The Experts below are selected from a list of 138 Experts worldwide ranked by ideXlab platform
Jo-anne Chuck - One of the best experts on this subject based on the ideXlab platform.
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Community structure and antibiotic production of Streptomyces Nodosus bioreactors cultured in liquid environments
Microbial biotechnology, 2008Co-Authors: Tanya Pereira, Jasmina Nikodinovic, Chojin Nakazono, Gary R. Dennis, Kevin D. Barrow, Jo-anne ChuckAbstract:Immobilized bacteria are being assessed by industry for drug delivery, novel fermentation systems and the protection of organisms in harsh environments. Alginate bioreactors containing Streptomyces Nodosus were examined for community structure, cell viability and amphotericin production under different growth conditions. When cell proliferation was encouraged, substrate hyphae were found inside the alginate matrix and within multicellular projections on the surface of the capsule. The periphery of these projections had erect and branched hyphae, morphologically identical to aerial hyphae. Antibiotic production from immobilized organisms was assessed using conditioned culture medium to eliminate the emergence of a free‐dwelling population. These organisms sporulated with reduced antibiotic production compared with free‐dwelling cultures. The commitment to sporulate was independent of a surface but dependent on community size and nutritional status. This is the first report of the sporulation of S. Nodosus in liquid cultures and description of the multicellular community the organism adopts at a solid–liquid interface.
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High frequency transformation of the Amphotericin-producing bacterium Streptomyces Nodosus
Journal of Microbiological Methods, 2003Co-Authors: Jasmina Nikodinovic, Kevin D. Barrow, Jo-anne ChuckAbstract:This study has investigated DNA transformation in the Amphotericin-producing organism Streptomyces Nodosus. Amphotericin B is an antifungal drug with severe side effects in humans and the availability of structural variants would aid investigations into the mode of action and cytotoxity of the drug. Analogs of related polyketide drugs have been rapidly made by genetic engineering of biosynthetic genes; however, this requires the introduction of foreign DNA into the host. Protocols for protoplast formation and regeneration were established; however, preparations were recalcitrant to DNA uptake. Electroporation-mediated methodologies also were not successful. Intergeneric conjugal transfer of DNA from E. coli demonstrated transformation efficiencies of 5 x 10(-5) exconjugants generated per recipient. Use of DNA methylation-impaired E. coli donor strains resulted in 100-fold higher transformation efficiencies, indicating that DNA methylation recognition systems are operable in the organism. This methodology will enable genetic and biochemical analysis of the gene cluster responsible for making Amphotericin B.
Caffrey Patrick - One of the best experts on this subject based on the ideXlab platform.
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Analysis and manipulation of amphotericin biosynthetic genes by means of modified phage KC515 transduction techniques
Elsevier, 2019Co-Authors: Carmody Maria, Byrne Barry, Murphy Barry, Breen Ciaran, Lynch Susan, Flood Elizabeth, Finnan Shirley, Caffrey PatrickAbstract: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.European Unio
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Biosynthesis of Deoxyamphotericins and Deoxyamphoteronolides by Engineered Strains of Streptomyces Nodosus
Elsevier, 2019Co-Authors: Byrne Barry, Rawlings Bernard, Carmody Maria, Gibson Emma, Caffrey PatrickAbstract: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.European Unio
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Biosynthesis of less toxic amphotericins
'American Society for Biochemistry & Molecular Biology (ASBMB)', 2019Co-Authors: Carmody Maria, Rawlings Bernard, Byrne Barry, Murphy Barry, Power Patrick, Rai, Dilip K., Caffrey PatrickAbstract: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.Higher Education AuthorityEuropean Unio
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Engineered biosynthesis and characterisation of disaccharide-modified 8-deoxyamphoteronolides
'Springer Science and Business Media LLC', 2017Co-Authors: Walmsley Simon, De Poire Eimear, Rawlings Bernard, Caffrey PatrickAbstract: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.Science Foundation Irelan
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Enzymes Catalyzing Late Steps in 67-121C Biosynthesis
'Japan Society for Bioscience Biotechnology and Agrochemistry', 2017Co-Authors: Stephens Niamh, Rawlings Bernard, Caffrey PatrickAbstract: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.Science Foundation Irelan
Jasmina Nikodinovic - One of the best experts on this subject based on the ideXlab platform.
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Community structure and antibiotic production of Streptomyces Nodosus bioreactors cultured in liquid environments
Microbial biotechnology, 2008Co-Authors: Tanya Pereira, Jasmina Nikodinovic, Chojin Nakazono, Gary R. Dennis, Kevin D. Barrow, Jo-anne ChuckAbstract:Immobilized bacteria are being assessed by industry for drug delivery, novel fermentation systems and the protection of organisms in harsh environments. Alginate bioreactors containing Streptomyces Nodosus were examined for community structure, cell viability and amphotericin production under different growth conditions. When cell proliferation was encouraged, substrate hyphae were found inside the alginate matrix and within multicellular projections on the surface of the capsule. The periphery of these projections had erect and branched hyphae, morphologically identical to aerial hyphae. Antibiotic production from immobilized organisms was assessed using conditioned culture medium to eliminate the emergence of a free‐dwelling population. These organisms sporulated with reduced antibiotic production compared with free‐dwelling cultures. The commitment to sporulate was independent of a surface but dependent on community size and nutritional status. This is the first report of the sporulation of S. Nodosus in liquid cultures and description of the multicellular community the organism adopts at a solid–liquid interface.
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High frequency transformation of the Amphotericin-producing bacterium Streptomyces Nodosus
Journal of Microbiological Methods, 2003Co-Authors: Jasmina Nikodinovic, Kevin D. Barrow, Jo-anne ChuckAbstract:This study has investigated DNA transformation in the Amphotericin-producing organism Streptomyces Nodosus. Amphotericin B is an antifungal drug with severe side effects in humans and the availability of structural variants would aid investigations into the mode of action and cytotoxity of the drug. Analogs of related polyketide drugs have been rapidly made by genetic engineering of biosynthetic genes; however, this requires the introduction of foreign DNA into the host. Protocols for protoplast formation and regeneration were established; however, preparations were recalcitrant to DNA uptake. Electroporation-mediated methodologies also were not successful. Intergeneric conjugal transfer of DNA from E. coli demonstrated transformation efficiencies of 5 x 10(-5) exconjugants generated per recipient. Use of DNA methylation-impaired E. coli donor strains resulted in 100-fold higher transformation efficiencies, indicating that DNA methylation recognition systems are operable in the organism. This methodology will enable genetic and biochemical analysis of the gene cluster responsible for making Amphotericin B.
Kevin D. Barrow - One of the best experts on this subject based on the ideXlab platform.
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Community structure and antibiotic production of Streptomyces Nodosus bioreactors cultured in liquid environments
Microbial biotechnology, 2008Co-Authors: Tanya Pereira, Jasmina Nikodinovic, Chojin Nakazono, Gary R. Dennis, Kevin D. Barrow, Jo-anne ChuckAbstract:Immobilized bacteria are being assessed by industry for drug delivery, novel fermentation systems and the protection of organisms in harsh environments. Alginate bioreactors containing Streptomyces Nodosus were examined for community structure, cell viability and amphotericin production under different growth conditions. When cell proliferation was encouraged, substrate hyphae were found inside the alginate matrix and within multicellular projections on the surface of the capsule. The periphery of these projections had erect and branched hyphae, morphologically identical to aerial hyphae. Antibiotic production from immobilized organisms was assessed using conditioned culture medium to eliminate the emergence of a free‐dwelling population. These organisms sporulated with reduced antibiotic production compared with free‐dwelling cultures. The commitment to sporulate was independent of a surface but dependent on community size and nutritional status. This is the first report of the sporulation of S. Nodosus in liquid cultures and description of the multicellular community the organism adopts at a solid–liquid interface.
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High frequency transformation of the Amphotericin-producing bacterium Streptomyces Nodosus
Journal of Microbiological Methods, 2003Co-Authors: Jasmina Nikodinovic, Kevin D. Barrow, Jo-anne ChuckAbstract:This study has investigated DNA transformation in the Amphotericin-producing organism Streptomyces Nodosus. Amphotericin B is an antifungal drug with severe side effects in humans and the availability of structural variants would aid investigations into the mode of action and cytotoxity of the drug. Analogs of related polyketide drugs have been rapidly made by genetic engineering of biosynthetic genes; however, this requires the introduction of foreign DNA into the host. Protocols for protoplast formation and regeneration were established; however, preparations were recalcitrant to DNA uptake. Electroporation-mediated methodologies also were not successful. Intergeneric conjugal transfer of DNA from E. coli demonstrated transformation efficiencies of 5 x 10(-5) exconjugants generated per recipient. Use of DNA methylation-impaired E. coli donor strains resulted in 100-fold higher transformation efficiencies, indicating that DNA methylation recognition systems are operable in the organism. This methodology will enable genetic and biochemical analysis of the gene cluster responsible for making Amphotericin B.
Patrick Caffrey - One of the best experts on this subject based on the ideXlab platform.
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Redesign of Polyene Macrolide Glycosylation: Engineered Biosynthesis of 19-(O)-Perosaminyl-Amphoteronolide B
Chemistry & biology, 2010Co-Authors: Eve Hutchinson, Barry Murphy, Terence Dunne, Ciaran Breen, Bernard J. Rawlings, Patrick CaffreyAbstract: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-catalyzed conversion of GDP-mannose to GDP-4-keto-6-deoxymannose. This is converted to GDP-3-keto-6-deoxymannose, which is transaminated to GDP-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 synthesized 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 glycorandomized amphoteronolides for drug discovery.
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amphotericin biosynthesis in Streptomyces Nodosus deductions from analysis of polyketide synthase and late genes
Chemistry & Biology, 2001Co-Authors: Patrick Caffrey, Susan V Lynch, Elizabeth Flood, Shirley Finnan, Markiyan OliynykAbstract:Abstract Background: The polyene macrolide amphotericin B is produced by Streptomyces Nodosus ATCC14899. Amphotericin B is a potent antifungal antibiotic and has activity against some viruses, protozoans and prions. Treatment of systemic fungal infections with amphotericin B is complicated by its low water-solubility and side effects which include severe nephrotoxicity. Analogues with improved properties could be generated by manipulating amphotericin biosynthetic genes in S. Nodosus . Results: A large polyketide synthase gene cluster was cloned from total cellular DNA of S. Nodosus . Nucleotide sequence analysis of 113 193 bp of this region revealed six large polyketide synthase genes as well as genes for two cytochrome P450 enzymes, two ABC transporter proteins, and genes involved in biosynthesis and attachment of mycosamine. Phage KC515-mediated gene disruption was used to show that this region is involved in amphotericin production. Conclusions: The availability of these genes and the development of a method for gene disruption and replacement in S. Nodosus should allow production of novel amphotericins. A panel of analogues could lead to identification of derivatives with increased solubility, improved biological activity and reduced toxicity.