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David Hopwood Lecture 1 (DH1)

David Hopwood Lecture 1 (DH1). Isolation of microbes from soil: fungi, actinomycetes, other bacteria (left); streptomycetes (right). Antibiotics. Other. Total. Actinomycetes. 7900*. 1220. 9120. Other bacteria. 1400. 240. 1640. Fungi. 2600. 1540. 4140. Total. 11,900. 3000.

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David Hopwood Lecture 1 (DH1)

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  1. David Hopwood Lecture 1 (DH1)

  2. Isolation of microbes from soil: fungi, actinomycetes, other bacteria (left); streptomycetes (right)

  3. Antibiotics Other Total Actinomycetes 7900* 1220 9120 Other bacteria 1400 240 1640 Fungi 2600 1540 4140 Total 11,900 3000 14,900 Bioactive compounds from microbes (2002) *70% from Streptomyces spp.

  4. Actinomycetes Other bacteria Fungi Diminishing returns in finding useful natural products

  5. Antibiotic producers are differentiating microbes

  6. Penicillium notatum (penicillin) Aspergillus terreus (lovastatin)

  7. Penicillins Cephalosporins Griseofulvin Valuable fungal metabolites Cyclosporin Lovastatin

  8. Some myxobacteria Myxococcus Stigmatella Sorangium (epothilone)

  9. Myxobacterial metabolites

  10. Epothilone Valuable myxobacterial metabolites Ambruticin

  11. A Streptomyces colony on an agar plate

  12. Streptomyces: scanning EM Transition stage: most antibiotic production Young vegetative hyphae Aerial hyphae, young spores Mature spores

  13. Reproduction Antibiotic production Apoptosis: nutrient release Feeding

  14. Fatal attraction

  15. Needs for new antibiotics • Overcome acquired resistance: • Staphylococcus aureus (MRSA) • Vancomycin-resistant Enterococcus • MDR and XDR Mycobacterium tuberculosis • Gram-negativerespiratory pathogens • Less toxic anti-viral or anti-cancer agents • Immunosuppressants, cholesterol lowerers…

  16. How to find new antibiotics • Novel natural products • Chemical synthesis combichem • Genetics, genetic engineering

  17. Genetics of antibiotic producers Filamentous fungi: gene replacements, genomics Myxobacteria: transduction, transposon libraries, gene replacements, genomics… Streptomyces: plasmid-mediated conjugation, protoplast fusion, autonomous and integrating plasmid and phage cloning vectors, gene replacements, transposon libraries, genomics…

  18. Streptomyces cloning vectors

  19. Streptomyces plasmid SCP2

  20. Streptomyces phage C31

  21. Streptomyces mycelium and protoplasts, light microscope

  22. Streptomyces mycelium and protoplasts, electron microscope

  23. The Streptomyces coelicolor genome

  24. 1958 First Streptomyces coelicolor linkage map

  25. First antibiotic gene (later named act) 1965

  26. antibiotic bald white 1990 1990

  27. 1993

  28. (325 clones) Http://jic-bioinfo.bbsrc.ac.uk/streptomyces -then click “ScoDB II”

  29. LH arm = 1.5 Mb RH arm = 2.3 Mb 9 May 2002 7825 ORFs (55 pseudogenes) 63 tRNA genes 6 rRNA operons 72.12% G+C Core = 4.9 Mb

  30. Isolation of antibiotic biosynthetic genes

  31. act mutant of Streptomyces coelicolor (Brian Rudd, 1976)

  32. act mutants of Streptomyces coelicolor

  33. The first act clone (Francisco Malpartida, 1984)

  34. The act genes of Streptomyces coelicolor Tailoring steps Regulation resistance Chain assembly (PKS) Actinorhodin

  35. Mederhodin First ‘hybrid’ antibiotic (1985) Medermycin (S. AM-7161) Actinorhodin (S. coelicolor)

  36. Manipulation of polyketide biosynthesis

  37. Some actinomycete antibiotics(Polyketides)Medicine Agriculture Application Examples Application Examples Anti-bacterial Erythromycin Livestock Monensin Tetracyclines rearing Tylosin Rifamycin Virginiamycin Anti-cancer Adriamycin Anti-parasitic Avermectin Immuno- FK 506 Fungicide Polyoxin suppression Kasugamycin Antifungal Candicidin Herbicide Bialaphos

  38. 6-MSA Cyanidin Aflatoxin Oxytetracycline Erythromycin Brevetoxin COOH Palmitic acid Polyketides A fatty acid

  39. Extender Chirality Side chains Starter R Chain length O H OH OH O H O Reduction level KR/DH/ER Variables in polyketides (‘Combinatorial biosynthesis’) O

  40. The act and ery PKS gene clusters Type II PKS act (simple) KS CLF Type I modular PKS ery (complex) * *

  41. The DEBS paradigm for complex polyketide biosynthesis

  42. Discovery of ‘cryptic’ secondary metabolites

  43. ‘Secondary metabolic’ gene clusters in Streptomyces coelicolor 3 antibiotics (type II PK, modular PK, NRP) 4 siderophores (2 NRP, 2 other) 3 pigments (type II PK, chalcone, carotenoid) 2 complex lipids (unsaturated FA, hopanoid) 2 signalling molecules (terpenoid, -butyrolactone) 8 other (2 modular PK, 1 NRP, 2 chalcones, 2 terpenoid, 1 deoxysugar) PK = polyketide, NRP = non-ribosomal peptide, FA = fatty acid Total length ~ 375 kb ~ 4.5% of the genome

  44. S. coelicolor v. S. avermitilis ClassS. coelicolorS. avermitilis Type I PK 3 8 Type II PK 2 (1) 3 (1) NRP 4 6 Carotenoid 1 1 Desferrioxamine 1 1 Chalcone 3 (1) 1 Others 9 4 Red = similar gene clusters

  45. Enediynes PKS Zazopoulos et al. (2003) Nature Biotech. 21:187

  46. Discoveries/year Cumulative discoveries Watve et al. (2001) Arch. Microbiol. 176:386 “How many antibiotics are produced by the genus Streptomyces?”

  47. Increased effort 500 current effort level 2003 Total may be 150,000!

  48. “Therefore, by genic manipulation of the cell we have a means for obtaining, in quantities sufficient for study, many of the metabolic products of the living organism that would be otherwise undetectable” Albert Kelner (1949)

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