The Discovery of Actinospene, a New Polyene Macrolide with Broad Activity against Plant Fungal Pathogens and Pathogenic Yeasts

Phytopathogenic fungi infect crops, presenting a worldwide threat to agriculture. Polyene macrolides are one of the most effective antifungal agents applied in human therapy and crop protection. In this study, we found a cryptic polyene biosynthetic gene cluster in Actinokineospora spheciospongiae by genome mining. Then, this gene cluster was activated via varying fermentation conditions, leading to the discovery of new polyene actinospene (1), which was subsequently isolated and its structure determined through spectroscopic techniques including UV, HR-MS, and NMR. The absolute configuration was confirmed by comparing the calculated and experimental electronic circular dichroism (ECD) spectra. Unlike known polyene macrolides, actinospene (1) demonstrated more versatile post-assembling decorations including two epoxide groups and an unusual isobutenyl side chain. In bioassays, actinospene (1) showed a broad spectrum of antifungal activity against several plant fungal pathogens as well as pathogenic yeasts with minimum inhibitory concentrations ranging between 2 and 10 μg/mL.

Identification and Predictions Regarding the Biosynthesis Pathway of Polyene Macrolides Produced by Streptomyces roseoflavus Men-myco-93-63

ABSTRACT A group of polyene macrolides mainly composed of two constituents was isolated from the fermentation broth of Streptomyces roseoflavus Men-myco-93-63, which was isolated from soil where potato scabs were repressed naturally. One of these macrolides was roflamycoin, which was first reported in 1968, and the other was a novel compound named Men-myco-A, which had one methylene unit more than roflamycoin. Together, they were designated RM. This group of antibiotics exhibited broad-spectrum antifungal activities in vitro against 17 plant-pathogenic fungi, with 50% effective concentrations (EC50) of 2.05 to 7.09 μg/ml and 90% effective concentrations (EC90) of 4.32 to 54.45 μg/ml, which indicates their potential use in plant disease control. Furthermore, their biosynthetic gene cluster was identified, and the associated biosynthetic assembly line was proposed based on a module and domain analysis of polyketide synthases (PKSs), supported by findings from gene inactivation experiments. IMPORTANCE Streptomyces roseoflavus Men-myco-93-63 is a biocontrol strain that has been studied in our laboratory for many years and exhibits a good inhibitory effect in many crop diseases. Therefore, the identification of antimicrobial metabolites is necessary and our main objective. In this work, chemical, bioinformatic, and molecular biological methods were combined to identify the structures and biosynthesis of the active metabolites. This work provides a new alternative agent for the biological control of plant diseases and is helpful for improving both the properties and yield of the antibiotics via genetic engineering.

Promoter Engineering Reveals the Importance of Heptameric Direct Repeats for DNA Binding byStreptomycesAntibiotic Regulatory Protein–Large ATP-Binding Regulator of the LuxR Family (SARP-LAL) Regulators inStreptomyces natalensis

ABSTRACTThe biosynthesis of small-size polyene macrolides is ultimately controlled by a couple of transcriptional regulators that act in a hierarchical way. AStreptomycesantibiotic 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,pimM. 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 PimRSARPappears to be the mechanism involved in the binding of regulator monomers to operators, and at least two protein monomers are required for efficient binding.IMPORTANCEHere, we have shown that a modulation of the production of the antifungal pimaricin inStreptomyces natalensiscan be accomplished via promoter engineering of the PAS-LuxR transcriptional activatorpimM. The expression of this gene is controlled by theStreptomycesantibiotic 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 inStreptomycesand 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 byStreptomyces natalensiscould be extrapolated to the production of different compounds by other species.

Engineered Biosynthesis of Disaccharide-Modified Polyene Macrolides

ABSTRACTRecent work has uncovered genes for two glycosyltransferases that are thought to catalyze mannosylation of mycosaminyl sugars of polyene macrolides. These two genes arenypYfromPseudonocardiasp. strain P1 andpegAfromActinoplanes caeruleus. Here we analyze these genes by heterologous expression in various strains ofStreptomyces nodosus, producer of amphotericins, and inStreptomyces albidoflavus, which produces candicidins. The NypY glycosyltransferase converted amphotericins A and B and 7-oxo-amphotericin B to disaccharide-modified formsin vivo. The enzyme did not act on amphotericin analogs lacking exocyclic carboxyl or mycosamine amino groups. Both NypY and PegA acted on candicidins. This work confirms the functions of these glycosyltransferases and provides insights into their acceptor substrate tolerance. Disaccharide-modified polyenes may have potential as less toxic antibiotics.