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Sergey B. Zotchev - One of the best experts on this subject based on the ideXlab platform.

  • new nystatin related antifungal polyene macrolides with altered polyol region generated via biosynthetic engineering of Streptomyces Noursei
    Applied and Environmental Microbiology, 2011
    Co-Authors: Trygve Brautaset, Håvard Sletta, Olga N. Sekurova, Trond E. Ellingsen, Kristin F Degnes, Ingrid Bakke, Olga Volokhan, Trygve Andreassen, Sergey B. Zotchev
    Abstract:

    Polyene macrolide antibiotics, including nystatin and amphotericin B, possess fungicidal activity and are being used as antifungal agents to treat both superficial and invasive fungal infections. Due to their toxicity, however, their clinical applications are relatively limited, and new-generation polyene macrolides with an improved therapeutic index are highly desirable. We subjected the polyol region of the heptaene nystatin analogue S44HP to biosynthetic engineering designed to remove and introduce hydroxyl groups in the C-9-C-10 region. This modification strategy involved inactivation of the P450 monooxygenase NysL and the dehydratase domain in module 15 (DH15) of the nystatin polyketide synthase. Subsequently, these modifications were combined with replacement of the exocyclic C-16 carboxyl with the methyl group through inactivation of the P450 monooxygenase NysN. Four new polyene macrolides with up to three chemical modifications were generated, produced at relatively high yields (up to 0.51 g/liter), purified, structurally characterized, and subjected to in vitro assays for antifungal and hemolytic activities. Introduction of a C-9 hydroxyl by DH15 inactivation also blocked NysL-catalyzed C-10 hydroxylation, and these modifications caused a drastic decrease in both antifungal and hemolytic activities of the resulting analogues. In contrast, single removal of the C-10 hydroxyl group by NysL inactivation had only a marginal effect on these activities. Results from the extended antifungal assays strongly suggested that the 9-hydroxy-10-deoxy S44HP analogues became fungistatic rather than fungicidal antibiotics.

  • analysis of the mycosamine biosynthesis and attachment genes in the nystatin biosynthetic gene cluster of Streptomyces Noursei atcc 11455
    Applied and Environmental Microbiology, 2007
    Co-Authors: Aina Nedal, Håvard Sletta, Sven Even F. Borgos, Olga N. Sekurova, Trond E. Ellingsen, Trygve Brautaset, Sergey B. Zotchev
    Abstract:

    The polyene macrolide antibiotic nystatin produced by Streptomyces Noursei contains a deoxyaminosugar mycosamine moiety attached to the C-19 carbon of the macrolactone ring through the beta-glycosidic bond. The nystatin biosynthetic gene cluster contains three genes, nysDI, nysDII, and nysDIII, encoding enzymes with presumed roles in mycosamine biosynthesis and attachment as glycosyltransferase, aminotransferase, and GDP-mannose dehydratase, respectively. In the present study, the functions of these three genes were analyzed. The recombinant NysDIII protein was expressed in Escherichia coli and purified, and its in vitro GDP-mannose dehydratase activity was demonstrated. The nysDI and nysDII genes were inactivated individually in S. Noursei, and analyses of the resulting mutants showed that both genes produced nystatinolide and 10-deoxynystatinolide as major products. Expression of the nysDI and nysDII genes in trans in the respective mutants partially restored nystatin biosynthesis in both cases, supporting the predicted roles of these two genes in mycosamine biosynthesis and attachment. Both antifungal and hemolytic activities of the purified nystatinolides were shown to be strongly reduced compared to those of nystatin, confirming the importance of the mycosamine moiety for the biological activity of nystatin.

  • characterization of the p450 monooxygenase nysl responsible for c 10 hydroxylation during biosynthesis of the polyene macrolide antibiotic nystatin in Streptomyces Noursei
    Applied and Environmental Microbiology, 2006
    Co-Authors: Olga Volokhan, Trond E. Ellingsen, Håvard Sletta, Sergey B. Zotchev
    Abstract:

    The nysL gene, encoding a putative P450 monooxygenase, was identified in the nystatin biosynthetic gene cluster of Streptomyces Noursei. Although it has been proposed that NysL is responsible for hydroxylation of the nystatin precursor, experimental evidence for this activity was lacking. The nysL gene was inactivated in S. Noursei by gene replacement, and the resulting mutant was shown to produce 10-deoxynystatin. Purification and an in vitro activity assay for 10-deoxynystatin demonstrated its antifungal activity being equal to that of nystatin. The NysL protein was expressed heterologously in Escherichia coli as a His-tagged protein and used in an enzyme assay with 10-deoxynystatin as a substrate. The results obtained clearly demonstrated that NysL is a hydroxylase responsible for the post-polyketide synthase modification of 10-deoxynystatin at position C-10. Kinetic studies with the purified recombinant enzyme allowed determination of Km and kcat and revealed no inhibition of recombinant NysL by either the substrate or the product. These studies open the possibility for in vitro evolution of NysL aimed at changing its specificity, thereby providing new opportunities for engineered biosynthesis of novel nystatin analogues hydroxylated at alternative positions of the macrolactone ring.

  • probing the structure function relationship of polyene macrolides engineered biosynthesis of soluble nystatin analogues
    Journal of Medicinal Chemistry, 2006
    Co-Authors: Sven Even F. Borgos, Håvard Sletta, Trond E. Ellingsen, Pascale Tsan, Jeanmarc Lancelin, Sergey B. Zotchev
    Abstract:

    Although polyene macrolides are efficient antifungal agents with fungicidal mode of action, their use in medical practice is problematic due to their low solubility and significant human toxicity. In an attempt to address the solubility problem, we have obtained two analogues of nystatin with hydroxy groups at positions C31 and C33 through manipulation of the nystatin polyketide synthase in the producing organism Streptomyces Noursei. Structures of the analogues were confirmed by nuclear magnetic resonance (NMR), and their solubility was found to be more than 2000 times higher than that of nystatin. However, both analogues were shown to have lost antifungal activity, implying that the integrity of the hydrophobic polyene region of the nystatin molecule is crucial for the fungicidal action. NMR data and computer modeling performed for the new analogues suggested conformational changes together with a significantly increased structural disorder, which may account for both increased solubility and the loss of activity.

  • Nystatin Biosynthesis and Transport: nysH and nysG Genes Encoding a Putative ABC Transporter System in Streptomyces Noursei ATCC 11455 Are Required for Efficient Conversion of 10-Deoxynystatin to Nystatin
    Antimicrobial agents and chemotherapy, 2005
    Co-Authors: Håvard Sletta, Sven Even F. Borgos, Per Bruheim, Olga N. Sekurova, Hans Grasdalen, Randi Aune, Trond E. Ellingsen, Sergey B. Zotchev
    Abstract:

    The genes nysH and nysG, encoding putative ABC-type transporter proteins, are located at the flank of the nystatin biosynthetic gene cluster in Streptomyces Noursei ATCC 11455. To assess the possible roles of these genes in nystatin biosynthesis, they were inactivated by gene replacements leading to in-frame deletions. Metabolite profile analysis of the nysH and nysG deletion mutants revealed that both of them synthesized nystatin at a reduced level and produced considerable amounts of a putative nystatin analogue. Liquid chromatography-mass spectrometry and nuclear magnetic resonance structural analyses of the latter metabolite confirmed its identity as 10-deoxynystatin, a nystatin precursor lacking a hydroxyl group at C-10. Washing experiments demonstrated that both nystatin and 10-deoxynystatin are transported out of cells, suggesting the existence of an alternative efflux system(s) for the transport of nystatin-related metabolites. This notion was further corroborated in experiments with the ATPase inhibitor sodium o-vanadate, which affected the production of nystatin and 10-deoxynystatin in the wild-type strain and transporter mutants in a different manner. The data obtained in this study suggest that the efflux of nystatin-related polyene macrolides occurs through several transporters and that the NysH-NysG efflux system provides conditions favorable for C-10 hydroxylation.

Håvard Sletta - One of the best experts on this subject based on the ideXlab platform.

  • new nystatin related antifungal polyene macrolides with altered polyol region generated via biosynthetic engineering of Streptomyces Noursei
    Applied and Environmental Microbiology, 2011
    Co-Authors: Trygve Brautaset, Håvard Sletta, Olga N. Sekurova, Trond E. Ellingsen, Kristin F Degnes, Ingrid Bakke, Olga Volokhan, Trygve Andreassen, Sergey B. Zotchev
    Abstract:

    Polyene macrolide antibiotics, including nystatin and amphotericin B, possess fungicidal activity and are being used as antifungal agents to treat both superficial and invasive fungal infections. Due to their toxicity, however, their clinical applications are relatively limited, and new-generation polyene macrolides with an improved therapeutic index are highly desirable. We subjected the polyol region of the heptaene nystatin analogue S44HP to biosynthetic engineering designed to remove and introduce hydroxyl groups in the C-9-C-10 region. This modification strategy involved inactivation of the P450 monooxygenase NysL and the dehydratase domain in module 15 (DH15) of the nystatin polyketide synthase. Subsequently, these modifications were combined with replacement of the exocyclic C-16 carboxyl with the methyl group through inactivation of the P450 monooxygenase NysN. Four new polyene macrolides with up to three chemical modifications were generated, produced at relatively high yields (up to 0.51 g/liter), purified, structurally characterized, and subjected to in vitro assays for antifungal and hemolytic activities. Introduction of a C-9 hydroxyl by DH15 inactivation also blocked NysL-catalyzed C-10 hydroxylation, and these modifications caused a drastic decrease in both antifungal and hemolytic activities of the resulting analogues. In contrast, single removal of the C-10 hydroxyl group by NysL inactivation had only a marginal effect on these activities. Results from the extended antifungal assays strongly suggested that the 9-hydroxy-10-deoxy S44HP analogues became fungistatic rather than fungicidal antibiotics.

  • improved antifungal polyene macrolides via engineering of the nystatin biosynthetic genes in Streptomyces Noursei
    Chemistry & Biology, 2008
    Co-Authors: Trygve Brautaset, Håvard Sletta, Sven Even F. Borgos, Olga N. Sekurova, Aina Nedal, Kristin F Degnes, Ingrid Bakke, Olga Volokhan, Ivan D Treshalin
    Abstract:

    Summary Seven polyene macrolides with alterations in the polyol region and exocyclic carboxy group were obtained via genetic engineering of the nystatin biosynthesis genes in Streptomyces Noursei . In vitro analyses of the compounds for antifungal and hemolytic activities indicated that combinations of several mutations caused additive improvements in their activity-toxicity properties. The two best analogs selected on the basis of in vitro data were tested for acute toxicity and antifungal activity in a mouse model. Both analogs were shown to be effective against disseminated candidosis, while being considerably less toxic than amphotericin B. To our knowledge, this is the first report on polyene macrolides with improved in vivo pharmacological properties obtained by genetic engineering. These results indicate that the engineered nystatin analogs can be further developed into antifungal drugs for human use.

  • analysis of the mycosamine biosynthesis and attachment genes in the nystatin biosynthetic gene cluster of Streptomyces Noursei atcc 11455
    Applied and Environmental Microbiology, 2007
    Co-Authors: Aina Nedal, Håvard Sletta, Sven Even F. Borgos, Olga N. Sekurova, Trond E. Ellingsen, Trygve Brautaset, Sergey B. Zotchev
    Abstract:

    The polyene macrolide antibiotic nystatin produced by Streptomyces Noursei contains a deoxyaminosugar mycosamine moiety attached to the C-19 carbon of the macrolactone ring through the beta-glycosidic bond. The nystatin biosynthetic gene cluster contains three genes, nysDI, nysDII, and nysDIII, encoding enzymes with presumed roles in mycosamine biosynthesis and attachment as glycosyltransferase, aminotransferase, and GDP-mannose dehydratase, respectively. In the present study, the functions of these three genes were analyzed. The recombinant NysDIII protein was expressed in Escherichia coli and purified, and its in vitro GDP-mannose dehydratase activity was demonstrated. The nysDI and nysDII genes were inactivated individually in S. Noursei, and analyses of the resulting mutants showed that both genes produced nystatinolide and 10-deoxynystatinolide as major products. Expression of the nysDI and nysDII genes in trans in the respective mutants partially restored nystatin biosynthesis in both cases, supporting the predicted roles of these two genes in mycosamine biosynthesis and attachment. Both antifungal and hemolytic activities of the purified nystatinolides were shown to be strongly reduced compared to those of nystatin, confirming the importance of the mycosamine moiety for the biological activity of nystatin.

  • characterization of the p450 monooxygenase nysl responsible for c 10 hydroxylation during biosynthesis of the polyene macrolide antibiotic nystatin in Streptomyces Noursei
    Applied and Environmental Microbiology, 2006
    Co-Authors: Olga Volokhan, Trond E. Ellingsen, Håvard Sletta, Sergey B. Zotchev
    Abstract:

    The nysL gene, encoding a putative P450 monooxygenase, was identified in the nystatin biosynthetic gene cluster of Streptomyces Noursei. Although it has been proposed that NysL is responsible for hydroxylation of the nystatin precursor, experimental evidence for this activity was lacking. The nysL gene was inactivated in S. Noursei by gene replacement, and the resulting mutant was shown to produce 10-deoxynystatin. Purification and an in vitro activity assay for 10-deoxynystatin demonstrated its antifungal activity being equal to that of nystatin. The NysL protein was expressed heterologously in Escherichia coli as a His-tagged protein and used in an enzyme assay with 10-deoxynystatin as a substrate. The results obtained clearly demonstrated that NysL is a hydroxylase responsible for the post-polyketide synthase modification of 10-deoxynystatin at position C-10. Kinetic studies with the purified recombinant enzyme allowed determination of Km and kcat and revealed no inhibition of recombinant NysL by either the substrate or the product. These studies open the possibility for in vitro evolution of NysL aimed at changing its specificity, thereby providing new opportunities for engineered biosynthesis of novel nystatin analogues hydroxylated at alternative positions of the macrolactone ring.

  • probing the structure function relationship of polyene macrolides engineered biosynthesis of soluble nystatin analogues
    Journal of Medicinal Chemistry, 2006
    Co-Authors: Sven Even F. Borgos, Håvard Sletta, Trond E. Ellingsen, Pascale Tsan, Jeanmarc Lancelin, Sergey B. Zotchev
    Abstract:

    Although polyene macrolides are efficient antifungal agents with fungicidal mode of action, their use in medical practice is problematic due to their low solubility and significant human toxicity. In an attempt to address the solubility problem, we have obtained two analogues of nystatin with hydroxy groups at positions C31 and C33 through manipulation of the nystatin polyketide synthase in the producing organism Streptomyces Noursei. Structures of the analogues were confirmed by nuclear magnetic resonance (NMR), and their solubility was found to be more than 2000 times higher than that of nystatin. However, both analogues were shown to have lost antifungal activity, implying that the integrity of the hydrophobic polyene region of the nystatin molecule is crucial for the fungicidal action. NMR data and computer modeling performed for the new analogues suggested conformational changes together with a significantly increased structural disorder, which may account for both increased solubility and the loss of activity.

Trond E. Ellingsen - One of the best experts on this subject based on the ideXlab platform.

  • new nystatin related antifungal polyene macrolides with altered polyol region generated via biosynthetic engineering of Streptomyces Noursei
    Applied and Environmental Microbiology, 2011
    Co-Authors: Trygve Brautaset, Håvard Sletta, Olga N. Sekurova, Trond E. Ellingsen, Kristin F Degnes, Ingrid Bakke, Olga Volokhan, Trygve Andreassen, Sergey B. Zotchev
    Abstract:

    Polyene macrolide antibiotics, including nystatin and amphotericin B, possess fungicidal activity and are being used as antifungal agents to treat both superficial and invasive fungal infections. Due to their toxicity, however, their clinical applications are relatively limited, and new-generation polyene macrolides with an improved therapeutic index are highly desirable. We subjected the polyol region of the heptaene nystatin analogue S44HP to biosynthetic engineering designed to remove and introduce hydroxyl groups in the C-9-C-10 region. This modification strategy involved inactivation of the P450 monooxygenase NysL and the dehydratase domain in module 15 (DH15) of the nystatin polyketide synthase. Subsequently, these modifications were combined with replacement of the exocyclic C-16 carboxyl with the methyl group through inactivation of the P450 monooxygenase NysN. Four new polyene macrolides with up to three chemical modifications were generated, produced at relatively high yields (up to 0.51 g/liter), purified, structurally characterized, and subjected to in vitro assays for antifungal and hemolytic activities. Introduction of a C-9 hydroxyl by DH15 inactivation also blocked NysL-catalyzed C-10 hydroxylation, and these modifications caused a drastic decrease in both antifungal and hemolytic activities of the resulting analogues. In contrast, single removal of the C-10 hydroxyl group by NysL inactivation had only a marginal effect on these activities. Results from the extended antifungal assays strongly suggested that the 9-hydroxy-10-deoxy S44HP analogues became fungistatic rather than fungicidal antibiotics.

  • analysis of the mycosamine biosynthesis and attachment genes in the nystatin biosynthetic gene cluster of Streptomyces Noursei atcc 11455
    Applied and Environmental Microbiology, 2007
    Co-Authors: Aina Nedal, Håvard Sletta, Sven Even F. Borgos, Olga N. Sekurova, Trond E. Ellingsen, Trygve Brautaset, Sergey B. Zotchev
    Abstract:

    The polyene macrolide antibiotic nystatin produced by Streptomyces Noursei contains a deoxyaminosugar mycosamine moiety attached to the C-19 carbon of the macrolactone ring through the beta-glycosidic bond. The nystatin biosynthetic gene cluster contains three genes, nysDI, nysDII, and nysDIII, encoding enzymes with presumed roles in mycosamine biosynthesis and attachment as glycosyltransferase, aminotransferase, and GDP-mannose dehydratase, respectively. In the present study, the functions of these three genes were analyzed. The recombinant NysDIII protein was expressed in Escherichia coli and purified, and its in vitro GDP-mannose dehydratase activity was demonstrated. The nysDI and nysDII genes were inactivated individually in S. Noursei, and analyses of the resulting mutants showed that both genes produced nystatinolide and 10-deoxynystatinolide as major products. Expression of the nysDI and nysDII genes in trans in the respective mutants partially restored nystatin biosynthesis in both cases, supporting the predicted roles of these two genes in mycosamine biosynthesis and attachment. Both antifungal and hemolytic activities of the purified nystatinolides were shown to be strongly reduced compared to those of nystatin, confirming the importance of the mycosamine moiety for the biological activity of nystatin.

  • characterization of the p450 monooxygenase nysl responsible for c 10 hydroxylation during biosynthesis of the polyene macrolide antibiotic nystatin in Streptomyces Noursei
    Applied and Environmental Microbiology, 2006
    Co-Authors: Olga Volokhan, Trond E. Ellingsen, Håvard Sletta, Sergey B. Zotchev
    Abstract:

    The nysL gene, encoding a putative P450 monooxygenase, was identified in the nystatin biosynthetic gene cluster of Streptomyces Noursei. Although it has been proposed that NysL is responsible for hydroxylation of the nystatin precursor, experimental evidence for this activity was lacking. The nysL gene was inactivated in S. Noursei by gene replacement, and the resulting mutant was shown to produce 10-deoxynystatin. Purification and an in vitro activity assay for 10-deoxynystatin demonstrated its antifungal activity being equal to that of nystatin. The NysL protein was expressed heterologously in Escherichia coli as a His-tagged protein and used in an enzyme assay with 10-deoxynystatin as a substrate. The results obtained clearly demonstrated that NysL is a hydroxylase responsible for the post-polyketide synthase modification of 10-deoxynystatin at position C-10. Kinetic studies with the purified recombinant enzyme allowed determination of Km and kcat and revealed no inhibition of recombinant NysL by either the substrate or the product. These studies open the possibility for in vitro evolution of NysL aimed at changing its specificity, thereby providing new opportunities for engineered biosynthesis of novel nystatin analogues hydroxylated at alternative positions of the macrolactone ring.

  • probing the structure function relationship of polyene macrolides engineered biosynthesis of soluble nystatin analogues
    Journal of Medicinal Chemistry, 2006
    Co-Authors: Sven Even F. Borgos, Håvard Sletta, Trond E. Ellingsen, Pascale Tsan, Jeanmarc Lancelin, Sergey B. Zotchev
    Abstract:

    Although polyene macrolides are efficient antifungal agents with fungicidal mode of action, their use in medical practice is problematic due to their low solubility and significant human toxicity. In an attempt to address the solubility problem, we have obtained two analogues of nystatin with hydroxy groups at positions C31 and C33 through manipulation of the nystatin polyketide synthase in the producing organism Streptomyces Noursei. Structures of the analogues were confirmed by nuclear magnetic resonance (NMR), and their solubility was found to be more than 2000 times higher than that of nystatin. However, both analogues were shown to have lost antifungal activity, implying that the integrity of the hydrophobic polyene region of the nystatin molecule is crucial for the fungicidal action. NMR data and computer modeling performed for the new analogues suggested conformational changes together with a significantly increased structural disorder, which may account for both increased solubility and the loss of activity.

  • Nystatin Biosynthesis and Transport: nysH and nysG Genes Encoding a Putative ABC Transporter System in Streptomyces Noursei ATCC 11455 Are Required for Efficient Conversion of 10-Deoxynystatin to Nystatin
    Antimicrobial agents and chemotherapy, 2005
    Co-Authors: Håvard Sletta, Sven Even F. Borgos, Per Bruheim, Olga N. Sekurova, Hans Grasdalen, Randi Aune, Trond E. Ellingsen, Sergey B. Zotchev
    Abstract:

    The genes nysH and nysG, encoding putative ABC-type transporter proteins, are located at the flank of the nystatin biosynthetic gene cluster in Streptomyces Noursei ATCC 11455. To assess the possible roles of these genes in nystatin biosynthesis, they were inactivated by gene replacements leading to in-frame deletions. Metabolite profile analysis of the nysH and nysG deletion mutants revealed that both of them synthesized nystatin at a reduced level and produced considerable amounts of a putative nystatin analogue. Liquid chromatography-mass spectrometry and nuclear magnetic resonance structural analyses of the latter metabolite confirmed its identity as 10-deoxynystatin, a nystatin precursor lacking a hydroxyl group at C-10. Washing experiments demonstrated that both nystatin and 10-deoxynystatin are transported out of cells, suggesting the existence of an alternative efflux system(s) for the transport of nystatin-related metabolites. This notion was further corroborated in experiments with the ATPase inhibitor sodium o-vanadate, which affected the production of nystatin and 10-deoxynystatin in the wild-type strain and transporter mutants in a different manner. The data obtained in this study suggest that the efflux of nystatin-related polyene macrolides occurs through several transporters and that the NysH-NysG efflux system provides conditions favorable for C-10 hydroxylation.

Olga N. Sekurova - One of the best experts on this subject based on the ideXlab platform.

  • new nystatin related antifungal polyene macrolides with altered polyol region generated via biosynthetic engineering of Streptomyces Noursei
    Applied and Environmental Microbiology, 2011
    Co-Authors: Trygve Brautaset, Håvard Sletta, Olga N. Sekurova, Trond E. Ellingsen, Kristin F Degnes, Ingrid Bakke, Olga Volokhan, Trygve Andreassen, Sergey B. Zotchev
    Abstract:

    Polyene macrolide antibiotics, including nystatin and amphotericin B, possess fungicidal activity and are being used as antifungal agents to treat both superficial and invasive fungal infections. Due to their toxicity, however, their clinical applications are relatively limited, and new-generation polyene macrolides with an improved therapeutic index are highly desirable. We subjected the polyol region of the heptaene nystatin analogue S44HP to biosynthetic engineering designed to remove and introduce hydroxyl groups in the C-9-C-10 region. This modification strategy involved inactivation of the P450 monooxygenase NysL and the dehydratase domain in module 15 (DH15) of the nystatin polyketide synthase. Subsequently, these modifications were combined with replacement of the exocyclic C-16 carboxyl with the methyl group through inactivation of the P450 monooxygenase NysN. Four new polyene macrolides with up to three chemical modifications were generated, produced at relatively high yields (up to 0.51 g/liter), purified, structurally characterized, and subjected to in vitro assays for antifungal and hemolytic activities. Introduction of a C-9 hydroxyl by DH15 inactivation also blocked NysL-catalyzed C-10 hydroxylation, and these modifications caused a drastic decrease in both antifungal and hemolytic activities of the resulting analogues. In contrast, single removal of the C-10 hydroxyl group by NysL inactivation had only a marginal effect on these activities. Results from the extended antifungal assays strongly suggested that the 9-hydroxy-10-deoxy S44HP analogues became fungistatic rather than fungicidal antibiotics.

  • improved antifungal polyene macrolides via engineering of the nystatin biosynthetic genes in Streptomyces Noursei
    Chemistry & Biology, 2008
    Co-Authors: Trygve Brautaset, Håvard Sletta, Sven Even F. Borgos, Olga N. Sekurova, Aina Nedal, Kristin F Degnes, Ingrid Bakke, Olga Volokhan, Ivan D Treshalin
    Abstract:

    Summary Seven polyene macrolides with alterations in the polyol region and exocyclic carboxy group were obtained via genetic engineering of the nystatin biosynthesis genes in Streptomyces Noursei . In vitro analyses of the compounds for antifungal and hemolytic activities indicated that combinations of several mutations caused additive improvements in their activity-toxicity properties. The two best analogs selected on the basis of in vitro data were tested for acute toxicity and antifungal activity in a mouse model. Both analogs were shown to be effective against disseminated candidosis, while being considerably less toxic than amphotericin B. To our knowledge, this is the first report on polyene macrolides with improved in vivo pharmacological properties obtained by genetic engineering. These results indicate that the engineered nystatin analogs can be further developed into antifungal drugs for human use.

  • analysis of the mycosamine biosynthesis and attachment genes in the nystatin biosynthetic gene cluster of Streptomyces Noursei atcc 11455
    Applied and Environmental Microbiology, 2007
    Co-Authors: Aina Nedal, Håvard Sletta, Sven Even F. Borgos, Olga N. Sekurova, Trond E. Ellingsen, Trygve Brautaset, Sergey B. Zotchev
    Abstract:

    The polyene macrolide antibiotic nystatin produced by Streptomyces Noursei contains a deoxyaminosugar mycosamine moiety attached to the C-19 carbon of the macrolactone ring through the beta-glycosidic bond. The nystatin biosynthetic gene cluster contains three genes, nysDI, nysDII, and nysDIII, encoding enzymes with presumed roles in mycosamine biosynthesis and attachment as glycosyltransferase, aminotransferase, and GDP-mannose dehydratase, respectively. In the present study, the functions of these three genes were analyzed. The recombinant NysDIII protein was expressed in Escherichia coli and purified, and its in vitro GDP-mannose dehydratase activity was demonstrated. The nysDI and nysDII genes were inactivated individually in S. Noursei, and analyses of the resulting mutants showed that both genes produced nystatinolide and 10-deoxynystatinolide as major products. Expression of the nysDI and nysDII genes in trans in the respective mutants partially restored nystatin biosynthesis in both cases, supporting the predicted roles of these two genes in mycosamine biosynthesis and attachment. Both antifungal and hemolytic activities of the purified nystatinolides were shown to be strongly reduced compared to those of nystatin, confirming the importance of the mycosamine moiety for the biological activity of nystatin.

  • Nystatin Biosynthesis and Transport: nysH and nysG Genes Encoding a Putative ABC Transporter System in Streptomyces Noursei ATCC 11455 Are Required for Efficient Conversion of 10-Deoxynystatin to Nystatin
    Antimicrobial agents and chemotherapy, 2005
    Co-Authors: Håvard Sletta, Sven Even F. Borgos, Per Bruheim, Olga N. Sekurova, Hans Grasdalen, Randi Aune, Trond E. Ellingsen, Sergey B. Zotchev
    Abstract:

    The genes nysH and nysG, encoding putative ABC-type transporter proteins, are located at the flank of the nystatin biosynthetic gene cluster in Streptomyces Noursei ATCC 11455. To assess the possible roles of these genes in nystatin biosynthesis, they were inactivated by gene replacements leading to in-frame deletions. Metabolite profile analysis of the nysH and nysG deletion mutants revealed that both of them synthesized nystatin at a reduced level and produced considerable amounts of a putative nystatin analogue. Liquid chromatography-mass spectrometry and nuclear magnetic resonance structural analyses of the latter metabolite confirmed its identity as 10-deoxynystatin, a nystatin precursor lacking a hydroxyl group at C-10. Washing experiments demonstrated that both nystatin and 10-deoxynystatin are transported out of cells, suggesting the existence of an alternative efflux system(s) for the transport of nystatin-related metabolites. This notion was further corroborated in experiments with the ATPase inhibitor sodium o-vanadate, which affected the production of nystatin and 10-deoxynystatin in the wild-type strain and transporter mutants in a different manner. The data obtained in this study suggest that the efflux of nystatin-related polyene macrolides occurs through several transporters and that the NysH-NysG efflux system provides conditions favorable for C-10 hydroxylation.

  • in vivo analysis of the regulatory genes in the nystatin biosynthetic gene cluster of Streptomyces Noursei atcc 11455 reveals their differential control over antibiotic biosynthesis
    Journal of Bacteriology, 2004
    Co-Authors: Olga N. Sekurova, Håvard Sletta, Sven Even F. Borgos, Trond E. Ellingsen, Trygve Brautaset, Oyvind M Jakobsen, Arne R Strom, Svein Valla, Sergey B. Zotchev
    Abstract:

    Six putative regulatory genes are located at the flank of the nystatin biosynthetic gene cluster in Streptomyces Noursei ATCC 11455. Gene inactivation and complementation experiments revealed that nysRI, nysRII, nysRIII, and nysRIV are necessary for efficient nystatin production, whereas no significant roles could be demonstrated for the other two regulatory genes. To determine the in vivo targets for the NysR regulators, chromosomal integration vectors with the xylE reporter gene under the control of seven putative promoter regions upstream of the nystatin structural and regulatory genes were constructed. Expression analyses of the resulting vectors in the S. Noursei wild-type strain and regulatory mutants revealed that the four regulators differentially affect certain promoters. According to these analyses, genes responsible for initiation of nystatin biosynthesis and antibiotic transport were the major targets for regulation. Data from cross-complementation experiments showed that nysR genes could in some cases substitute for each other, suggesting a functional hierarchy of the regulators and implying a cascade-like mechanism of regulation of nystatin biosynthesis.

Trygve Brautaset - One of the best experts on this subject based on the ideXlab platform.

  • new nystatin related antifungal polyene macrolides with altered polyol region generated via biosynthetic engineering of Streptomyces Noursei
    Applied and Environmental Microbiology, 2011
    Co-Authors: Trygve Brautaset, Håvard Sletta, Olga N. Sekurova, Trond E. Ellingsen, Kristin F Degnes, Ingrid Bakke, Olga Volokhan, Trygve Andreassen, Sergey B. Zotchev
    Abstract:

    Polyene macrolide antibiotics, including nystatin and amphotericin B, possess fungicidal activity and are being used as antifungal agents to treat both superficial and invasive fungal infections. Due to their toxicity, however, their clinical applications are relatively limited, and new-generation polyene macrolides with an improved therapeutic index are highly desirable. We subjected the polyol region of the heptaene nystatin analogue S44HP to biosynthetic engineering designed to remove and introduce hydroxyl groups in the C-9-C-10 region. This modification strategy involved inactivation of the P450 monooxygenase NysL and the dehydratase domain in module 15 (DH15) of the nystatin polyketide synthase. Subsequently, these modifications were combined with replacement of the exocyclic C-16 carboxyl with the methyl group through inactivation of the P450 monooxygenase NysN. Four new polyene macrolides with up to three chemical modifications were generated, produced at relatively high yields (up to 0.51 g/liter), purified, structurally characterized, and subjected to in vitro assays for antifungal and hemolytic activities. Introduction of a C-9 hydroxyl by DH15 inactivation also blocked NysL-catalyzed C-10 hydroxylation, and these modifications caused a drastic decrease in both antifungal and hemolytic activities of the resulting analogues. In contrast, single removal of the C-10 hydroxyl group by NysL inactivation had only a marginal effect on these activities. Results from the extended antifungal assays strongly suggested that the 9-hydroxy-10-deoxy S44HP analogues became fungistatic rather than fungicidal antibiotics.

  • improved antifungal polyene macrolides via engineering of the nystatin biosynthetic genes in Streptomyces Noursei
    Chemistry & Biology, 2008
    Co-Authors: Trygve Brautaset, Håvard Sletta, Sven Even F. Borgos, Olga N. Sekurova, Aina Nedal, Kristin F Degnes, Ingrid Bakke, Olga Volokhan, Ivan D Treshalin
    Abstract:

    Summary Seven polyene macrolides with alterations in the polyol region and exocyclic carboxy group were obtained via genetic engineering of the nystatin biosynthesis genes in Streptomyces Noursei . In vitro analyses of the compounds for antifungal and hemolytic activities indicated that combinations of several mutations caused additive improvements in their activity-toxicity properties. The two best analogs selected on the basis of in vitro data were tested for acute toxicity and antifungal activity in a mouse model. Both analogs were shown to be effective against disseminated candidosis, while being considerably less toxic than amphotericin B. To our knowledge, this is the first report on polyene macrolides with improved in vivo pharmacological properties obtained by genetic engineering. These results indicate that the engineered nystatin analogs can be further developed into antifungal drugs for human use.

  • analysis of the mycosamine biosynthesis and attachment genes in the nystatin biosynthetic gene cluster of Streptomyces Noursei atcc 11455
    Applied and Environmental Microbiology, 2007
    Co-Authors: Aina Nedal, Håvard Sletta, Sven Even F. Borgos, Olga N. Sekurova, Trond E. Ellingsen, Trygve Brautaset, Sergey B. Zotchev
    Abstract:

    The polyene macrolide antibiotic nystatin produced by Streptomyces Noursei contains a deoxyaminosugar mycosamine moiety attached to the C-19 carbon of the macrolactone ring through the beta-glycosidic bond. The nystatin biosynthetic gene cluster contains three genes, nysDI, nysDII, and nysDIII, encoding enzymes with presumed roles in mycosamine biosynthesis and attachment as glycosyltransferase, aminotransferase, and GDP-mannose dehydratase, respectively. In the present study, the functions of these three genes were analyzed. The recombinant NysDIII protein was expressed in Escherichia coli and purified, and its in vitro GDP-mannose dehydratase activity was demonstrated. The nysDI and nysDII genes were inactivated individually in S. Noursei, and analyses of the resulting mutants showed that both genes produced nystatinolide and 10-deoxynystatinolide as major products. Expression of the nysDI and nysDII genes in trans in the respective mutants partially restored nystatin biosynthesis in both cases, supporting the predicted roles of these two genes in mycosamine biosynthesis and attachment. Both antifungal and hemolytic activities of the purified nystatinolides were shown to be strongly reduced compared to those of nystatin, confirming the importance of the mycosamine moiety for the biological activity of nystatin.

  • in vivo analysis of the regulatory genes in the nystatin biosynthetic gene cluster of Streptomyces Noursei atcc 11455 reveals their differential control over antibiotic biosynthesis
    Journal of Bacteriology, 2004
    Co-Authors: Olga N. Sekurova, Håvard Sletta, Sven Even F. Borgos, Trond E. Ellingsen, Trygve Brautaset, Oyvind M Jakobsen, Arne R Strom, Svein Valla, Sergey B. Zotchev
    Abstract:

    Six putative regulatory genes are located at the flank of the nystatin biosynthetic gene cluster in Streptomyces Noursei ATCC 11455. Gene inactivation and complementation experiments revealed that nysRI, nysRII, nysRIII, and nysRIV are necessary for efficient nystatin production, whereas no significant roles could be demonstrated for the other two regulatory genes. To determine the in vivo targets for the NysR regulators, chromosomal integration vectors with the xylE reporter gene under the control of seven putative promoter regions upstream of the nystatin structural and regulatory genes were constructed. Expression analyses of the resulting vectors in the S. Noursei wild-type strain and regulatory mutants revealed that the four regulators differentially affect certain promoters. According to these analyses, genes responsible for initiation of nystatin biosynthesis and antibiotic transport were the major targets for regulation. Data from cross-complementation experiments showed that nysR genes could in some cases substitute for each other, suggesting a functional hierarchy of the regulators and implying a cascade-like mechanism of regulation of nystatin biosynthesis.

  • biosynthesis of the polyene antifungal antibiotic nystatin in Streptomyces Noursei atcc 11455 analysis of the gene cluster and deduction of the biosynthetic pathway
    Chemistry & Biology, 2000
    Co-Authors: Trygve Brautaset, Håvard Sletta, Olga N. Sekurova, Trond E. Ellingsen, Arne R Strom, Svein Valla, Sergey B. Zotchev
    Abstract:

    Abstract Background: The polyene macrolide antibiotic nystatin produced by Streptomyces Noursei ATCC 11455 is an important antifungal agent. The nystatin molecule contains a polyketide moiety represented by a 38-membered macrolactone ring to which the deoxysugar mycosamine is attached. Molecular cloning and characterization of the genes governing the nystatin biosynthesis is of considerable interest because this information can be used for the generation of new antifungal antibiotics. Results: A DNA region of 123,580 base pairs from the S. Noursei ATCC 11455 genome was isolated, sequenced and shown by gene disruption to be involved in nystatin biosynthesis. Analysis of the DNA sequence resulted in identification of six genes encoding a modular polyketide synthase (PKS), genes for thioesterase, deoxysugar biosynthesis, modification, transport and regulatory proteins. One of the PKS-encoding genes, nysC , was found to encode the largest (11,096 amino acids long) modular PKS described to date. Analysis of the deduced gene products allowed us to propose a model for the nystatin biosynthetic pathway in S. Noursei . Conclusions: A complete set of genes responsible for the biosynthesis of the antifungal polyene antibiotic nystatin in S. Noursei ATCC 11455 has been cloned and analyzed. This represents the first example of the complete DNA sequence analysis of a polyene antibiotic biosynthetic gene cluster. Manipulation of the genes identified within the cluster may potentially lead to the generation of novel polyketides and yield improvements in the production strains.