possibilities for the generation of biologically active filipin derivatives at high yield and with improved properties.
PAS-LuxR transcriptional regulators are conserved proteins governing polyene antifungal biosynthesis. PteF is the regulator of filipin biosynthesis from Streptomyces avermitilis. Its mutation ...drastically abates filipin, but also oligomycin production, a macrolide ATP-synthase inhibitor, and delays sporulation; thus, it has been considered a transcriptional activator. Transcriptomic analyses were performed in S. avermitilis ΔpteF and its parental strain. Both strains were grown in a YEME medium without sucrose, and the samples were taken at exponential and stationary growth phases. A total of 257 genes showed an altered expression in the mutant, most of them at the exponential growth phase. Surprisingly, despite PteF being considered an activator, most of the genes affected showed overexpression, thereby suggesting a negative modulation. The affected genes were related to various metabolic processes, including genetic information processing; DNA, energy, carbohydrate, and lipid metabolism; morphological differentiation; and transcriptional regulation, among others, but were particularly related to secondary metabolite biosynthesis. Notably, 10 secondary metabolite gene clusters out of the 38 encoded by the genome showed altered expression profiles in the mutant, suggesting a regulatory role for PteF that is wider than expected. The transcriptomic results were validated by quantitative reverse-transcription polymerase chain reaction. These findings provide important clues to understanding the intertwined regulatory machinery that modulates antibiotic biosynthesis in Streptomyces.
Control of polyene macrolide production in Streptomyces natalensis is mediated by the transcriptional activator PimR. This regulator combines an N-terminal domain corresponding to the Streptomyces ...antibiotic regulatory protein (SARP) family of transcriptional activators with a C-terminal half homologous to guanylate cyclases and large ATP-binding regulators of the LuxR family. The PimR SARP domain (PimR(SARP)) was expressed in Escherichia coli as a glutathione S-transferase (GST)-fused protein. Electrophoretic mobility shift assays showed that GST-PimR(SARP) binds a single target, the intergenic region between the regulatory genes pimR and pimMs in the pimaricin cluster. The PimR(SARP)-binding site was investigated by DNaseI protection studies, revealing that it contains three heptameric direct repeats adjusting to the consensus 5'-CGGCAAG-3'. Transcription start points of pimM and pimR promoters were identified by 5'-RACE, revealing that unlike other SARPs, PimR(SARP) does not interact with the -35 region of its target promoter. Quantitative transcriptional analysis of these regulatory genes on mutants on each of them has allowed the identification of the pimM promoter as the transcriptional target for PimR. Furthermore, the constitutive expression of pimM restored pimaricin production in a pimaricin-deficient strain carrying a deletion mutant of pimR. These results reveal that PimR exerts its positive effect on pimaricin production by controlling pimM expression level, a regulator whose gene product activates transcription from eight different promoters of pimaricin structural genes directly.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Published data on vitamin D and the quality of life of asthma patients are scarce and disparate. The ACVID clinical trial, published in 2020, showed the efficacy of calcifediol in improving asthma ...control in asthma patients with vitamin D deficiency. Data on vitamin D and quality of life measured by the Mini-AQLQ questionnaire were analysed: supplemented patients showed improved quality of life compared with a placebo group, and the initial mini-AQLQ scores were improved for both groups.
•Calcifediol supplementation improved quality of life in asthmatics patients.•A positive correlation was observed between vitamin D levels and quality of life.•Calcifediol improves symptoms, activity limitation and emotional sphere in asthma.
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