Now showing 1 - 10 of 12
  • 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.
      267Scopus© Citations 13
  • Publication
    New insights into polyene macrolide biosynthesis in Couchioplanes caeruleus
    (Royal Society of Chemistry, 2017-05-01) ; ;
    Couchioplanes caeruleus DSM43634 synthesises 67–121C, an aromatic heptaene macrolide that contains a mannosyl-mycosaminyl disaccharide. An improved draft genome sequence was used to obtain the biosynthetic gene cluster for this antifungal. Bioinformatic analysis of the polyketide synthase indicated that extension modules 7 and 8 contain A-type ketoreductase and dehydratase domains. These modules are therefore predicted to form cis double bonds. The deduced stereostructure of the 67–121C macrolactone is identical to that experimentally determined for the partricin subgroup of aromatic heptaenes. Some of these polyenes are N-methylated on the aminoacetophenone moiety. The C. caeruleus AceS protein was shown to methylate 4-aminoacetophenone and esters of 4-aminobenzoate, but not 4-aminobenzoate. This suggests that the substrate specificity of AceS prevents it from interfering with folate biosynthesis. The methyltransferase should be valuable for chemoenzymatic alkylation of compounds that contain aminobenzoyl moieties.
      605Scopus© Citations 9
  • 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.
      481Scopus© Citations 36
  • Publication
    Polyene macrollide biosynthesis in streptomycetes and related bacteria: recent advances from genome sequencing and experimental studies
    The polyene macrolide group includes important antifungal drugs, to which resistance does not arise readily. Chemical and biological methods have been used in attempts to make polyene antibiotics with fewer toxic side effects. Genome sequencing of producer organisms is contributing to this endeavour, by providing access to new compounds and by enabling yield improvement for polyene analogues obtained by engineered biosynthesis. This recent work is also enhancing bioinformatic methods for deducing the structures of cryptic natural products from their biosynthetic enzymes. The stereostructure of candicidin D has recently been determined by NMR spectroscopy. Genes for the corresponding polyketide synthase have been uncovered in several different genomes. Analysis of this new information strengthens the view that protein sequence motifs can be used to predict double bond geometry in many polyketides. Chemical studies have shown that improved polyenes can be obtained by modifying the mycosamine sugar that is common to most of these compounds. Glycoengineered analogues might be produced by biosynthetic methods, but polyene glycosyltransferases show little tolerance for donors other than GDP-α-D-mycosamine. Genome sequencing has revealed extending glycosyltransferases that add a second sugar to the mycosamine of some polyenes. NppY of Pseudonocardia autotrophica uses UDP-N-acetyl-α-D-glucosamine as donor whereas PegA from Actinoplanes caeruleus uses GDP-α-D-mannose. These two enzymes show 51 % sequence identity and are also closely related to mycosaminyltransferases. These findings will assist attempts to construct glycosyltransferases that transfer alternative UDP- or (d)TDP-linked sugars to polyene macrolactones.
      639Scopus© Citations 34
  • Publication
    Analysis and manipulation of amphotericin biosynthetic genes by means of modified phage KC515 transduction techniques
    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.
      441Scopus© Citations 55
  • 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.
      354Scopus© Citations 101
  • 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.
      408Scopus© Citations 13
  • Publication
    Biosynthetic engineering of polyene macrolides for generation of improved antifungal and antiparasitic agents
    Polyene macrolides are potent antifungal agents that are also active against parasites, enveloped viruses and prion diseases. They are medically important as antifungal antibiotics but their therapeutic use is limited by serious side effects. In recent years there has been considerable progress in genetic analysis and manipulation of the streptomycetes that produce nystatin, amphotericin B, candicidin, pimaricin and rimocidin/CE-108-related polyenes. This has led to engineered biosynthesis of several new polyenes that are not easily obtained as semi-synthetic derivatives. This review summarises recent advances made since the subject was last reviewed in 2003. Polyene biosynthesis generally involves assembly and cyclisation of a polyketide chain, followed by oxidative modifications and glycosylation of the macrolactone ring. New derivatives have been obtained by engineering both early and late stages of polyene biosynthetic pathways. These compounds have allowed more detailed investigations of structure-activity relationships and some are likely to show improvements in therapeutic index. The biosynthetic approach is already yielding sufficient material for testing the toxicity and activity of new compounds, thus opening possibilities for discovery of leads for development of effective and safe antifungal and antiparasitic agents.
      553Scopus© Citations 68
  • Publication
    Exploiting the genome sequence of Streptomyces nodosus for enhanced antibiotic production
    The genome of the amphotericin producer Streptomyces nodosus was sequenced. A single scaffold of 7,714,110 bp was obtained. Biosynthetic genes were identified for several natural products including polyketides, peptides, siderophores and terpenes. The majority of these clusters specified known compounds. Most were silent or expressed at low levels and unlikely to compete with amphotericin production. Biosynthesis of a skyllamycin analogue was activated by introducing expression plasmids containing either a gene for a LuxR transcriptional regulator or genes for synthesis of the acyl moiety of the lipopeptide. In an attempt to boost amphotericin production, genes for acyl CoA carboxylases, a phosphopantetheinyl transferase and the AmphRIV transcriptional activator were overexpressed, and the effects on yields were investigated. This study provides the groundwork for metabolic engineering of S. nodosus strains to produce high yields of amphotericin analogues.
      785Scopus© Citations 21
  • Publication
    Phosphomannose isomerase and phosphomannomutase gene disruptions in Streptomyces nodosus: impact on amphotericin biosynthesis and implications for glycosylation engineering
    Streptomycetes synthesise several bioactive natural products that are modified with sugar residues derived from GDP-mannose. These include the antifungal polyenes, the antibacterial antibiotics hygromycin A and mannopeptimycins, and the anticancer agent bleomycin. Three enzymes function in biosynthesis of GDP-mannose from the glycolytic intermediate fructose 6-phosphate: phosphomannose isomerase (PMI), phosphomannomutase (PMM) and GDP-mannose pyrophosphorylase (GMPP). Synthesis of GDP-mannose from exogenous mannose requires hexokinase or phosphotransferase enzymes together with PMM and GMPP. In this study, a region containing genes for PMI, PMM and GMPP was cloned from Streptomyces nodosus, producer of the polyenes amphotericins A and B. Inactivation of the manA gene for PMI resulted in production of amphotericins and their aglycones, 8-deoxyamphoteronolides. A double mutant lacking the PMI and PMM genes produced 8-deoxyamphoteronolides in good yields along with trace levels of glycosylated amphotericins. With further genetic engineering these mutants may activate alternative hexoses as GDP-sugars for transfer to aglycones in vivo.
      626Scopus© Citations 32