Disease control efficacy of 32,33-didehydroroflamycoin produced by Streptomyces rectiviolaceus strain DY46 against gray mold of tomato fruit.

Despite the efficacy of synthetic fungicides in controlling postharvest diseases, public concerns regarding chemical residues in food and an increase in drug-resistant strains of pathogens have led to a need for new agents to control postharvest diseases. The current study was performed to find control agents of microbial origin that are effective on gray mold of tomato fruits. We recently isolated Streptomyces rectiviolaceus DY46, which has antagonistic activity against various plant pathogenic fungi. The incidence of gray mold of tomato fruits was markedly reduced by 80.0% in tomatoes treated with the cell extract of Streptomyces rectiviolaceus DY46 compared with the control tomatoes. The active ingredient was purified from the cell extract of DY46 and identified to be 32,33-didehydroroflamycoin (DDHR). DDHR displayed MICs (minimal inhibitory concentrations) against the mycelial growth of various plant pathogenic fungi at concentrations of 8-64 mg L-1. The incidence of gray mold in tomato fruits inoculated with conidial suspension (104 conidia mL-1) of Botrytis cinerea was markedly reduced by 88.9% in tomatoes treated with DDHR (100 mg L-1) compared with the control. The DDHR residue in tomato fruit was significantly diminished 2 d after treatment. These results show that DDHR would be relatively safe for use as a postharvest fungicide.

Functional analysis of filipin tailoring genes from Streptomyces filipinensis reveals alternative routes in filipin III biosynthesis and yields bioactive derivatives.

BACKGROUND: 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. RESULTS: 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. CONCLUSIONS: 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.

Membrane activity of the pentaene macrolide didehydroroflamycoin in model lipid bilayers.

Didehydroroflamycoin (DDHR), a recently isolated member of the polyene macrolide family, was shown to have antibacterial and antifungal activity. However, its mechanism of action has not been investigated. Antibiotics from this family are amphiphilic; thus, they have membrane activity, their biological action is localized in the membrane, and the membrane composition and physical properties facilitate the recognition of a particular compound by the target organism. In this work, we use model lipid membranes comprised of giant unilamellar vesicles (GUVs) for a systematic study of the action of DDHR. In parallel, experiments are conducted using filipin III and amphotericin B, other members of the family, and the behavior observed for DDHR is described in the context of that of these two heavily studied compounds. The study shows that DDHR disrupts membranes via two different mechanisms and that the involvement of these mechanisms depends on the presence of cholesterol. The leakage assays performed in GUVs and the conductance measurements using black lipid membranes (BLM) reveal that the pores that develop in the absence of cholesterol are transient and their size is dependent on the DDHR concentration. In contrast, cholesterol promotes the formation of more defined structures that are temporally stable.