Please note that the following presentation may change slightly

1. Please note that the following presentation may change slightly before the Summer School starts, but it has been provided now for those of you leaving home soon, and who need to print it out. If you print out at three slides per page, you will have space adjacent to each slide to add notes. Looking forward to seeing you all in Split. Best regards, Mervyn Bibb

2. Mining Biodiversity for New Antibiotics Mervyn Bibb Department of Molecular Microbiology John Innes Centre, Norwich

3. There is a very real and urgent need for new antibiotics New approaches are needed Metagenomics High-throughput culturing

4. A wealth of unexplored microbial diversity Microorganisms in the Environment HabitatCultured (%) Seawater 0.001 - 0.1 Freshwater 0.25 Soil 0.3 Activated Sludge 1.0 - 15.0 Gut 1.0 – 50.0

5. Hot springs Soil Deep ocean tube worms Sponges • Awealth of unexplored microbial genomes • Less than 1% of all microorganisms have been cultured under laboratory conditions: wealth of unexplored biochemistry and enzymology • Many of these uncultivated microbes exist in complex, competitive communities - potentially source of novel biologically active compounds • How can these organisms and communities be explored and potentially exploited? Metagenomics

6. What is metagenomics? Academic Culture-independent genomic analysis of microbial communities and their physiology

7. Environmental or Community Genomics Nature 428: 37-47 (2004) • Produces acid mine drainage • Worldwide environmental problem • Acidophilic biofilm • pH 0.5, 40oC, FeS2 • Bacteria (Leptospirillum) • Archaea (Ferroplasma)

8. Environmental genomics • Shotgun sequence • 2 “complete” and 3 partial genomes of uncultivated • microbes • Reconstruct metabolism • Shared metabolic roles • in biofilm formation and • sustainability • Explore community metabolic network • Culture and explore uncultivated microbes

9. Environmental or Community Genomics Agricultural soil Deep-sea whale fall carcasses Human gut Science 312: 1355-1359 (2006) Science 308: 554-557 (2005)

10. What is metagenomics? Industrial Culture-independent exploitation of untapped microbial biodiversity Works well for enzyme discovery

11. Metagenomics for enzyme discovery Environmental DNA Cloned (small inserts) in E. coli HT screen for enzyme activity GigaMatrix™ 100,000 wells in the footprint of a 96-well plate Gene/enzyme readily characterised and manipulated

12. Nitrilases in the Public Database Eukaryotic nitrilases Fungal cyanide hydratases 24 Bacterial nitrilases Nitriles Acids Diversa Corporation

13. New Nitrilase Diversity Eukaryotic Microbial Fungal cyanide hydratases 236 Bacterial nitrilases Robertson et al. (2004) AEM 70:2429/2436 Diversa Corporation

14. What is metagenomics? Industrial Culture-independent exploitation of untapped microbial biodiversity Works well for enzyme discovery Can it work effectively for small molecule (antibiotic) discovery?

15. Traditional approach to antibiotic discovery New clinically useful antibiotics

16. Accessing uncultured microbial diversity - Metagenomics Isolate environmental DNA (e-DNA) Insert e-DNA into convenient bacterial host Streptomyces coelicolor Screen for antibiotic activity Readily characterized and manipulated

17. Metagenomics can yield new natural products ☺ ☺ Terragine A Turbomycin A ☺ Turbomycin B Indirubin Deoxyviolacein Violacein ☺ Long chain N-acyl amino acids Fatty dienic alcohol isomers Can it be de done on an industrial scale and yield complex, biologically active molecules in the numbers required to support drug discovery? What is needed?

18. What do you need for an efficient metagenomic approach to new natural products? • “Secondary metabolites” are products of complex pathways, derived from common precursors • Biosynthetic genes clustered • Biosynthetic clusters range from ca. 15 kb to >120 kb • Cloning requires specialized fosmid and BAC/PAC vectors • Expression of pathway genes requires appropriate host (e.g. Streptomyces sp.) • Relaxed expression requirements • Genetically manipulable – enhance natural product biosynthesis • Understanding of the physiology of the organism (for precursor supply) • Understanding of the regulatory circuitry to effect efficient expression • High-throughput screening • Requires expertise in natural product isolation and characterisation

19. The host is critical: knowledge-based improvement of antibiotic production in S. diversaTM Used to express large gene clusters from a variety of actinomycetes and pseudomads Actinorhodin is a blue-pigmented Type II polyketide – ca. 25 kb cluster with ca. 20 genes Directed mutations enhanced productivity 60 to 250 fold above wild type (depending on heterologous pathway expressed) Diversa Corporation

20. Screening metagenomic libraries for anti-microbial activity E. coli conjugative fosmid/PAC library “Environmental” sample Agar plug assay Pintool Colony pick Master plate 96 well agar plate Expression host e.g. Streptomyces diversaTM Liquid: solvent extracts Screen against, e.g. E. coli,Str. pneumoniae Sta. aureus and C. albicans Diversa Corporation

21. Issues: • High molecular weight and digestible DNA • Representative libraries – dominant species – normalised libraries? • Large numbers to screen Where should we look? • Unbiased approach – environmental DNA • Targeted approach • Proven, but intractable sources of novel chemical diversity • - quasi-metagenomics (Quasi: Resembling, seeming, virtual)

22. Antibiotics from microbes Origin Number Actinomycetes 9120 Other bacteria 1640 Fungi (moulds) 4140

23. Anti-cancer Doxorubicin Immuno- FK 506 suppression Anti-fungal Canesten Antibiotics made by Actinomycetes Agriculture Medicine Application Examples Application Examples Livestock Monensin Anti-bacterial Erythromycin Tylosin rearing Tetracyclines Virginiamycin Kanamycin Chloramphenicol Cephamycin Vancomycin Anti-parasitic Avermectin Fungicide Polyoxin Kasugamycin Herbicide Basta

24. Where should we look? • Unbiased approach – environmental DNA – issues: • High molecular weight and digestible DNA • Representative libraries – dominant species • Large numbers to screen • Proven, but intractable sources of novel chemical diversity – quasi-metagenomics • Rare (difficult to culture) actinomycetes - rifamycins, erythromycin, teichoplanin, vancomycin, gentamicin, ramoplanin, dalbavancin • Marine actinomycetes

25. PAC Library Screening in Streptomyces diversaTM

26. PAC Library Screening in Streptomyces diversaTM E. coli hit S. aureus hit

27. Novel metabolites made by “marine” actinomycetes 2003–2005 Compound Source Activity Abyssomicins Verrucosispora sp. AB Aureoverticillactam Streptomyces aureoverticillatus AC Bonactin Streptomyces sp. AB, AF Caprolactones Streptomyces sp. AC Chandrananimycins Actinomadura sp. AA, AB, AC, AF Chinikomycins Streptomyces sp. AC Chloro-dihydroquinones Novel actinomycete AB, AC Diazepinomicin (ECO-4601) Micromonospora sp. AB, AC, AI 3,6-disubstituted indoles Streptomyces sp. AC Frigocyclinone Streptomyces griseus AB Glaciapyrroles Streptomyces sp. AB Gutingimycin Streptomyces sp. AB Helquinoline Janibacter limosus AB Himalomycins Streptomyces sp. AB IB-00208 Actinomadura sp. AC Komodoquinone A Streptomyces sp. NA Lajollamycin Streptomyces nodosus AB Marinomycins ‘Marinispora’ AB, AC Mechercharmycins Thermoactinomyces sp. AC MKN-349A Nocardiopsis sp. Unknown Salinosporamide A (NPI-0052) Salinispora tropica AC Sporolides Salinispora tropica Unknown Trioxacarcins Streptomyces sp. AB, AC, AM AB antibacterial, AC anticancer, AF antifungal, AI anti-inflamatory, AM antimalarial, NA Neuritogenic activity Lam - Current Opinion in Microbiology 9: 245-251 (2006)

28. Journal of Industrial Microbiology & Biotechnology (1999) 23, 178–187 Where should we look? Marine actinomycetes Mining marine microorganisms as a source of new antimicrobials and antifungals Bernan VS, Greenstein M, Carter GT Curr Med Chem – Ant-Infective Agents 3: 181-195 (2004) Tropical terrestrial actinomycetes

29. Antibiotic-producing ability by representatives of a newly discovered lineage of actinomycetes Busti E, Monciardini P, Cavaletti L, Bamonte R, Lazzarini A, Sosio M, Donadio S Vicuron Pharmaceuticals, via R. Lepetit 34, 21040 Gerenzano, Italy The discovery of new antibiotics and other bioactive microbial metabolites continues to be an important objective in new drug research. Since extensive screening has led to the discovery of thousands of bioactive microbial molecules, new approaches must be taken in order to reduce the probability of rediscovering known compounds. The authors have recently isolated slow-growing acidophiles belonging to the novel genera Catenulispora and Actinospica within the order Actinomycetales. These strains, which likely belong to a new suborder, grow as filamentous mycelia, have a genome size around 8 Mb, and produce antimicrobial activities. In addition, a single strain harbours simultaneously genes encoding type I and type II polyeketide synthases, as well as non-ribosomal peptide synthetases. The metabolite produced by one strain was identified as a previously reported dimeric isochromanequinone. In addition, at least the Catenulispora strains appear globally distributed, since a PCR-specific signal could be detected in a significant fraction of acidic soils from different continents, and similar strains have been independently isolated from an Australian soil (Jospeh et al., Appl Environ Microbiol 69, 7210–7215, 2003). Thus, these previously uncultured actinomycetes share several features with Streptomyces and related antibiotic-producing genera, and represent a promising source of novel antibiotics. Microbiology 152: 675–683 (2006) Where should we look? Back garden? Diversity of Actinoplanes and related genera isolated from an Italian soil Mazza P, Monciardini P, Cavaletti L, Sosio M, Donadio S Vicuron Pharmaceuticals, via R. Lepetit 34, 21040 Gerenzano (VA), Italy.Actinoplanes and related genera are good producers of bioactive secondary metabolites. However, many strains within these genera present similar morphological characteristics, and this prevents an effective discrimination of replicate strains during industrial isolation and screening programs. Using PCR-RFLP analysis of the 23S rDNA gene and of the 16S-23S intergenic spacer, we have analyzed 182 strains of Actinoplanes and related genera obtained through a selective isolation method from a single Italian soil. Combining the 23S and IGS data, 99 unique profiles were observed, and morphologically undistinguishable strains were discriminated. Further analyses on a restricted number of strains through 16S sequencing and hybridization to a probe for secondary metabolism established a good correlation between strain diversity seen by PCR-RFLP and that seen by the other methods. Overall, the data indicate the presence of a high diversity of Actinoplanes and related genera isolated from a single Italian soil. Microbial Ecology 45: 362-372 (2003)

30. Where should we look? Sources of polyketides and non-ribosomal peptides Donadio S, Busti E, Monciardini P, Bamonte R, Mazza P, Sosio M, Cavaletti L Vicuron Pharmaceutical, Gerenzano, Italy Ernst Schering Research Foundation Workshop 51:19-41 (2005)

31. Uncultured Pseudomonas sp. (beetle symbiont) Uncultured sponge symbionts Where should we look? • Microbial symbionts – sources of potent anti-tumour compounds (PKS-NRPS) Joern Piel Piel et al. (2004) PNAS 101:16222/16227 • Myxobacteria – many difficult to culture

32. Tc+ Tc- Sensitive high-throughput screening of metagenomic libraries for biological activity • Use a gene reporter system to detect a transcriptional response to an anti- bacterial agent • Greater sensitivity than growth inhibition assay • Amenable to high-throughput FACS-based screening • Screens can be directed towards particular aspects of cell physiology • (mode of action) e.g. cell wall biosynthesis – fuse specific stress-induced • promoters to GFP Co-encapsulation of a tetracycline (Tc) producer with a Tc-responsive GFP reporter Diversa Corporation

33. What is metagenomics? Industrial Culture-independent exploitation of untapped microbial biodiversity Works well for enzyme discovery Can it work effectively for small molecule (antibiotic) discovery? Yes – requires technology development, concerted multi-disciplinary effort, and financial and lengthy commitment

34. Metagenomics can yield new natural products ☺ ☺ Terragine A Turbomycin A ☺ Turbomycin B Indirubin Deoxyviolacein Violacein ☺ Long chain N-acyl amino acids Fatty dienic alcohol isomers Many more to come

35. Genome scanning – the Ecopia way • Focus on cryptic pathways in actinomycetes • Construct small and large (cosmid and BAC) insert libraries • Sequence one end of small inserts (up to 700 nt) – Genome Sequence Tags • 1000 GSTs of 8.5 Mb genome – sample every 8.5 kb on average • 20 – 200 kb cluster represented 2 – 20 times • Compare sequences to microbial natural product (NP) gene/enzyme database • Use small inserts containing NP biosynthetic gene(s) to probe large insert library • Sequence large insert(s) • Reconstruct biosynthetic gene cluster in silico and predict structure • Screen for growth conditions that induce expression of novel compounds

36. Genome scanning – the Ecopia way • Used to identify over 450 actinomycete natural product gene clusters : • Enediyne anti-tumour compounds (Type 1 PKS) • Dynemicin (Micromonospora chersina) • Macromomycin (Streptomyces macromyceticus) (8/50 actinomycetes not known to make enediynes contained enediyne-like gene clusters – widely distributed across many genera – likely to encode new classes of enediynes) Calicheamicin Dynemicin C-1027

37. Genome scanning – the Ecopia way • Used to identify over 450 actinomycete natural product gene clusters : • Anti-fungal compounds • ECO-02301 (Streptomyces aizunensis) – novel linear Type I PKS derived compound • E-837 (Streptomyces aculeolatus) and E-492 & E-975 (Streptomyces sp Eco86) – novel alkenyl furanones (Type I PKS-derived)

38. Genome scanning – the Ecopia way References Zazopoulos E, Huang K, Staffa A, Liu W, Bachmann BO, Nonaka K, Ahlert J, Thorson JS, Shen B, Farnet CM. A genomics-guided approach for discovering and expressing cryptic metabolic pathways. Nat Biotechnol 21: 187–190 (2003) McAlpine JB, Bachmann BO, Piraee M, Tremblay S, Alarco AM, Zazopoulos E, Farnet CM. Microbial genomics as a guide to drug discovery and structural elucidation: ECO-02301, a novel antifungal agent, as an example. J Nat Prod 68: 493–496 (2005) Banskota AH, McAlpine JB, Sørensen D, Aouidate M, Piraee M, Alarco AM, Omura S, Shiomi K, Farnet CM, Zazopoulos E. Isolation and identification of three new 5-Alkenyl-3,3(2H)-furanones from two Streptomyces species using a genomic screening approach. J Antibiot 59: 168–176 (2006) Banskota AH, Mcalpine JB, Sørensen D, Ibrahim A, Aouidate M, Piraee M, Alarco AM, Farnet CM, Zazopoulos E. Genomic analyses lead to novel secondary metabolites. Part 3. ECO-0501, a novel antibacterial of a new class. J Antibiot 59:533-42 (2006)

39. There is a very real and urgent need for new antibiotics New approaches are needed Metagenomics High-throughput culturing

40. High throughput cultivation • Growth of previously uncultivated microorganisms under natural conditions • Throughput of 5-10,000 isolated strains per month • Yields novel strains AND novel biologically active molecules • Provides material for the metagenomic approach

41. High throughput cultivation • FACS-based technology • Gel micro-droplet (GMD) encapsulation • Screening rates of 5,000 GMDs/second • Enables HTP isolation and cultivation of previously uncultured microorganisms Screen for biological activity

42. Sorting of gel micro-droplets containing micro-colonies GMD with micro-colony GMD with single cell Empty micro-droplets Free bacteria 5,000 GMDs /second

43. Model of the experimental setup

44. 4 Different Media Cultivation of Sargasso Sea microbes • 4 different types of media: • Filtered sea water + rich marine medium (1/100) • Filtered sea water + amino acids • Filtered sea water + inorganic nutrients • Filtered sea water

45. Cultivation of Sargasso Sea microbes • Encapsulated single cells in a GMD • Grown for 5 weeks • 1200 GMDs with micro-colonies FACS sorted into 96 well plates (marine medium) • 16S rRNA gene library made by PCR; 140 clones characterised by RFLP • 50 16S rRNA genes sequenced to determine nature of micro-colonies

46. Cultivation of Sargasso Sea microbes • Four different types of media, 50 16S rRNA gene sequences: • Sea water + rich marine medium (1/100) 4 species: • Vibrio • Marinobacter • Cytophaga • Sea water + amino acids 12 species • Sea water + inorganic nutrients 11 species • Sea water 39 species More extensive analysis of 500 GMDs 16 new clades Rich marine medium Zengler et al. (2002) PNAS 99:15681/6 Filtered seawater

47. Mining Biodiversity for New Antibiotics Supplementary Slides

48. There is a very real and urgent need for new antibiotics Emergence of antibiotic resistant pathogens

49. Experts fear superbug pandemic 1 April 2005 A strain of the MRSA superbug caught in public places as opposed to hospitals could spread faster and wider than first thought, experts say. Superbug deaths up by nearly a quarter in year 24 February 2006· NHS hospitals the most likely source of infection The number of deaths related to MRSA, the so- called hospital superbug, increased by almost a quarter, according to the latest figures. MRSA is now six times more likely to be a factor in the deaths of people in NHS hospitals than anywhere else, the Office for National Statistics said yesterday. New superbug outbreak sweeps southern England 20 July 2005 An outbreak of a superbug resistant to antibiotics has infected more than 1,000 people and caused dozens of deaths. The bug, which can lead to blood poisoning, is spreading in southern England and is more serious than Clostridium difficile, which hit the headlines last month after a virulent strain infected 15 hospitals. Superbug that moves faster than science 1 March 2005 The world may run out of effective antibiotics by the end of this decade and faces a gap of at least five years before new drugs can be developed to combat superbugs, according to one of the world's most influential scientists.

50. 'Superbug' infections spiralling in Canadian hospitals 23 March 2005 Toronto - Hospitals are failing to control antibiotic- resistant "superbug" infections that kill as many as 8,000 patients each year and cost health-care systems at least $100 million annually, a CBC News investigation has learned. Marine killed by scratch and superbug 24 May 2005 A SUPERFIT Royal Marine collapsed and died within days of scratching his leg on a bush while on a training run — victim of a mutated superbug one doctor described as the worst she had ever seen. New strains of superbug can kill in 24 hours 20 February 2005 Highly virulent strains of the superbug MRSA which infect healthy young people with no connection to hospitals are appearing in the UK. The new varieties cause skin and soft tissue infections such as boils, abscesses and inflammation and, in rare cases so far only seen in other countries, a severe pneumonia that can kill in 24 hours. Lethal superbug hits 44,000 elderly patients 27 August 2005 Record numbers of elderly people fell victim last year to a potentially lethal superbug which is plaguing Britain's hospitals, according to details of the first complete survey of the disease. Concerns about the bug, Clostridium difficile, were first revealed in The Independent in June following an outbreak of a lethal strain at Stoke Mandeville Hospital. The figures yesterday showed that there were 44,488 cases of the bug among people over 65.