Isolation and screening of secondary metabolite producing microrganisms

Transcription

1 Department of Biotechnology and Molecular Sciences Microbial Biotechnology Lab FLAVIA MARINELLI University of Insubria, Varese. Italy Isolation and screening of secondary metabolite producing microrganisms Split Summer School Microbial Genomics and Secondary Metabolites

2 MICROBIAL DIVERSITY VALUABLE PRODUCTS Biomass Products from anaerobic metabolism Products from incomplete oxidations Products from secondary metabolism Enzymes Polysaccharides Heterologous proteins

3 WHAT ARE SECONDARY METABOLITES? Bu Lock 1961: Non essential for growth and reproduction Each of them is formed by only a few organisms, but some microbes (es.actinomycetes) form a variety of different chemical classes Their formation is extremely dependent on the cultural conditions Some secondary metabolites are produced as a group of closely related structures

4 HOW MANY MICROBIAL SECONDARY METABOLITES ARE KNOWN? ANTIBIOTIC LITERATURE DATABASE: Collecting more than entries describing bioactive metabolites of microbial origin from literature and patents since 1950 till microbial secondary metabolites possessing some activity in vitro

5 HOW MANY MICROBIAL SECONDARY METABOLITES ARE KNOWN? ANTIBIOTIC LITERATURE DATABASE: Collecting more than entries describing bioactive metabolites of microbial origin from literature and patents since 1950 till 2006 Immunomodulator Pharmacological Antiparasitic Algicides Antiviral Herbicides Siderophore Antibacterial Antitumor Antifungal microbial secondary metabolites possessing some activity in vitro

6 WHO ARE THE MICROBIAL PRODUCERS? ANTIBIOTIC LITERATURE DATABASE: Collecting more than entries describing bioactive metabolites of microbial origin from literature and patents since 1950 till 2006 Distribution of the microbial producers of the active secondary metabolites

7 Products from Ascomycota GENUS Penicillium Acremonium Aspergillus Zalerion Tolypocladium Fusarium Cepahlosporium PRODUCTS Penicillin, Griseofulvin Cephalosporin Fusidic acid Echinocandins Lovastatin Pneumocandins Cyclosporin Zearalenone Cerulenin USE Antibacterial Antifungal Antibacterial Antibacterial Antifungal Hypocholesterolemic Antifungal Immunomodulator Anabolic Hypolypidic

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9 Promising bacterial groups difficult to isolate and cultivate Discovery events M yxobacteria Cyanobacteria

10 ACTINOMYCETES (order Actinomycetales) Soil Gram-positives generating aerial mycelium and spores High GC content (60-78%) Producing more than 70% of the known antibiotics

11 A C T I N O M Y C E T E S ß-lactams (thienamycin) Aminoglycosides (streptomycin) Macrolides (tylosin) Tetracyclines Streptogramins Cloramphenicol Lincomycin Fosfomycin Novobiocin Polyenes (amphotericin) Antracyclines (adriamycin) Bleomycins, Tacrolimus&Sirolimus, Clavulanic acid, Avermectins Ionophoric polyethers, Bialaphos, Polyoxins

12 rare actinos Glycopeptides (teicoplanin) A C T I N O M Y C E T E S Acarbose Glycopeptides (vancomycin) Ansamycins (rifamycins)

13 A C T I N O M Y C E T E S Aminoglycosides (gentamicin, sisomicin) Macrolides (erytrhomycin)

14 Bacilli s metabolites: Peptides such as gramicidin, bacitracin,polymyxin

15 Chemical diversity from Actinomycetes

16 Notes: Even if actinomycetes and fungi have been massively isolated and screened since 1950, their versatile secondary metabolism still provides unique lead compounds for drug and other developments *Searching novel species both by accessing poorly expolited niches and by developing new isolation/cultivation methods *Triggering the expression of cryptic genes for novel enzymes and metabolic pathways

17 Streptomyces coelicolor Model organism ca kbp: genes including more than 20 clusters for secondary metabolite production Streptomyces avermitilis Producer of avermectin, used to treat parasitic infections in livestock and people ca kbp: genes including 30 clusters for secondary metabolite production Saccharopolyspora erythrea Producer of erythromycin that is used to treat pneumonia, bronchitis, and other infections. More than kbp: ca genes

18 Prospecting Microbial Diversity Microbial Sources Pure Strain Selective Media Fermentation Sample treatment Strain Collection Extract Library

19 Antarctic lakes hot-spot for microbial diversity and biotechnological screening MICROMAT (BIO4-CT98-040) Isolation of more than 723 bacteria and 150 fungi from benthic mats in 13 lakes Description of many new bacterial phylotypes Only few actinomycetes Microbial mat

20 Selective isolation of actinomycetes - Spore resistance to dryness and heat - Resistance profile to antibiotics and organic solvents - Metabolic profile (i.e growth on chitin) - Filamentous growth - Spore motility in some genera

21 Isolation plate without selection Isolation plate with selection

22 ISOLATION METHODS FOR SOIL ACTINOMYCETES Actinomadura: heat treatment and a medium containing rifampicin selective agar media (es.chitin based) Actinoplanes: chemotactic methods Kitasatospora: use of novobiocin Micromonospora: alkaline treatment and use of tunicamycin Streptosporangium: heat treatment and vitamins Thermophilic actinomycetes: growth at 50 C

23 QLK3-CT BAC technology environmental DNA library environmental sample Classical isolation methods environmental DNA molecular probes Directed isolation methods description of microbial diversity

24 1900 isolates by selective methods from 50 samples Taxonomic probes: 16S rdna specific primers Metabolic probes: PCR primers to known pathway genes (es.non ribosomal peptide synthetase, isopennicillin N synthase,type II polyketide synthase) environmental DNA Optimization of protocols for DNA extraction and library construction from 4 selected habitats Chemical and biologically guided screening (100 reference strains for dereplication of most common metabolites)

25 An example of industrial collection: VICURON STRAIN COLLECTION: proprietary strains Streptomyces 15% Other bacteria 1% Fungi 35% Other actinomycetes 48% Myxobacteria 0.4%

26 VICURON STRAIN COLLECTION: Non Streptomyces actinomycetes strains 1% 33% 43% classified 0% Streptosporangineae 4% Micromonosporineae 11% Pseudonocardineae Streptomycineae Frankineae (Geo-like) Bacteria-like 51% Ca strains

27 Extract Generation Process Supernatant Replicas [Solid phase] Mycelium Replicas [Solvent]

28 Strain Collection Extract Bank Assay Target Screening HIT Lead Biological & Chemical Profiling, Patent Supply In vivo efficacy, PK, Tox Optimized Lead Clinical candidate

29 Two 'case histories' Novel tetrapeptide inhibitors of bacterial protein synthesis produced by a Streptomyces sp. L.Brandi et al. Biochemistry 2006, 45: A Novel Lantibiotic Acting on Bacterial Cell Wall Synthesis Produced by the Uncommon Actinomycete Planomonospora sp. F.Castiglione et al. Biochemistry 2007, 46: