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Thomas C. Sparks - One of the best experts on this subject based on the ideXlab platform.
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The Spinosyns, spinosad, spinetoram, and synthetic Spinosyn mimics - discovery, exploration, and evolution of a natural product chemistry and the impact of computational tools.
Pest management science, 2020Co-Authors: Thomas C. Sparks, Gary D. Crouse, Zoltan L. Benko, Demeter David A, Natalie C. Giampietro, William Thomas Lambert, Brown AnnetteAbstract:Natural products (NPs) have long been a source of insecticidal crop protection products. Like many macrolide NPs, the Spinosyns originated from a soil inhibiting microorganism (Saccharopolyspora spinosa). More than 20 years after initial registration, the Spinosyns remain a unique class of NP-based insect control products that presently encompass two insecticidal active ingredients, spinosad, a naturally occurring mixture of Spinosyns, and spinetoram, a semi-synthetic Spinosyn product. The exploration and exploitation of the Spinosyns has, unusually, been tied to an array of computational tools including artificial intelligence-based quantitative structure activity relationship (QSAR) and most recently computer-aided modeling and design (CAMD). The AI-based QSAR directly lead to the discovery of spinetoram, while the CAMD studies have recently resulted in the discovery and building of a series of synthetic Spinosyn mimics. The most recent of these synthetic Spinosyn mimics show promise as insecticides targeting lepidopteran insect pests as demonstrated by field studies wherein the efficacy has been shown to be comparable to spinosad and spinetoram. These and a range of other aspects related to the exploration of the Spinosyns over the past 30 years are reviewed herein. This article is protected by copyright. All rights reserved.
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De Novo Design of Potent, Insecticidal Synthetic Mimics of the Spinosyn Macrolide Natural Products.
Scientific reports, 2018Co-Authors: Gary D. Crouse, Demeter David A, Geno Samaritoni, Mcleod Casandra Lee, Thomas C. SparksAbstract:New insect pest control agents are needed to meet the demands to feed an expanding global population, to address the desire for more environmentally-friendly insecticide tools, and to fill the loss of control options in some crop-pest complexes due to development of insecticide resistance. The Spinosyns are a highly effective class of naturally occurring, fermentation derived insecticides, possessing a very favorable environmental profile. Chemically, the Spinosyns are composed of a large complex macrolide tetracycle coupled to two sugars. As a means to further exploit this novel class of natural product-based insecticides, molecular modeling studies coupled with bioactivity-directed chemical modifications were used to define a less complex, synthetically accessible replacement for the Spinosyn tetracycle. These studies lead to the discovery of highly insecticidal analogs, possessing a simple tri-aryl ring system as a replacement for the complex macrolide tetracycle.
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Synthesis and Insecticidal Activity of Spinosyns with C9-O-Benzyl Bioisosteres in Place of the 2',3',4'-Tri-O-methyl Rhamnose.
Journal of agricultural and food chemistry, 2015Co-Authors: M. Paige Oliver, Demeter David A, Gary D. Crouse, Thomas C. SparksAbstract:The Spinosyns are fermentation-derived natural products active against a wide range of insect pests. They are structurally complex, consisting of two sugars (forosamine and rhamnose) coupled to a macrocyclic tetracycle. Removal of the rhamnose sugar results in a >100-fold reduction in insecticidal activity. C9-O-benzyl analogues of Spinosyn D were synthesized to determine if the 2′,3′,4′-tri-O-methyl rhamnose moiety could be replaced with a simpler, synthetic bioisostere. Insecticidal activity was evaluated against larvae of Spodoptera exigua (beet armyworm) and Helicoverpa zea (corn earworm). Whereas most analogues were far less active than Spinosyn D, a few of the C9-O-benzyl analogues, such as 4-CN, 4-Cl, 2-isopropyl, and 3,5-diOMe, were within 3–15 times the activity of Spinosyn D for larvae of S. exigua and H. zea. Thus, although not yet quite as effective, synthetic bioisosteres can substitute for the naturally occurring 2′,3′,4′-tri-O-methyl rhamnose moiety.
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Synthesis and Insecticidal Activity of Spinosyns with C9‑O‑Benzyl Bioisosteres in Place of the 2′,3′,4′-Tri‑O‑methyl Rhamnose
2015Co-Authors: Paige M. Oliver, Gary D. Crouse, David A. Demeter, Thomas C. SparksAbstract:The Spinosyns are fermentation-derived natural products active against a wide range of insect pests. They are structurally complex, consisting of two sugars (forosamine and rhamnose) coupled to a macrocyclic tetracycle. Removal of the rhamnose sugar results in a >100-fold reduction in insecticidal activity. C9-O-benzyl analogues of Spinosyn D were synthesized to determine if the 2′,3′,4′-tri-O-methyl rhamnose moiety could be replaced with a simpler, synthetic bioisostere. Insecticidal activity was evaluated against larvae of Spodoptera exigua (beet armyworm) and Helicoverpa zea (corn earworm). Whereas most analogues were far less active than Spinosyn D, a few of the C9-O-benzyl analogues, such as 4-CN, 4-Cl, 2-isopropyl, and 3,5-diOMe, were within 3–15 times the activity of Spinosyn D for larvae of S. exigua and H. zea. Thus, although not yet quite as effective, synthetic bioisosteres can substitute for the naturally occurring 2′,3′,4′-tri-O-methyl rhamnose moiety
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Nicotinic Acetylcholine Receptors as Spinosyn Targets for Insect Pest Management
Advances in Insect Physiology, 2013Co-Authors: Chaoxian Geng, Gerald B. Watson, Thomas C. SparksAbstract:Abstract The Spinosyns are insecticidal natural products originated from Saccharopolyspora spinosa. Spinosad, the first commercial product, is a mixture of two naturally occurring Spinosyns (A & D). A second product, spinetoram, is a mixture composed of two synthetically modified Spinosyns that, compared to spinosad, provides improved insecticidal potency and a broader pest insect spectrum. The Spinosyns act on a subgroup of insect nicotinic acetylcholine receptors (nAChR), which are distinct from those targeted by the neonicotinoid insecticides. Selection of Drosophila for resistance to spinosad helped pinpoint the Dα6 as the nAChR subunit involved in the insecticidal action of the Spinosyns. Cases of both target-site- and metabolism-based Spinosyn resistance in insect pests in the field have been reported, and programmes have been developed to manage Spinosyn resistance. This review highlights the discovery of the Spinosyns, elucidation of the Spinosyn target site, Spinosyn–receptor interaction, and progress in Spinosyn resistance management.
Richard H Baltz - One of the best experts on this subject based on the ideXlab platform.
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genes for the biosynthesis of Spinosyns applications for yield improvement in saccharopolyspora spinosa
Journal of Industrial Microbiology & Biotechnology, 2001Co-Authors: Krishnamurthy Madduri, M C Broughton, Clive Waldron, Donald J Merlo, Patti Matsushima, Kathryn P Crawford, Richard H BaltzAbstract:Spinosyns A and D are the active ingredients in an insect control agent produced by fermentation of Saccharopolyspora spinosa. Spinosyns are macrolides with a 21-carbon, tetracyclic lactone backbone to which the deoxysugars forosamine and tri-O-methylrhamnose are attached. The Spinosyn biosynthesis genes, except for the rhamnose genes, are located in a cluster that spans 74 kb of the S. spinosa genome. DNA sequence analysis, targeted gene disruptions and bioconversion studies identified five large genes encoding type I polyketide synthase subunits, and 14 genes involved in sugar biosynthesis, sugar attachment to the polyketide or cross-bridging of the polyketide. Four rhamnose biosynthetic genes, two of which are also necessary for forosamine biosynthesis, are located outside the Spinosyn gene cluster. Duplication of the Spinosyn genes linked to the polyketide synthase genes stimulated the final step in the biosynthesis — the conversion of the forosamine-less pseudoaglycones to endproducts. Duplication of genes involved in the early steps of deoxysugar biosynthesis increased Spinosyn yield significantly. Journal of Industrial Microbiology & Biotechnology (2001) 27, 399–402.
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Genes for the biosynthesis of Spinosyns: applications for yield improvement in Saccharopolyspora spinosa
Journal of industrial microbiology & biotechnology, 2001Co-Authors: Krishnamurthy Madduri, M C Broughton, Clive Waldron, Donald J Merlo, Patti Matsushima, Kathryn P Crawford, Richard H BaltzAbstract:Spinosyns A and D are the active ingredients in an insect control agent produced by fermentation of Saccharopolyspora spinosa. Spinosyns are macrolides with a 21-carbon, tetracyclic lactone backbone to which the deoxysugars forosamine and tri-O-methylrhamnose are attached. The Spinosyn biosynthesis genes, except for the rhamnose genes, are located in a cluster that spans 74 kb of the S. spinosa genome. DNA sequence analysis, targeted gene disruptions and bioconversion studies identified five large genes encoding type I polyketide synthase subunits, and 14 genes involved in sugar biosynthesis, sugar attachment to the polyketide or cross-bridging of the polyketide. Four rhamnose biosynthetic genes, two of which are also necessary for forosamine biosynthesis, are located outside the Spinosyn gene cluster. Duplication of the Spinosyn genes linked to the polyketide synthase genes stimulated the final step in the biosynthesis--the conversion of the forosamine-less pseudoaglycones to endproducts. Duplication of genes involved in the early steps of deoxysugar biosynthesis increased Spinosyn yield significantly.
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cloning and analysis of the spinosad biosynthetic gene cluster of saccharopolyspora spinosa
Chemistry & Biology, 2001Co-Authors: Clive Waldron, M C Broughton, Krishnamurthy Madduri, Donald J Merlo, Patti Matsushima, Kathryn P Crawford, Jan R Turner, Paul Robert Rosteck, Richard H BaltzAbstract:Abstract Background: Spinosad is a mixture of novel macrolide secondary metabolites produced by Saccharopolyspora spinosa . It is used in agriculture as a potent insect control agent with exceptional safety to non-target organisms. The cloning of the Spinosyn biosynthetic gene cluster provides the starting materials for the molecular genetic manipulation of spinosad yields, and for the production of novel derivatives containing alterations in the polyketide core or in the attached sugars. Results: We cloned the spinosad biosynthetic genes by molecular probing, complementation of blocked mutants, and cosmid walking, and sequenced an 80 kb region. We carried out gene disruptions of some of the genes and analyzed the mutants for product formation and for the bioconversion of intermediates in the Spinosyn pathway. The Spinosyn gene cluster contains five large open reading frames that encode a multifunctional, multi-subunit type I polyketide synthase (PKS). The PKS cluster is flanked on one side by genes involved in the biosynthesis of the amino sugar forosamine, in O -methylations of rhamnose, in sugar attachment to the polyketide, and in polyketide cross-bridging. Genes involved in the early common steps in the biosynthesis of forosamine and rhamnose, and genes dedicated to rhamnose biosynthesis, were not located in the 80 kb cluster. Conclusions: Most of the S. spinosa genes involved in Spinosyn biosynthesis are found in one 74 kb cluster, though it does not contain all of the genes required for the essential deoxysugars. Characterization of the clustered genes suggests that the Spinosyns are synthesized largely by mechanisms similar to those used to assemble complex macrolides in other actinomycetes. However, there are several unusual genes in the Spinosyn cluster that could encode enzymes that generate the most striking structural feature of these compounds, a tetracyclic polyketide aglycone nucleus.
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A cluster of genes for the biosynthesis of Spinosyns, novel macrolide insect control agents produced by Saccharopolyspora spinosa.
Antonie van Leeuwenhoek, 2000Co-Authors: Clive Waldron, M C Broughton, Krishnamurthy Madduri, Donald J Merlo, K Crawford, P Treadway, Richard H BaltzAbstract:Spinosyns A and D are the active ingredients in a family of insect control agents produced by fermentation of Saccharopolyspora spinosa. Spinosyns are 21-carbon tetracyclic lactones to which are attached two deoxysugars. Most of the genes involved in Spinosyn biosynthesis are clustered in an 74 kb region of the S. spinosa genome. This region has been characterized by DNA sequence analysis and by targeted gene disruptions. The Spinosyn biosynthetic gene cluster contains five large genes encoding a type I polyketide synthase, and 14 genes involved in modification of the macrolactone, or in the synthesis, modification and attachment of the deoxysugars. Four genes required for rhamnose biosynthesis (two of which are also required for forosamine biosynthesis) are not present in the cluster. A pathway for the biosynthesis of Spinosyns is proposed.
Hungwen Liu - One of the best experts on this subject based on the ideXlab platform.
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Structural Studies of the Spinosyn Forosaminyltransferase, SpnP
2015Co-Authors: Eta A. Isiorho, Hungwen Liu, Byung Sun Jeon, Namho Kim, Adrian T. Keatinge-clayAbstract:Spinosyns A and D (spinosad) are complex polyketide natural products biosynthesized through the cooperation of a modular polyketide synthase and several tailoring enzymes. SpnP catalyzes the final tailoring step, transferring forosamine from a TDP-d-forosamine donor substrate to a Spinosyn pseudoaglycone acceptor substrate. Sequence analysis indicated that SpnP belongs to a small group of glycosyltransferases (GTs) that require an auxiliary protein for activation. However, unlike other GTs in this subgroup, no putative auxiliary protein gene could be located in the biosynthetic gene cluster. To learn more about SpnP, the structures of SpnP and its complex with TDP were determined to 2.50 and 3.15 Å resolution, respectively. Binding of TDP causes the reordering of several residues in the donor substrate pocket. SpnP possesses a structural feature that has only been previously observed in the related glycosyltransferase EryCIII, in which it mediates association with the auxiliary protein EryCII. This motif, H-X-R-X5-D-X5-R-X12–20-D-P-X3-W-L-X12–18-E-X4-G, may be predictive of glycosyltransferases that interact with an auxiliary protein. A reverse glycosyl transfer assay demonstrated that SpnP possesses measurable activity in the absence of an auxiliary protein. Our data suggest that SpnP can bind its donor substrate by itself but that the glycosyl transfer reaction is facilitated by an auxiliary protein that aids in the correct folding of a flexible loop surrounding the pseudoaglycone acceptor substrate-binding pocket
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Structural Studies of the Spinosyn Forosaminyltransferase, SpnP
Biochemistry, 2014Co-Authors: Eta A. Isiorho, Hungwen Liu, Byung Sun Jeon, Namho Kim, Adrian T. Keatinge-clayAbstract:Spinosyns A and D (spinosad) are complex polyketide natural products biosynthesized through the cooperation of a modular polyketide synthase and several tailoring enzymes. SpnP catalyzes the final tailoring step, transferring forosamine from a TDP-d-forosamine donor substrate to a Spinosyn pseudoaglycone acceptor substrate. Sequence analysis indicated that SpnP belongs to a small group of glycosyltransferases (GTs) that require an auxiliary protein for activation. However, unlike other GTs in this subgroup, no putative auxiliary protein gene could be located in the biosynthetic gene cluster. To learn more about SpnP, the structures of SpnP and its complex with TDP were determined to 2.50 and 3.15 A resolution, respectively. Binding of TDP causes the reordering of several residues in the donor substrate pocket. SpnP possesses a structural feature that has only been previously observed in the related glycosyltransferase EryCIII, in which it mediates association with the auxiliary protein EryCII. This motif,...
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Structural studies of the Spinosyn rhamnosyltransferase, SpnG.
Biochemistry, 2012Co-Authors: Eta A. Isiorho, Hungwen Liu, Adrian T. Keatinge-clayAbstract:Spinosyns A and D (spinosad), like many other complex polyketides, are tailored near the end of their biosyntheses through the addition of sugars. SpnG, which catalyzes their 9-OH rhamnosylation, is also capable of adding other monosaccharides to the Spinosyn aglycone (AGL) from TDP-sugars; however, the substitution of UDP-d-glucose for TDP-d-glucose as the donor substrate is known to result in a >60000-fold reduction in kcat. Here, we report the structure of SpnG at 1.65 A resolution, SpnG bound to TDP at 1.86 A resolution, and SpnG bound to AGL at 1.70 A resolution. The SpnG–TDP complex reveals how SpnG employs N202 to discriminate between TDP- and UDP-sugars. A conformational change of several residues in the active site is promoted by the binding of TDP. The SpnG–AGL complex shows that the binding of AGL is mediated via hydrophobic interactions and that H13, the potential catalytic base, is within 3 A of the nucleophilic 9-OH group of AGL. A model for the Michaelis complex was constructed to reveal th...
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Structural Studies of the Spinosyn Rhamnosyltransferase, SpnG
2012Co-Authors: Eta A. Isiorho, Hungwen Liu, Adrian T. Keatinge-clayAbstract:Spinosyns A and D (spinosad), like many other complex polyketides, are tailored near the end of their biosyntheses through the addition of sugars. SpnG, which catalyzes their 9-OH rhamnosylation, is also capable of adding other monosaccharides to the Spinosyn aglycone (AGL) from TDP-sugars; however, the substitution of UDP-d-glucose for TDP-d-glucose as the donor substrate is known to result in a >60000-fold reduction in kcat. Here, we report the structure of SpnG at 1.65 Å resolution, SpnG bound to TDP at 1.86 Å resolution, and SpnG bound to AGL at 1.70 Å resolution. The SpnG–TDP complex reveals how SpnG employs N202 to discriminate between TDP- and UDP-sugars. A conformational change of several residues in the active site is promoted by the binding of TDP. The SpnG–AGL complex shows that the binding of AGL is mediated via hydrophobic interactions and that H13, the potential catalytic base, is within 3 Å of the nucleophilic 9-OH group of AGL. A model for the Michaelis complex was constructed to reveal the features that allow SpnG to transfer diverse sugars; it also revealed that the rhamnosyl moiety is in a skew-boat conformation during the transfer reaction
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enzyme catalysed 4 2 cycloaddition is a key step in the biosynthesis of Spinosyn a
Nature, 2011Co-Authors: Hak Joong Kim, Mark W Ruszczycky, Sei Hyun Choi, Yung Nan Liu, Hungwen LiuAbstract:The Diels-Alder reaction is a [4+2] cycloaddition reaction in which a cyclohexene ring is formed between a 1,3-diene and an electron-deficient alkene via a single pericyclic transition state. This reaction has been proposed as a key transformation in the biosynthesis of many cyclohexene-containing secondary metabolites. However, only four purified enzymes have thus far been implicated in biotransformations that are consistent with a Diels-Alder reaction, namely solanapyrone synthase, LovB, macrophomate synthase, and riboflavin synthase. Although the stereochemical outcomes of these reactions indicate that the product formation could be enzyme-guided in each case, these enzymes typically demonstrate more than one catalytic activity, leaving their specific influence on the cycloaddition step uncertain. In our studies of the biosynthesis of Spinosyn A, a tetracyclic polyketide-derived insecticide from Saccharopolyspora spinosa, we identified a cyclase, SpnF, that catalyses a transannular [4+2] cycloaddition to form the cyclohexene ring in Spinosyn A. Kinetic analysis demonstrates that SpnF specifically accelerates the ring formation reaction with an estimated 500-fold rate enhancement. A second enzyme, SpnL, was also identified as responsible for the final cross-bridging step that completes the tetracyclic core of Spinosyn A in a manner consistent with a Rauhut-Currier reaction. This work is significant because SpnF represents the first example characterized in vitro of a stand-alone enzyme solely committed to the catalysis of a [4+2] cycloaddition reaction. In addition, the mode of formation of the complex perhydro-as-indacene moiety in Spinosyn A is now fully established.
Clive Waldron - One of the best experts on this subject based on the ideXlab platform.
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Butenyl-Spinosyns, a natural example of genetic engineering of antibiotic biosynthetic genes
Journal of Industrial Microbiology and Biotechnology, 2006Co-Authors: Donald R. Hahn, Clive Waldron, Gary Gustafson, Brian Bullard, James D. Jackson, Jon MitchellAbstract:Spinosyns, a novel class of insect active macrolides produced by Saccharopolyspora spinosa , are used for insect control in a number of commercial crops. Recently, a new class of Spinosyns was discovered from S. pogona NRRL 30141. The butenyl-Spinosyns, also called pogonins, are very similar to Spinosyns, differing in the length of the side chain at C-21 and in the variety of novel minor factors. The butenyl-Spinosyn biosynthetic genes ( bus ) were cloned on four cosmids covering a contiguous 110-kb region of the NRRL 30141 chromosome. Their function in butenyl-Spinosyn biosynthesis was confirmed by a loss-of-function deletion, and subsequent complementation by cloned genes. The coding sequences of the butenyl-Spinosyn biosynthetic genes and the Spinosyn biosynthetic genes from S. spinosa were highly conserved. In particular, the PKS-coding genes from S. spinosa and S. pogona have 91–94% nucleic acid identity, with one notable exception. The butenyl-Spinosyn gene sequence codes for one additional PKS module, which is responsible for the additional two carbons in the C-21 tail. The DNA sequence of Spinosyn genes in this region suggested that the S. spinosa spnA gene could have been the result of an in-frame deletion of the S. pogona busA gene. Therefore, the butenyl-Spinosyn genes represent the putative parental gene structure that was naturally engineered by deletion to create the Spinosyn genes.
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genes for the biosynthesis of Spinosyns applications for yield improvement in saccharopolyspora spinosa
Journal of Industrial Microbiology & Biotechnology, 2001Co-Authors: Krishnamurthy Madduri, M C Broughton, Clive Waldron, Donald J Merlo, Patti Matsushima, Kathryn P Crawford, Richard H BaltzAbstract:Spinosyns A and D are the active ingredients in an insect control agent produced by fermentation of Saccharopolyspora spinosa. Spinosyns are macrolides with a 21-carbon, tetracyclic lactone backbone to which the deoxysugars forosamine and tri-O-methylrhamnose are attached. The Spinosyn biosynthesis genes, except for the rhamnose genes, are located in a cluster that spans 74 kb of the S. spinosa genome. DNA sequence analysis, targeted gene disruptions and bioconversion studies identified five large genes encoding type I polyketide synthase subunits, and 14 genes involved in sugar biosynthesis, sugar attachment to the polyketide or cross-bridging of the polyketide. Four rhamnose biosynthetic genes, two of which are also necessary for forosamine biosynthesis, are located outside the Spinosyn gene cluster. Duplication of the Spinosyn genes linked to the polyketide synthase genes stimulated the final step in the biosynthesis — the conversion of the forosamine-less pseudoaglycones to endproducts. Duplication of genes involved in the early steps of deoxysugar biosynthesis increased Spinosyn yield significantly. Journal of Industrial Microbiology & Biotechnology (2001) 27, 399–402.
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Genes for the biosynthesis of Spinosyns: applications for yield improvement in Saccharopolyspora spinosa
Journal of industrial microbiology & biotechnology, 2001Co-Authors: Krishnamurthy Madduri, M C Broughton, Clive Waldron, Donald J Merlo, Patti Matsushima, Kathryn P Crawford, Richard H BaltzAbstract:Spinosyns A and D are the active ingredients in an insect control agent produced by fermentation of Saccharopolyspora spinosa. Spinosyns are macrolides with a 21-carbon, tetracyclic lactone backbone to which the deoxysugars forosamine and tri-O-methylrhamnose are attached. The Spinosyn biosynthesis genes, except for the rhamnose genes, are located in a cluster that spans 74 kb of the S. spinosa genome. DNA sequence analysis, targeted gene disruptions and bioconversion studies identified five large genes encoding type I polyketide synthase subunits, and 14 genes involved in sugar biosynthesis, sugar attachment to the polyketide or cross-bridging of the polyketide. Four rhamnose biosynthetic genes, two of which are also necessary for forosamine biosynthesis, are located outside the Spinosyn gene cluster. Duplication of the Spinosyn genes linked to the polyketide synthase genes stimulated the final step in the biosynthesis--the conversion of the forosamine-less pseudoaglycones to endproducts. Duplication of genes involved in the early steps of deoxysugar biosynthesis increased Spinosyn yield significantly.
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rhamnose biosynthesis pathway supplies precursors for primary and secondary metabolism in saccharopolyspora spinosa
Journal of Bacteriology, 2001Co-Authors: Krishnamurthy Madduri, Clive Waldron, Donald J MerloAbstract:Rhamnose is an essential component of the insect control agent spinosad. However, the genes coding for the four enzymes involved in rhamnose biosynthesis in Saccharopolyspora spinosa are located in three different regions of the genome, all unlinked to the cluster of other genes that are required for Spinosyn biosynthesis. Disruption of any of the rhamnose genes resulted in mutants with highly fragmented mycelia that could survive only in media supplemented with an osmotic stabilizer. It appears that this single set of genes provides rhamnose for cell wall synthesis as well as for secondary metabolite production. Duplicating the first two genes of the pathway caused a significant improvement in the yield of Spinosyn fermentation products.
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cloning and analysis of the spinosad biosynthetic gene cluster of saccharopolyspora spinosa
Chemistry & Biology, 2001Co-Authors: Clive Waldron, M C Broughton, Krishnamurthy Madduri, Donald J Merlo, Patti Matsushima, Kathryn P Crawford, Jan R Turner, Paul Robert Rosteck, Richard H BaltzAbstract:Abstract Background: Spinosad is a mixture of novel macrolide secondary metabolites produced by Saccharopolyspora spinosa . It is used in agriculture as a potent insect control agent with exceptional safety to non-target organisms. The cloning of the Spinosyn biosynthetic gene cluster provides the starting materials for the molecular genetic manipulation of spinosad yields, and for the production of novel derivatives containing alterations in the polyketide core or in the attached sugars. Results: We cloned the spinosad biosynthetic genes by molecular probing, complementation of blocked mutants, and cosmid walking, and sequenced an 80 kb region. We carried out gene disruptions of some of the genes and analyzed the mutants for product formation and for the bioconversion of intermediates in the Spinosyn pathway. The Spinosyn gene cluster contains five large open reading frames that encode a multifunctional, multi-subunit type I polyketide synthase (PKS). The PKS cluster is flanked on one side by genes involved in the biosynthesis of the amino sugar forosamine, in O -methylations of rhamnose, in sugar attachment to the polyketide, and in polyketide cross-bridging. Genes involved in the early common steps in the biosynthesis of forosamine and rhamnose, and genes dedicated to rhamnose biosynthesis, were not located in the 80 kb cluster. Conclusions: Most of the S. spinosa genes involved in Spinosyn biosynthesis are found in one 74 kb cluster, though it does not contain all of the genes required for the essential deoxysugars. Characterization of the clustered genes suggests that the Spinosyns are synthesized largely by mechanisms similar to those used to assemble complex macrolides in other actinomycetes. However, there are several unusual genes in the Spinosyn cluster that could encode enzymes that generate the most striking structural feature of these compounds, a tetracyclic polyketide aglycone nucleus.
Adrian T. Keatinge-clay - One of the best experts on this subject based on the ideXlab platform.
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Structural Studies of the Spinosyn Forosaminyltransferase, SpnP
2015Co-Authors: Eta A. Isiorho, Hungwen Liu, Byung Sun Jeon, Namho Kim, Adrian T. Keatinge-clayAbstract:Spinosyns A and D (spinosad) are complex polyketide natural products biosynthesized through the cooperation of a modular polyketide synthase and several tailoring enzymes. SpnP catalyzes the final tailoring step, transferring forosamine from a TDP-d-forosamine donor substrate to a Spinosyn pseudoaglycone acceptor substrate. Sequence analysis indicated that SpnP belongs to a small group of glycosyltransferases (GTs) that require an auxiliary protein for activation. However, unlike other GTs in this subgroup, no putative auxiliary protein gene could be located in the biosynthetic gene cluster. To learn more about SpnP, the structures of SpnP and its complex with TDP were determined to 2.50 and 3.15 Å resolution, respectively. Binding of TDP causes the reordering of several residues in the donor substrate pocket. SpnP possesses a structural feature that has only been previously observed in the related glycosyltransferase EryCIII, in which it mediates association with the auxiliary protein EryCII. This motif, H-X-R-X5-D-X5-R-X12–20-D-P-X3-W-L-X12–18-E-X4-G, may be predictive of glycosyltransferases that interact with an auxiliary protein. A reverse glycosyl transfer assay demonstrated that SpnP possesses measurable activity in the absence of an auxiliary protein. Our data suggest that SpnP can bind its donor substrate by itself but that the glycosyl transfer reaction is facilitated by an auxiliary protein that aids in the correct folding of a flexible loop surrounding the pseudoaglycone acceptor substrate-binding pocket
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Structural Studies of the Spinosyn Forosaminyltransferase, SpnP
Biochemistry, 2014Co-Authors: Eta A. Isiorho, Hungwen Liu, Byung Sun Jeon, Namho Kim, Adrian T. Keatinge-clayAbstract:Spinosyns A and D (spinosad) are complex polyketide natural products biosynthesized through the cooperation of a modular polyketide synthase and several tailoring enzymes. SpnP catalyzes the final tailoring step, transferring forosamine from a TDP-d-forosamine donor substrate to a Spinosyn pseudoaglycone acceptor substrate. Sequence analysis indicated that SpnP belongs to a small group of glycosyltransferases (GTs) that require an auxiliary protein for activation. However, unlike other GTs in this subgroup, no putative auxiliary protein gene could be located in the biosynthetic gene cluster. To learn more about SpnP, the structures of SpnP and its complex with TDP were determined to 2.50 and 3.15 A resolution, respectively. Binding of TDP causes the reordering of several residues in the donor substrate pocket. SpnP possesses a structural feature that has only been previously observed in the related glycosyltransferase EryCIII, in which it mediates association with the auxiliary protein EryCII. This motif,...
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Structural studies of the Spinosyn rhamnosyltransferase, SpnG.
Biochemistry, 2012Co-Authors: Eta A. Isiorho, Hungwen Liu, Adrian T. Keatinge-clayAbstract:Spinosyns A and D (spinosad), like many other complex polyketides, are tailored near the end of their biosyntheses through the addition of sugars. SpnG, which catalyzes their 9-OH rhamnosylation, is also capable of adding other monosaccharides to the Spinosyn aglycone (AGL) from TDP-sugars; however, the substitution of UDP-d-glucose for TDP-d-glucose as the donor substrate is known to result in a >60000-fold reduction in kcat. Here, we report the structure of SpnG at 1.65 A resolution, SpnG bound to TDP at 1.86 A resolution, and SpnG bound to AGL at 1.70 A resolution. The SpnG–TDP complex reveals how SpnG employs N202 to discriminate between TDP- and UDP-sugars. A conformational change of several residues in the active site is promoted by the binding of TDP. The SpnG–AGL complex shows that the binding of AGL is mediated via hydrophobic interactions and that H13, the potential catalytic base, is within 3 A of the nucleophilic 9-OH group of AGL. A model for the Michaelis complex was constructed to reveal th...
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Structural Studies of the Spinosyn Rhamnosyltransferase, SpnG
2012Co-Authors: Eta A. Isiorho, Hungwen Liu, Adrian T. Keatinge-clayAbstract:Spinosyns A and D (spinosad), like many other complex polyketides, are tailored near the end of their biosyntheses through the addition of sugars. SpnG, which catalyzes their 9-OH rhamnosylation, is also capable of adding other monosaccharides to the Spinosyn aglycone (AGL) from TDP-sugars; however, the substitution of UDP-d-glucose for TDP-d-glucose as the donor substrate is known to result in a >60000-fold reduction in kcat. Here, we report the structure of SpnG at 1.65 Å resolution, SpnG bound to TDP at 1.86 Å resolution, and SpnG bound to AGL at 1.70 Å resolution. The SpnG–TDP complex reveals how SpnG employs N202 to discriminate between TDP- and UDP-sugars. A conformational change of several residues in the active site is promoted by the binding of TDP. The SpnG–AGL complex shows that the binding of AGL is mediated via hydrophobic interactions and that H13, the potential catalytic base, is within 3 Å of the nucleophilic 9-OH group of AGL. A model for the Michaelis complex was constructed to reveal the features that allow SpnG to transfer diverse sugars; it also revealed that the rhamnosyl moiety is in a skew-boat conformation during the transfer reaction
