Now showing 1 - 6 of 6
  • Publication
    Biosynthesis of Deoxyamphotericins and Deoxyamphoteronolides by Engineered Strains of Streptomyces nodosus
    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.
  • Publication
    Engineered biosynthesis and characterisation of disaccharide-modified 8-deoxyamphoteronolides
    Several polyene macrolides are potent antifungal agents that have severe side effects. Increased glycosylation of these compounds can improve water solubility and reduce toxicity. Three extending glycosyltransferases are known to add hexoses to the mycosaminyl sugar residues of polyenes. The Actinoplanes caeruleus PegA enzyme catalyses attachment of a D-mannosyl residue in a β-1,4 linkage to the mycosamine of the aromatic heptaene 67-121A to form 67-121C. NppY from Pseudonocardia autotrophica adds an N-acetyl-D-glucosamine to the mycosamine of 10-deoxynystatin. NypY from Pseudonocardia sp. P1 adds an extra hexose to a nystatin, but the identity of the sugar is unknown. Here, we express the nypY gene in Streptomyces nodosus amphL and show that NypY modifies 8-deoxyamphotericins more efficiently than C-8 hydroxylated forms. The modified heptaene was purified and shown to be mannosyl-8-deoxyamphotericin B. This had the same antifungal activity as amphotericin B but was slightly less haemolytic. Chemical modification of this new disaccharide polyene could give better antifungal antibiotics.
      411Scopus© Citations 6
  • Publication
    Redesign of Polyene Macrolide Glycosylation: Engineered Biosynthesis of 19-(O)-Perosaminyl-Amphoteronolide B
    Most polyene macrolide antibiotics are glycosylated with mycosamine (3,6-dideoxy-3-aminomannose). In the amphotericin B producer, Streptomyces nodosus, mycosamine biosynthesis begins with AmphDIII-catalysed conversion of GDP-mannose to GDP-4-keto-6-deoxymannose. This is converted to GDP-3-keto-6-deoxymannose, which is transaminated to mycosamine by the AmphDII protein. The glycosyltransferase AmphDI transfers mycosamine to amphotericin aglycones (amphoteronolides). The aromatic heptaene perimycin is unusual among polyenes in that the sugar is perosamine (4,6-dideoxy-4-aminomannose), which is synthesised by direct transamination of GDP-4-keto-6-deoxymannose. Here we use the Streptomyces aminophilus perDII perosamine synthase and perDI perosaminyltransferase genes to engineer biosynthesis of perosaminyl-amphoteronolide B in S. nodosus. Efficient production required a hybrid glycosyltransferase containing an N-terminal region of AmphDI and a C-terminal region of PerDI. This work will assist efforts to generate glycorandomised amphoteronolides for drug discovery.
      323Scopus© Citations 36
  • Publication
    Streptomyces nodosus Host Strains Optimised for Glycosylation Engineering
    The AmphDI glycosyltransferase transfers a mycosaminyl sugar residue from GDP onto 8-deoxyamphoteronolide B, the aglycone of the antifungal amphotericin B. In this study the amphDI gene was inactivated in Streptomyces nodosus strains lacking the AmphN cytochrome P450. The new mutants produced 8-deoxy-16-methyl-16-descarboxyl amphoteronolides in high yield. These strains and aglycones should prove valuable for in vivo and in vitro glycosylation engineering.
      182Scopus© Citations 13
  • Publication
    Versatility of enzymes catalyzing late steps in polyene 67-121C biosynthesis
    (Taylor and Francis, 2013-05-22) ; ;
    Actinoplanes caeruleus produces 67-121C, a heptaene macrolide modified with a D-mannosyl-D-mycosaminyl disaccharide. Draft genome sequencing revealed genes encoding mycosaminyltransferase, mycosamine synthase, a cytochrome P450 that modifies the macrolactone core, and the extending mannosyltransferase. Only the mycosamine synthase and P450 were active in the biosynthesis of amphotericins in Streptomyces nodosus, the amphotericin producer.
      330Scopus© Citations 12
  • Publication
    Biosynthesis of amphotericin derivatives lacking exocyclic carboxyl groups
    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.
      239Scopus© Citations 93