Please note that the following presentation may change slightlybefore 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 adjacentto each slide to add notes.

Looking forward to seeing you all in Split.

Best regards,

Mervyn Bibb

Mining Biodiversity for New Antibiotics

Mervyn Bibb

Department of Molecular MicrobiologyJohn Innes Centre, Norwich

There is a very real and urgent need for new antibiotics

Metagenomics

New approaches are needed

High-throughput culturing

A wealth of unexplored microbial diversity

Microorganisms in the Environment

Habitat Cultured (%) Seawater 0.001 - 0.1 Freshwater 0.25 Soil 0.3 Activated Sludge 1.0 - 15.0 Gut 1.0 – 50.0

A wealth 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?

Sponges

Hot springsSoil

Deep ocean tube worms

Metagenomics

What is metagenomics?

Culture-independent genomic analysis of microbial communities and their physiology

Academic

Nature 428: 37-47 (2004)

• Acidophilic biofilm• pH 0.5, 40oC, FeS2

• Bacteria (Leptospirillum)• Archaea (Ferroplasma)

• Produces acid mine drainage• Worldwide environmental problem

Environmental or Community Genomics

• Environmental genomics

• Shotgun sequence

• 2 “complete” and 3 partial genomes of uncultivatedmicrobes

• Reconstruct metabolism

• Shared metabolic rolesin biofilm formation andsustainability

• Explore community metabolic network

• Culture and explore uncultivated microbes

Science 308: 554-557 (2005)

Agricultural soilDeep-sea whale fall carcasses

Environmental or Community Genomics

Science 312: 1355-1359 (2006)

Human gut

What is metagenomics?

Works well for enzyme discovery

Culture-independent exploitation of untapped microbial biodiversity

Industrial

Metagenomics for enzyme discovery

Environmental DNA

Cloned (small inserts) in E. coli

HT screen for enzyme activity

Gene/enzyme readily characterised and manipulated

GigaMatrix™100,000 wells in the

footprint of a 96-well plate

Nitrilases in the Public Database

Eukaryoticnitrilases

Bacterialnitrilases

Fungalcyanide

hydratases

24

Nitriles Acids Diversa Corporation

236

New Nitrilase Diversity

Robertson et al. (2004)AEM 70:2429/2436

Eukaryotic

Microbial

Fungalcyanide

hydratases

Bacterialnitrilases

Diversa Corporation

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?

Traditional approach to antibiotic discovery

New clinically useful antibiotics

Accessing uncultured microbial diversity - Metagenomics

Isolate environmental DNA (e-DNA)

Insert e-DNA into convenient bacterial host

Screen for antibiotic activity

Readily characterized and manipulated

Streptomycescoelicolor

Metagenomics can yield new natural products

☺ ☺

☺

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?

Indirubin

Terragine ATurbomycin ATurbomycin B☺

Violacein Deoxyviolacein

Long chain N-acyl amino acidsFatty dienic alcohol isomers

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

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)

The host is critical: knowledge-based improvement of antibiotic production in S. diversaTM

Diversa Corporation

Used to express large gene clusters froma variety of actinomycetes

and pseudomads

Colonypick

Agar plug assay

Expression hoste.g. Streptomyces

diversaTM

Master plate96 well agar plate

Pintool

Liquid: solvent extracts

E. coli conjugativefosmid/PAC library

Screen against, e.g. E. coli, Str. pneumoniaeSta. aureus and C. albicans

Screening metagenomic libraries for anti-microbial activity

“Environmental” sample

Diversa Corporation

• Unbiased approach – environmental DNA

Where should we look?

• Targeted approach

• High molecular weight and digestible DNA

• Representative libraries – dominant species – normalised libraries?

• Large numbers to screen

• Issues:

• Proven, but intractable sources of novel chemical diversity- quasi-metagenomics (Quasi: Resembling, seeming, virtual)

Antibiotics from microbes

Origin Number

Actinomycetes 9120

Other bacteria 1640

Fungi (moulds) 4140

Antibiotics made by Actinomycetes

Application Examples

Anti-bacterial ErythromycinTetracyclinesKanamycin

Anti-cancer Doxorubicin

Immuno-

suppression

FK 506

Anti-fungal Canesten

Application Examples

Livestock

rearing

MonensinTylosinVirginiamycin

Anti-parasitic Avermectin

Fungicide PolyoxinKasugamycin

Herbicide Basta

AgricultureMedicine

ChloramphenicolCephamycinVancomycin

• 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

Where should we look?

PAC Library Screening in Streptomyces diversaTM

Strain Total Clones

Mean Insert Size (kb)

>100kb

Actinomycete (unknown) 1536 120 85%

Pseudonocardia 2304 76 45%

Saccharopolyspora 1920 95 65%

Streptosporangium 1536 90 50%

Microbispora 1536 110 75%

Saccharothrix 1536 125 90%

Amycolatopsis 1920 83 40%

Actinoplanes 2304 80 40%

Amycolatopsis 1920 125 N.D.

Saccharopolyspora 1920 95 N.D.

Actinomycete mixture 4608 115 75%

Actinomycete mixture 4608 91 70%

PAC Library Screening in Streptomyces diversaTM

Strain Clones screened

E. coli (ESS) hits

S. aureushits

Pseudonocardia 1500 23 2

Saccharopolyspora 1500 3 0

Streptosporangium 1500 20 1

Microbispora 1500 1 N.D.

Amycolatopsis 1300 16 N.D.

E. coli hit S. aureus hit

Compound Source Activity

Abyssomicins Verrucosispora sp. ABAureoverticillactam Streptomyces aureoverticillatus ACBonactin Streptomyces sp. AB, AFCaprolactones Streptomyces sp. ACChandrananimycins Actinomadura sp. AA, AB, AC, AFChinikomycins Streptomyces sp. ACChloro-dihydroquinones Novel actinomycete AB, ACDiazepinomicin (ECO-4601) Micromonospora sp. AB, AC, AI3,6-disubstituted indoles Streptomyces sp. ACFrigocyclinone Streptomyces griseus ABGlaciapyrroles Streptomyces sp. ABGutingimycin Streptomyces sp. ABHelquinoline Janibacter limosus ABHimalomycins Streptomyces sp. ABIB-00208 Actinomadura sp. ACKomodoquinone A Streptomyces sp. NALajollamycin Streptomyces nodosus ABMarinomycins ‘Marinispora’ AB, ACMechercharmycins Thermoactinomyces sp. ACMKN-349A Nocardiopsis sp. UnknownSalinosporamide A (NPI-0052) Salinispora tropica ACSporolides Salinispora tropica UnknownTrioxacarcins Streptomyces sp. AB, AC, AM

AB antibacterial, AC anticancer, AF antifungal, AI anti-inflamatory, AM antimalarial, NA Neuritogenic activity

Novel metabolites made by “marine” actinomycetes 2003–2005

Lam - Current Opinion in Microbiology 9: 245-251 (2006)

Marine actinomycetes

Mining marine microorganisms as a source of new antimicrobials and antifungalsBernan VS, Greenstein M, Carter GTCurr Med Chem – Ant-Infective Agents 3: 181-195 (2004)

Journal of Industrial Microbiology & Biotechnology (1999) 23, 178–187

Where should we look?

Tropical terrestrial actinomycetes

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)

Where should we look?Back garden?

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 bedetected 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)

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)

Where should we look?

• Microbial symbionts – sources of potent anti-tumour compounds (PKS-NRPS)

Where should we look?

Uncultured sponge symbionts

Uncultured Pseudomonas sp.(beetle symbiont)

• Myxobacteria – many difficult to culture

Piel et al. (2004) PNAS 101:16222/16227

Joern Piel

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

Tc+ Tc-

Diversa Corporation

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

Metagenomics can yield new natural products

☺ ☺

☺

Indirubin

Terragine ATurbomycin ATurbomycin B☺

Violacein Deoxyviolacein

Long chain N-acyl amino acidsFatty dienic alcohol isomers

Many more to come

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

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

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)

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)

There is a very real and urgent need for new antibiotics

Metagenomics

New approaches are needed

High-throughput culturing

• 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

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

High throughput cultivation

GMD with micro-colony

GMD withsingle cell

Free bacteria

Sorting of gel micro-droplets containing micro-colonies

Empty micro-droplets 5,000 GMDs/second

Model of the experimental setup

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

4 DifferentMedia

• 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

Cultivation of Sargasso Sea microbes

Four different types of media, 50 16S rRNA gene sequences:

• Sea water + rich marine medium (1/100) 4 species:VibrioMarinobacterCytophaga

• Sea water + amino acids 12 species• Sea water + inorganic nutrients 11 species• Sea water 39 species

Rich marine medium

Filtered seawater

16 new clades

Zengler et al. (2002)PNAS 99:15681/6

Cultivation of Sargasso Sea microbes

More extensiveanalysis of 500 GMDs

Mining Biodiversity for New AntibioticsSupplementary Slides

There is a very real and urgent need for new antibiotics

Emergence of antibiotic resistantpathogens

Experts fear superbug pandemic1 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.

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.

Superbug deaths up by nearly a quarterin 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.

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.

'Superbug' infections spiralling in Canadian hospitals23 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.

Lethal superbug hits 44,000 elderly patients27 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.

Hospital and Community Based Problem

Hospital-Acquired Infections

• Methicillin resistant Staphylococcus aureus (MRSA) – 37%• Vancomycin resistant Enterococci (VRE) – 65%• Vancomycin resistant Staphylococcus aureus (VISA and VRSA)• Cephalosporin resistant Gram-negative bacteria• Azole resistant Candida albicans

Community-Acquired Infections

• Penicillin resistant Streptococcus pneumoniae (PRSP) – 25%• Multidrug resistant S. pneumoniae• Multidrug resistant Salmonella, e.g.Salmonella DT104

(AmpR, CmlR, StrR, SulR, TmpR, TetR, KanR, CipR)• Multidrug resistant Shigella• Fluoroquinolone resistant gonococci• Multidrug resistant Mycobacterium tuberculosis X

• Infectious disease mortality • 1900 to1980: declined 20 fold• 1980 to1995: increased two fold

• Antibiotic resistance is the major contributor

Increasing incidence of antibiotic resistance

1980 1985 1990 1995 2000

60

50

40

30

20

10

0

% in

cide

nce

MRSA

VRE

• Few new antibiotics marketed in the last 40 years

• And yet large pharmaceutical companies are curtailing anti-infective research and antibiotic discovery programmes

• Why?

Declining numbers of new antibiotics entering the clinic

Demise of antibiotic discovery in the pharmaceutical industry

• Development of a new antibiotic is expensive and risky: how long will the antibiotic remain effective and will it be profitable?

• Competition from highly profitable drugs for chronic diseases (high blood pressure, cholesterol lowering agents, depression etc)

• Led to reduced investment in or elimination of many antibiotic drug discovery programs

• Discovery of new antibiotics from traditional sources (microbes) has become increasingly difficult - have they all been found already?

• No – but new and imaginative approaches are required to find them