1. David Hopwood Lecture 1 (DH1)

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

3. Bioactive compounds from microbes (2002) Antibiotics Other Total Actinomycetes 7900* 1220 9120 Other bacteria 1400 240 1640 Fungi 2600 1540 4140 Total 11,900 300014,900 *70% from Streptomyces spp.

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

5. Antibiotic producers are differentiating microbes

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

7. PenicillinsCephalosporins Griseofulvin CyclosporinLovastatin Valuable fungal metabolites

8. Myxococcus Sorangium (epothilone) Stigmatella Some myxobacteria

9. Myxobacterial metabolites

10. Epothilone Ambruticin Valuable myxobacterial metabolites

11. A Streptomyces colony on an agar plate

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

13. Apoptosis: nutrient release Feeding Antibiotic production Reproduction

14. Fatal attraction

15. Needs for new antibiotics Overcome acquired resistance: Staphylococcus aureus (MRSA) Vancomycin-resistant Enterococcus MDR and XDR Mycobacterium tuberculosis Gram-negative respiratory 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. 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… Genetics of antibiotic producers

19. Streptomyces cloning vectors

20. Streptomyces plasmid SCP2

21. Streptomyces phage  C31

22. Streptomyces mycelium and protoplasts, light microscope

23. Streptomyces mycelium and protoplasts, electron microscope

24. The Streptomyces coelicolor genome

25. 1958 First Streptomyces coelicolor linkage map

26. 1965 First antibiotic gene (later named act)

27. 1990 antibiotic bald white

28. 1993

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

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

31. Isolation of antibiotic biosynthetic genes

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

33. act mutants of Streptomyces coelicolor

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

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

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

37. Manipulation of polyketide biosynthesis

38. Some actinomycete antibiotics (Polyketides) Medicine Agriculture ApplicationExamplesApplicationExamples Anti-bacterialErythromycin Tetracyclines Rifamycin Livestock rearing Monensin Tylosin Virginiamycin Anti-cancerAdriamycinAnti-parasiticAvermectin Immuno- suppression FK 506FungicidePolyoxin Kasugamycin AntifungalCandicidinHerbicideBialaphos

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

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

41. Type II PKS act (simple) Type I modular PKS ery (complex) The act and ery PKS gene clusters * * CLFKS

43. The DEBS paradigm for complex polyketide biosynthesis

44. Discovery of ‘cryptic’ secondary metabolites

45. ‘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

46. S. coelicolor v. S. avermitilis ClassS. coelicolorS. avermitilis Type I PK38 Type II PK2 (1)3 (1) NRP46 Carotenoid11 Desferrioxamine11 Chalcone3 (1)1 Others 94 Red = similar gene clusters

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

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

49. 500 current effort level Increased effort 2003 Total may be 150,000!

50. “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)

51. Improvement of productivity

52. Regulation + - Environmental factors Cofactor availability Shunt products Feedback inhibition Pathway genes Substrate Product Uptake Export Competing pathways Synthesis Undesired substrate Some targets for influencing antibiotic productivity Need for functional genomics

53. Many genes with cumulative effects!

55. 22,000 survivors of mutagenesis Pick 11 best strains and fuse protoplasts Pick 7 best from 1000 progeny and fuse protoplasts Screen 1000 progeny: 2 as good as the best from 1 million cultures screened over 20 year [Zhang, y. et al. (2002) Nature 415: 644] Increased productivity of S. fradiae for tylosin (24,000 colonies screened over 1 year)