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Steven G Kendrew,
Hrvoje Petkovic,
Sabine Gaisser,
Sarah J Ready,
Matthew A Gregory,
Nigel J Coates,
Mohammad Nur-E-Alam,
Tony Warneck,
Dipen Suthar,
Teresa A Foster,
Leonard McDonald,
Gerhard Schlingman,
Frank E Koehn,
Jerauld S Skotnicki,
Guy T Carter,
Steven J Moss,
Ming-Qiang Zhang, Christine J Martin,
Rose M Sheridan,
Barrie Wilkinson
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ABSTRACT: The rapK gene required for biosynthesis of the DHCHC starter acid that initiates rapamycin biosynthesis was deleted from strain BIOT-3410, a derivative of S. rapamycinicus which had been subjected to classical strain and process development and capable of robust rapamycin production at titres up to 250mg/L. The resulting strain BIOT-4010 could no longer produce rapamycin, but when supplied exogenously with DHCHC produced rapamycin at titres equivalent to its parent strain. This strain enabled mutasynthetic access to new rapalogs that could not readily be isolated from lower titre strains when fed DHCHC analogs. Mutasynthesis of some rapalogs resulted predominantly in compounds lacking late post polyketide synthase biosynthetic modifications. To enhance the relative production of fully elaborated rapalogs, genes encoding late-acting biosynthetic pathway enzymes which failed to act efficiently on the novel compounds were expressed ectopically to give strain BIOT-4110. Strains BIOT-4010 & BIOT-4110 represent valuable tools for natural product lead optimization using biosynthetic medicinal chemistry and for the production of rapalogs for pre-clinical and early stage clinical trials.
Metabolic Engineering 11/2012; · 5.61 Impact Factor
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Jennifer N Andexer,
Steven G Kendrew,
Mohammad Nur-e-Alam,
Orestis Lazos,
Teresa A Foster,
Anna-Sophie Zimmermann,
Tony D Warneck,
Dipen Suthar,
Nigel J Coates,
Frank E Koehn,
Jerauld S Skotnicki,
Guy T Carter,
Matthew A Gregory, Christine J Martin,
Steven J Moss,
Peter F Leadlay,
Barrie Wilkinson
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ABSTRACT: The macrocyclic polyketides FK506, FK520, and rapamycin are potent immunosuppressants that prevent T-cell proliferation through initial binding to the immunophilin FKBP12. Analogs of these molecules are of considerable interest as therapeutics in both metastatic and inflammatory disease. For these polyketides the starter unit for chain assembly is (4R,5R)-4,5-dihydroxycyclohex-1-enecarboxylic acid derived from the shikimate pathway. We show here that the first committed step in its formation is hydrolysis of chorismate to form (4R,5R)-4,5-dihydroxycyclohexa-1,5-dienecarboxylic acid. This chorismatase activity is encoded by fkbO in the FK506 and FK520 biosynthetic gene clusters, and by rapK in the rapamycin gene cluster of Streptomyces hygroscopicus. Purified recombinant FkbO (from FK520) efficiently catalyzed the chorismatase reaction in vitro, as judged by HPLC-MS and NMR analysis. Complementation using fkbO from either the FK506 or the FK520 gene cluster of a strain of S. hygroscopicus specifically deleted in rapK (BIOT-4010) restored rapamycin production, as did supplementation with (4R,5R)-4,5-dihydroxycyclohexa-1,5-dienecarboxylic acid. Although BIOT-4010 produced no rapamycin, it did produce low levels of BC325, a rapamycin analog containing a 3-hydroxybenzoate starter unit. This led us to identify the rapK homolog hyg5 as encoding a chorismatase/3-hydroxybenzoate synthase. Similar enzymes in other bacteria include the product of the bra8 gene from the pathway to the terpenoid natural product brasilicardin. Expression of either hyg5 or bra8 in BIOT-4010 led to increased levels of BC325. Also, purified Hyg5 catalyzed the predicted conversion of chorismate into 3-hydroxybenzoate. FkbO, RapK, Hyg5, and Bra8 are thus founder members of a previously unrecognized family of enzymes acting on chorismate.
Proceedings of the National Academy of Sciences 03/2011; 108(12):4776-81. · 9.68 Impact Factor
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Jeanette Woolard,
William Vousden,
Steven J Moss,
Arjun Krishnakumar,
Melissa Vr Gammons,
David G Nowak,
Neil Dixon,
Jason Micklefield,
Astrid Spannhoff,
Mark T Bedford,
Matthew A Gregory, Christine J Martin,
Peter F Leadlay,
Ming Q Zhang,
Steven J Harper,
David O Bates,
Barrie Wilkinson
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ABSTRACT: The polyketide natural product borrelidin 1 is a potent inhibitor of angiogenesis and spontaneous metastasis. Affinity biopanning of a phage display library of colon tumor cell cDNAs identified the tandem WW domains of spliceosome-associated protein formin binding protein 21 (FBP21) as a novel molecular target of borrelidin, suggesting that borrelidin may act as a modulator of alternative splicing. In support of this idea, 1, and its more selective analog 2, bound to purified recombinant WW domains of FBP21. They also altered the ratio of vascular endothelial growth factor (VEGF) isoforms in retinal pigmented endothelial (RPE) cells in favour of anti-angiogenic isoforms. Transfection of RPE cells with FBP21 altered the ratio in favour of pro-angiogenic VEGF isoforms, an effect inhibited by 2. These data implicate FBP21 in the regulation of alternative splicing and suggest the potential of borrelidin analogs as tools to deconvolute key steps of spliceosome function.
Chemical science (Royal Society of Chemistry) 02/2011; 2011(2):273-278. · 7.53 Impact Factor
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ABSTRACT: Biosynthetic genes encoding proteins involved in the first steps of deoxyhexose biosynthesis from D-glucose-1-phosphate were expressed in Saccharopolyspora erythraea. The resulting mutant was able to accumulate and utilise TDP-L-olivose. Co-expression of the spinosyn glycosyl transferase SpnP in the resulting mutant endowed upon it the ability to biotransform exogenously added spinosyn aglycones to yield novel spinosyn analogues.
Organic & Biomolecular Chemistry 05/2009; 7(8):1705-8. · 3.70 Impact Factor
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Ming-Qiang Zhang,
Sabine Gaisser,
Mohammad Nur-E-Alam,
Lesley S Sheehan,
William A Vousden,
Nikolaos Gaitatzis,
Gerrard Peck,
Nigel J Coates,
Steven J Moss,
Markus Radzom,
Teresa A Foster,
Rose M Sheridan,
Matthew A Gregory,
S Mark Roe,
Chrisostomos Prodromou,
Laurence Pearl,
Susan M Boyd,
Barrie Wilkinson, Christine J Martin
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ABSTRACT: A biosynthetic medicinal chemistry approach was applied to the optimization of the natural product Hsp90 inhibitor macbecin. By genetic engineering, mutants have been created to produce novel macbecin analogues including a nonquinone compound (5) that has significantly improved binding affinity to Hsp90 (Kd 3 nM vs 240 nM for macbecin) and reduced toxicity (MTD > or = 250 mg/kg). Structural flexibility may contribute to the preorganization of 5 to exist in solution in the Hsp90-bound conformation.
Journal of Medicinal Chemistry 10/2008; 51(18):5494-7. · 4.80 Impact Factor
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Christine J Martin,
Sabine Gaisser,
Iain R Challis,
Isabelle Carletti,
Barrie Wilkinson,
Matthew Gregory,
Chrisostomos Prodromou,
S Mark Roe,
Laurence H Pearl,
Susan M Boyd,
Ming-Qiang Zhang
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ABSTRACT: Macbecin compares favorably to geldanamycin as an Hsp90 inhibitor, being more soluble, stable, more potently inhibiting ATPase activity (IC50 = 2 microM) and binding with higher affinity (Kd = 0.24 microM). Structural studies reveal significant differences in their Hsp90 binding characteristics, and macbecin-induced tumor cell growth inhibition is accompanied by characteristic degradation of Hsp90 client proteins. Macbecin significantly reduced tumor growth rates (minimum T/C: 32%) in a DU145 murine xenograft. Macbecin thus represents an attractive lead for further optimization.
Journal of Medicinal Chemistry 06/2008; 51(9):2853-7. · 5.25 Impact Factor
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Lesley S Sheehan,
Rachel E Lill,
Barrie Wilkinson,
Rose M Sheridan,
William A Vousden,
Andrew L Kaja,
Gary D Crouse,
James Gifford,
Paul R Graupner,
Laura Karr,
Paul Lewer,
Thomas C Sparks,
Peter F Leadlay,
Clive Waldron, Christine J Martin
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ABSTRACT: The spinosyns are a family of potent and highly selective insect control agents that display a favorable environmental profile. As some regions of the spinosyn molecule are recalcitrant to chemical modification, a targeted genetic approach was carried out to generate new analogues. The polyketide synthase (PKS) loading modules from the avermectin PKS of Streptomyces avermitilis and the erythromcyin PKS of Saccharopolyspora erythraea were each used to replace the spinosyn PKS loading module. Both of the resulting strains containing hybrid PKS pathways produced the anticipated spinosyn analogues. Supplementation of the culture media with a range of exogenous carboxylic acids led to the successful incorporation of these novel elements to yield further novel spinosyn molecules, some of which demonstrated potent and new insecticidal activities. Furthermore, it has been demonstrated that semisynthesis of such novel metabolites can then be used to generate active analogues, demonstrating the effectiveness of utilizing these complementary methods to search the chemical space around this template.
Journal of Natural Products 01/2007; 69(12):1702-10. · 3.13 Impact Factor
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Matthew A Gregory,
Hui Hong,
Rachel E Lill,
Sabine Gaisser,
Hrvoje Petkovic,
Lindsey Low,
Lesley S Sheehan,
Isabelle Carletti,
Sarah J Ready,
Michael J Ward,
Andrew L Kaja,
Alison J Weston,
Iain R Challis,
Peter F Leadlay, Christine J Martin,
Barrie Wilkinson,
Rose M Sheridan
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ABSTRACT: The function of gene products involved in the biosynthesis of the clinically important polyketide rapamycin were elucidated by biotransformation and gene complementation.
Organic & Biomolecular Chemistry 11/2006; 4(19):3565-8. · 3.70 Impact Factor
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ABSTRACT: Modular polyketide synthases biosynthesise natural products through successive Claisen-type condensations, where one module is responsible for one round of chain extension. This review describes recent findings where this rule of co-linearity is broken, either by one module being bypassed (skipping) or through one module being used for multiple chain extension events (stuttering).
Natural Product Reports 11/2004; 21(5):575-93. · 9.79 Impact Factor
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Carlos Olano,
Barrie Wilkinson,
César Sánchez,
Steven J Moss,
Rose Sheridan,
Vidya Math,
Alison J Weston,
Alfredo F Braña, Christine J Martin,
Markiyan Oliynyk,
Carmen Méndez,
Peter F Leadlay,
José A Salas
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ABSTRACT: The biosynthetic gene cluster for the angiogenesis inhibitor borrelidin has been cloned from Streptomyces parvulus Tü4055. Sequence analysis indicates that the macrolide ring of borrelidin is formed by a modular polyketide synthase (PKS) (borA1-A6), a result that was confirmed by disruption of borA3. The borrelidin PKS is striking because only seven rather than the nine modules expected for a nonaketide product are encoded by borA1-A6. The starter unit of the PKS has been verified as trans-cyclopentane-1,2-dicarboxylic acid (trans-1,2-CPDA), and the genes involved in its biosynthesis identified. Other genes responsible for biosynthesis of the nitrile moiety, regulation, and self-resistance were also identified.
Chemistry & Biology 02/2004; 11(1):87-97. · 5.83 Impact Factor
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ABSTRACT: A truncated version of the spinosyn polyketide synthase comprising the loading module and the first four extension modules fused to the erythromycin thioesterase domain was expressed in Saccharopolyspora erythraea. A novel pentaketide lactone product was isolated, identifying cryptic steps of spinosyn biosynthesis and indicating the potential of this approach for the biosynthetic engineering of spinosyn analogues. A pathway for the formation of the tetracyclic spinosyn aglycone is proposed.
Organic & Biomolecular Chemistry 01/2004; 1(23):4144-7. · 3.70 Impact Factor
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ABSTRACT: A tetraketide synthase containing a loading module (LM), the extension modules erythromycin module 1, rapamycin module 2, and erythromycin module 2 (LM-Ery1-Rap2-Ery2-TE), when expressed in Saccharopolyspora erythraea strain JC2, produced as previously reported a mixture of tetraketide lactones (minor products) and triketide lactones (major products). Several alternative plausible mechanisms by which this "skipping" phenomenon might occur may be proposed. Site-directed mutagenesis of the ketosynthase (KS) and acylcarrier protein (ACP) domains in the interpolated module has shown that skipping within the hybrid PKS involves passage of the growing polyketide through the interpolated module, by direct ACP-to-ACP transfer of the polyketide chain.
Chemistry & Biology 08/2002; 9(7):781-7. · 5.83 Impact Factor
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ABSTRACT: An Escherichia coli-actinomycete shuttle vector, pCJW93, was constructed which places cloned genes under the control of the thiostrepton-inducible tip promoter from Streptomyces lividans. We also constructed expression vectors bearing the actII-ORF4/PactI activator-promoter system of the actinorhodin biosynthetic pathway of Streptomyces coelicolor. With both types of vector, levels of expression varied widely in different actinomycete strains, indicating different levels of the host factors needed for optimal expression. Deletion of the actII-ORF4 activator gene from one such plasmid in Saccharopolyspora erythraea drastically reduced expression from the cognate actI promoter, showing that host factors are required for optimal production of the activator protein itself. However, a low copy number expression vector pWIZ1 for the polyketide synthase DEBS1-TE, in which the promoter for the activator gene was replaced by the strong heterologous ermE* promoter of S. erythraea directed highly efficient production of polyketide synthase protein in Streptomyces cinnamonensis; and the levels of triketide lactone product found were up to 100-fold greater than were produced by the same plasmid in which actII-ORF4 was expressed from its own promoter. Ensuring appropriate expression of a specific activator protein should enable more convenient and consistent heterologous expression of genes in a broad range of actinomycete hosts.
Journal of Molecular Microbiology and Biotechnology 08/2002; 4(4):417-26. · 1.95 Impact Factor
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Sabine Gaisser, Christine J Martin,
Barrie Wilkinson,
Rose M Sheridan,
Rachel E Lill,
Alison J Weston,
Sarah J Ready,
Clive Waldron,
Gary D Crouse,
Peter F Leadlay,
James Staunton
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ABSTRACT: Novel spinosyns have been prepared by biotransformation, using a genetically engineered strain of Saccharopolyspora erythraea, in which the beta-D-forosamine moiety in glycosidic linkage to the hydroxy group at C17 is replaced by alpha-L-mycarose.
Chemical Communications 04/2002; · 6.17 Impact Factor
