Amphotericin B is a medically important antifungal antibiotic that is also active against human immunodeficiency virus, Leishmania parasites, and prion diseases. The therapeutic use of amphotericin B is restricted by severe side effects that can be moderated by liposomal formulation or structural alteration. Chemical modification has shown that suppression of charge on the exocyclic carboxyl group of amphotericin B substantially reduces toxicity. We report targeted deletions of the amphN cytochrome P450 gene from the chromosome of the amphotericin-producing bacterium Streptomyces nodosus. The mutant strains produced amphotericin analogues in which methyl groups replace the exocyclic carboxyl groups. These compounds retained antifungal activity and had reduced hemolytic activity.There are increasingly urgent requirements for new antibiotics to treat infectious disease (1). The need is especially acute with systemic fungal infections that are increasing in incidence and are often fatal (2). Of the few antifungals available at present, the most reliable is amphotericin B (compound 1 in Fig. 1), a polyene macrolide synthesized by Streptomyces nodosus (3). Amphotericin B disrupts ergosterol-containing fungal membranes and has a broad spectrum of activity. Whereas resistance to most non-polyene antifungals has appeared rapidly, the most prevalent fungal pathogens of humans have been slow to develop resistance to amphotericin B, even after more than three decades of clinical use (4). The advantages of amphotericin B are offset by toxicity that results from low water solubility and interactions with cholesterol in mammalian membranes. Toxicity has also prevented full exploitation of the antiviral, antiprion, and antiparasitic properties of amphotericin B (3). The adverse effects of the drug can be reduced by liposomal formulation, heat-induced superaggregation, or structural alteration (5). Chemical modification has shown that suppression of charge on the exocyclic carboxyl group reduces toxicity and improves antifungal specificity (6). Further improvements have been made by derivatization with additional sugars (7).Substantial reduction in amphotericin B toxicity would minimize adverse side effects on patients and also allow high dose treatment of infections by emerging fungal pathogens that are sensitive only to high concentrations of the drug. Non-toxic amphotericin analogues and formulations may also be useful in other therapeutic areas. Polyene antibiotics delay the onset of prion diseases in animal models (8). These effects are thought to result from interactions of polyenes with cholesterol-rich membrane microdomains that contain glycosylphosphatidylinositolanchored prion proteins (3, 9). Amphotericin B formulations are gaining favor for treatment of cutaneous and visceral leishmaniasis, particularly as resistance to other anti-Leishmania drugs continues to increase (10). Polyenes may also become important in treatment of viral infections. Polyenes interfere with the sterol-rich membranes of enveloped viruses suc...

Amphotericin B is an antifungal antibiotic produced by Streptomyces nodosus. During biosynthesis of amphotericin, the macrolactone core undergoes three modifications: oxidation of a methyl branch to a carboxyl group, mycosaminylation, and hydroxylation. Gene disruption was undertaken to block two of these modifications. Initial experiments targeted the amphDIII gene, which encodes a GDP-D-mannose 4,6-dehydratase involved in biosynthesis of mycosamine. Analysis of products by mass spectrometry and NMR indicated that the amphDIII mutant produced 8-deoxyamphoteronolides A and B. This suggests that glycosylation with mycosamine normally precedes C-8 hydroxylation and that formation of the exocyclic carboxyl group can occur prior to both these modifications. Inactivation of the amphL cytochrome P450 gene led to production of novel polyenes with masses appropriate for 8-deoxyamphotericins A and B. These compounds retained antifungal activity and may be useful new antibiotics.

Amphotericin B is a medically important antifungal antibiotic that is produced by Streptomyces nodosus. Genetic manipulation of this organism has led to production of the first amphotericin analogues by engineered biosynthesis. Here, these studies were extended by sequencing the chromosomal regions flanking the amphotericin polyketide synthase genes, and by refining the phage KC515 transduction method for disruption and replacement of S. nodosus genes. A hybrid vector was constructed from KC515 DNA and the Escherichia coli plasmid pACYC177. This vector replicated as a plasmid in E. coli and the purified DNA yielded phage plaques on Streptomyces lividans after polyethylene glycol (PEG)-mediated transfection of protoplasts. The left flank of the amphotericin gene cluster was found to include amphRI, RII, RIII and RIV genes that are similar to regulatory genes in other polyene biosynthetic gene clusters. One of these regulatory genes, amphRI, was found to have a homologue, amphRVI, located in the right flank at a distance of 127 kbp along the chromosome. However, disruption of amphRVI using the hybrid vector had no effect on the yield of amphotericin obtained from cultures grown on production medium. The hybrid vector was also used for precise deletion of the DNA coding for two modules of the AmphC polyketide synthase protein. Analysis by UV spectrophotometry revealed that the deletion mutant produced a novel pentaene, with reduced antifungal activity but apparently greater water-solubility than amphotericin B. This shows the potential for use of the new vector in engineering of this and other biosynthetic pathways in Streptomyces.