flavia marinelli university of insubria, varese. italy · university of insubria, varese. italy ....
TRANSCRIPT
Isolation, screening and cultivation of microbial producers of
“small bioactive molecules”
Dubrovnik Summer School 26-8-2012
Department of Biotechnology and Life Sciences
Microbial Biotechnology Lab FLAVIA MARINELLI
University of Insubria, Varese. Italy
MICROBIAL DIVERSITYVALUABLE PRODUCTS
Biomass
Products fromanaerobic metabolism
Products fromincomplete oxidations
Products fromsecondary metabolism
Enzymes
Polysaccharides
Heterologous proteins
WHAT ARE SECONDARY METABOLITES?
Bu’Lock 1961: not essential for cell life and not found in every growingcell
Low molecular weight (<3000 Da) biosynthesized from one or more general (primary) metabolites by a wider variety of pathways than those involved in general (primary) metabolism.
Each of them is formed by only a few organisms, but some microbes(es.actinomycetes or ascomycota) form a variety of different chemicalclasses.
The onset of morphological differentiation usually coincides with the production of secondary metabolites
Streptomyces coelicolor
By fermentation of producingmicrobes
In liquid cultures, secondarymetabolite production is extremely
dependent on the cultural conditions and generally confined to
the stationary phase of growth
Most of the secondary metabolites are produced as a group of closely related
structures (complex)
HOW SECONDARY METABOLITES ARE PRODUCED?
ANTIBIOTICS
“A chemical substance of microbial origin that possess antimicrobial
activities”
“Low molecular weight chemicalsubstances produced by
microorganisms, which at lowconcentrations inhibit the growth
of other microorganisms”Selman Waksman (left) and
Alexander Fleming (right) in 1951. Science Source / Science
Photo Library
The original approach of “screening” of Selman Waksman 1940
• 1) systematically collect soil microorganims
• 2) growing them in axenic cultures
• 3) testing the culture broths for their ability toinhibit the growth of pathogens
• 4) recovering the active substances produced
Most of antibiotics were discovered during the Golden Age (1940-1950) from soil actinomycetes and fungi by classical screening
Marinelli F and Marcone GL, Small Molecules | Microbial Secondary Metabolites. In: Comprehensive Biotechnology, 2011
ANTIBIOTIC LITERATURE DATABASE:Collecting more than 31.600 entries describing bioactive metabolites of microbial origin from literature and patents since 1950 till 2006
HOW MANY MICROBIAL SECONDARY METABOLITES
ARE KNOWN ?
20.200 microbial secondary metabolites possessing some activity in vitro
F.Marinelli, Methods in Enzymology,2009
ANTIBIOTIC LITERATURE DATABASE*:Collecting more than 31.600 entries describing bioactive metabolites of microbial origin from literature and patents since 1950 till 2006
HOW MANY MICROBIAL SECONDARY METABOLITES
ARE KNOWN ?
Algicides
HerbicidesAntiviralAntiparasitic
Immunomodulator
Pharmacological
Antitumor
Antifungal
AntibacterialSiderophore
20.200 microbial secondary metabolites possessing some activity in vitro
F.Marinelli, Methods in Enzymology,2009
Introduction of novel assays for discovering antitumors, immunomodulators, anti-inflammatory and antiviral drugs, insecticide
and antiparasitic compounds
Class of activityAssay system Examples References
Anticancer Cell-based: cytotoxicityversus rapidly proliferating cells /tumoral cell lines
Cytotoxicity/growth inhibition versus mouse L929 fibroblasts, human T-24 bladder carcinoma cells, leukaemia L1210 cells
(Gerth et al., 1996; Newman and Shapiro, 2008; Reichenbach and Hofle, 2008; Wani et al., 1971)
Immunosuppressant In vivo in animalsCell-based: inhibition of immune responseCell-free: inhibition of receptor-ligand binding
Haemagglutinin test in miceSuppression of mixed lymphocyte reaction in mouse cell linesInhibition of interleukin-2 production in mouse cell lines Immunoassay based on the cyclosporine binding to cyclophilin
(Borel et al., 1976; Mann, 2001; Quesniaux et al., 1987; Reynolds and Demain, 1997)
Anticholesterolemic Cell-free: enzyme inhibition
Inhibition of 3-hydroxy-3-methylglutaryl-coenzyme A reductase
(Alberts et al., 1980; Endo, 1980; Endo et al., 1976)
Anti inflammatory Cell-free: inhibition of receptor ligandbinding
Immobilized-ligand IL-1 receptor binding assay Particle concentration fluorescence receptor binding assay
(Stefanelli et al., 1997)
Antihyperglycemic Cell-free:enzymeinhibition;genetic screening
Inhibition of α-glucosidasesPCR based method to detect sedo-heptulose 7-phosphate cyclase
(Hyun et al., 2005)
Antiviral Cell-free: enzyme inhibition of viral enzymes
Inhibiton of HIV-1 integrase (Singh et al., 2003)
Antiparasitic In vivo in animals Activity in mice against the nematode Nematosporides dubius
(Vining, 1990).
Insecticide In vivo in insects Microbial solid cultures supplied as food to larvae and to adults of Muscadomestica
(Fabre et al., 1988)
Secondary metabolites today: not only antibiotics
Antibiotics, pigments, toxins, siderophores, effectors of
ecological competition and symbiosis, pheromones,
enzyme inhibitors, immunomodulating agents, receptor
antagonists or agonists, pesticides, antitumor agents,
growth promotants of animals and plants …………………..
WHO ARE THE MICROBIAL PRODUCERS ?
Distribution of the microbial producers of the 20.200 active secondary metabolites
Soil Ascomycota
Soil high G+Cgram-positives
Bacillus, Pseudomonas, Myxobacteria, Cyanobacteria
F.Marinelli, Methods in Enzymology,2009
GENUS PRODUCTS USEPenicillium Penicillin,
GriseofulvinAntibacterialAntifungal
Acremonium CephalosporinFusidic acid
AntibacterialAntibacterial
Aspergillus EchinocandinsLovastatin
AntifungalHypocholesterolemic
Zalerion Pneumocandins AntifungalTolypocladium Cyclosporin ImmunomodulatorFusarium Zearalenone AnabolicCepahlosporium Cerulenin Hypolypidic
Products from Ascomycota
CANCIDAS (Merck)2001
ECALTA (Pfizer)2007
““NovelNovel”” antifungalantifungal agentsagents
((launchedlaunched in the in the lastlast ten ten yearsyears))
Novel mechanism of action
KnownKnown antifungalsantifungals’’mechanismsmechanisms of actionof action
StatinsStatins, fungal metabolites acting as cholesterol, fungal metabolites acting as cholesterol--lowering lowering agents due to their inhibition of eukaryotic 3agents due to their inhibition of eukaryotic 3--hydroxyhydroxy--33--
methylglutarylmethylglutaryl--coenzyme A coenzyme A reductasereductase, , discovered and discarded as antifungal agentsdiscovered and discarded as antifungal agents
ß-lactams (thienamycin) Aminoglycosides (streptomycin) Macrolides (tylosin) TetracyclinesStreptogramins CloramphenicolLincomycin FosfomycinNovobiocin DaptomycinFidaxomicinPolyenes (amphotericin) Antracyclines (adriamycin) BleomycinsTacrolimus&SirolimusClavulanic acid AvermectinsIonophoric polyethersBialaphosPolyoxinsLipstatin
AACCTTIINNOOMMYYCCEETTEESS
Streptomyces’sproducts on the market
Starting in the 1960s and 1970s to the present, a considerable proportion of toxic antibiotics were retested as anticancer drugs because of their inhibitory effect toward rapid proliferating eukaryotic cells.
Another group of important metabolites initially often isolated in antibiotic screening and then “rediscovered” between the 1970 and 1990s for other useful pharmacological activity in animals are those
showing an immunomodulating property, such as agents for organ transplantation (i.e. Tacrolimus and Sirolimus).
Screening for metabolites inhibiting nematodes, cestodes, and protozoa found new products such as Avermectins, which are potent and specific inhibitors of invertebrates and lack antibiotic activity
Discovery of novel drugsby developing innovative screening
systems
Mining and improvingthe drugs’ microbial
producers
Baltz’s analyses of old & new screening campaigns
104 screened soil actinomycetes: 2,500 antibiotic producers including2,100 making streptothricin (2x10-1)
125 making streptomycin (1x10-2)40 making tetracycline (4x10-3)
..................vancomycin producers (1.5x10-5)erythromycin producers (5 X10-6)
.............daptomycin producers (1x10-7)
SIM news, 2005J Ind Microbiol Biotechnol, 2006
The problem of finding and re-finding the same molecules
Daptomycin (cubicin) launched in 2003: a novel mechanism of action vs. multi-resistant
Gram positive pathogens
NH
NH
HN (CH2)8CH3
O
OCONH2
COOH
NH
O
HNNH
HN
O
COOH
O
O
NH
HN
NH2
O
O
ONH
O
O
H2N
O
NH
HN
COOH
O
OH
HN
HOOC
O
O
DaptomycinDaptomycin produced from produced from StreptomycesStreptomyces roseosporusroseosporus
Chances of finding new bioactivesmall molecules
Searching novel species both by accessing poorly expolited niches and by developing new isolation/cultivation methods
Genome mining and heterologous expression of secondary metaboliteclusters
Triggering the expression of cryptic genes by optimizing medium and fermentation conditions
Chemical screening by novel techniques
Metagenomic approach to uncultivable microbial diversity
“Dereplication” of identical/similar strains usingtools ranging from morphology to chemotaxonomic
markers and diverse molecular and chemicalapproaches
The The assumptionassumption isis thatthat differentdifferent strainsstrainsmaymay produce produce differentdifferent chemicalschemicals
Microbial Microbial diversitydiversity asas anan imperfectimperfectsurrogate of surrogate of chemicalchemical diversitydiversity
IsolationIsolation plateplate withoutwithout
selectionselectionIsolationIsolation plateplate withwith
selectionselection
Each gram of soil contains 106-108
bacteria, 104-106 actinomycetesspores, 102-104 fungal spores
Selection methods: media, temperature, pH,resistanceprofile
Selective isolation of actinomycetesfrom soil
- Resistance to antifungal agents
- Spore resistance to dryness and heat
- Resistance profile to antibiotics and organic solvents
- Metabolic profile (i.e growth on chitin or xylose/xylans)
- Filamentous growth
-Spore motility and chemotaxy in some genera
- Resistance to phages
- Resistance to NaCl and different pHs
Isolation methods for uncommon actinomycetesActinoplanes: chemotactic methods (mobile spores)resistance to novobiocinMicromonospora: alkaline treatment and use of tunicamycin, resistance to CO2Actinomadura: heat treatment and a medium containing rifampicin and/or streptomycin and /or kanamycinselective agar media (es.chitin-based) Thermophilic actinomycetesgrowth at 50°CStreptosporangium: heat treatment and vitamins, resistance to solvents
LazzariniLazzarini etet al, 2000 al, 2000
Distribution of the actinomycete producers
on a sample of 5200 antibacterial/antifungal molecules
WHICH IS THE CONTRIBUTION FROM DIFFERENT FAMILIES OF FILAMENTOUS ACTINOMYCETES?
Streptomycetaceae 70%
OtherStreptosporangiaceae
Micromonosporaceae
Thermomonosporaceae
Pseudonocardiaceae
WHICH IS THE CONTRIBUTION FROM DIFFERENT FAMILIES OF FILAMENTOUS ACTINOMYCETES?
F.Marinelli & L.Marcone, Comprehensive Biotechnology 2° ed. 2011
AACCTTIINNOOMMYYCCEETTEESS
PseudonocardiaceaePseudonocardiaceae
Micromonosporaceae
Streptosporangiaceae
Thermomonosporaceae
Streptomycetaceae
Glycopeptides (teicoplanin) Lantibiotics (actagardine)Ramoplanin FidaxomycinAcarboseAminoglycosides (gentamicin)
Glycopeptides (vancomycin)RifamycinsMacrolides
(erytrhomycin,spinosyns)ThermorubinGE23077
Glycopeptides (A40926)
Thiazolylpeptides(GE2270)
Lantibiotics(planosporicin, microbisporicin)
Alternative sources other than soil samples
Endophytic fungi and actinomycetes
nonpathogenicnonpathogenic organismsorganisms thatthat residereside withinwithin plantplanttissuestissues ((slow slow growersgrowers) and ) and provideprovide theirtheir hostshostswithwith biochemicalbiochemical protectionprotection againstagainst otherothermicrorganismsmicrorganisms and and insectsinsects
Marine Marine actinomycetesactinomycetes and fungiand fungi
obligateobligate or or facultativefacultative marine marine microbesmicrobes leavingleaving in in marine marine sedimentssediments or or asas epiepi-- or or endobiontsendobionts in in marine marine invertebratesinvertebrates ((spongessponges, , tunicatestunicates, , bryozoansbryozoans))
SymbioticSymbiotic bacteriabacteria and fungi and fungi in in insectsinsects, , wapswaps, , antsants, , lichenslichens ………………………………..
………………………………………………………………cultivatingcultivating the the unculturableunculturable??
DeterminedDetermined microbiologistsmicrobiologists bringbringculture culture toto the the ““uncultureduncultured”” !!
OligotrophicOligotrophic isolationisolation mediamedia
EncapsulationEncapsulation in in polymerpolymer gelsgels or or membranemembrane
Microbial Microbial consortiaconsortia
AdditionAddition of of elicitorselicitors
AnalysisAnalysis of of physiologicalphysiological//enviromentalenviromentalparametersparameters
Streptomyces coelicolorModel organism
•ca. 8.66 Mb: 7.825 genes including more than 20 clusters forsecondary metabolite production
Streptomyces avermitilisProducer of avermectin, used to treat parasitic infections in livestock and people
Saccharopolyspora erythreaProducer of erythromycin that is used to treatpneumonia, bronchitis, and other infections.
•ca. 9.02 Mb: 7.575 genes including 25 clusters for secondarymetabolite production
•More than 8.2 Mb: ca. 7.264 genes including at least 25 clusters forsecondary metabolites
ObligateObligate marine marine actinomycetesactinomycetes belongingbelonging totoMicromonosporaceaeMicromonosporaceae and and producingproducing
manymany secondarysecondary metabolitesmetabolites
5.18 Mb, 4,539 predicted genes,
17 secondary metabolite gene clusters
WhatWhat news?news?More More thanthan 250 250 genomesgenomes fromfrom actinobacteriaactinobacteria sequencedsequenced includingincluding manymany antibioticantibiotic
producingproducing actynomycetesactynomycetes ::
--DraftDraft genomegenome sequencesequence of of anan efficientefficient antibioticantibiotic--producingproducing industrialindustrial strainstrain of of SaccharomonosporaSaccharomonosporaazureaazurea, SZMC 14600, SZMC 14600
--GenomeGenome sequencessequences of of threethree tunicamycintunicamycin--producingproducing StreptomycesStreptomyces StrainsStrains, S. , S. chartreusischartreusis NRRL NRRL 12338, S. 12338, S. chartreusischartreusis NRRL 3882, and S. NRRL 3882, and S. lysosuperificuslysosuperificus ATCC 31396. ATCC 31396.
--DraftDraft genomegenome sequencesequence of of StreptomycesStreptomyces clavuligerusclavuligerus NRRL 3585, a NRRL 3585, a producerproducer of diverse of diverse secondarysecondarymetabolitesmetabolites
--DraftDraft genomegenome sequencesequence of marine of marine StreptomycesStreptomyces sp. sp. strainstrain W007, W007, whichwhich producesproduces angucyclinoneangucyclinoneantibioticsantibiotics withwith a a benzbenz[a]anthraceneanthracene skeletonskeleton
--WholeWhole GenomeGenome SequenceSequence of the of the RifamycinRifamycin BB--ProducingProducing StrainStrain S699S699
--Complete Complete genomegenome sequencesequence of of StreptomycesStreptomyces cattleyacattleya NRRL 8057, a NRRL 8057, a producerproducer of of antibioticsantibiotics and and fluorometabolitesfluorometabolites
-Genome sequence of the abyssomicin- and proximicin-producing marine actinomycete Verrucosisporamaris AB-18-032.
-Genome sequence of the Spinosyns-producing bacterium Saccharopolyspora spinosa NRRL 18395
--DraftDraft GenomeGenome of of StreptomycesStreptomyces tsukubaensistsukubaensis NRRL 18488, the NRRL 18488, the ProducerProducer of the of the ClinicallyClinically ImportantImportantImmunosuppressantImmunosuppressant TacrolimusTacrolimus (FK506) (FK506)
Insights into genomes of secondary metaboliteproducing strains
0
100
200
300
400
500
600
700
1980 1990 2000
MyxobacteriaCyanobacteria
Discovery events
OtherOther promisingpromising bacterialbacterial groupsgroups
difficultdifficult toto isolate and isolate and cultivatecultivate
Cell cycle of Myxococcus xanthus
Morphological diversity of myxobacteria
MyxobacteriaMyxobacteria::
Social Social glidinggliding predatorpredatorgramgram--negativenegative bacteriabacteria
((δδ--groupgroup of of proteobacteriaproteobacteria))
Genome size of Myxococcusxanthus: 9.1 Mb
GoldmanGoldman etet alal.PNAS.PNAS 20062006
AntineoplasticAntineoplastic agentsagents possessingpossessing antitubulinantitubulinactivityactivity producedproduced bybySorangiumSorangium cellulosumcellulosum
A derivative (A derivative (ixabepiloneixabepilone) ) approvedapprovedbyby FDA (FDA (OctoberOctober 2007) 2007)
forfor refractoryrefractory breastbreast cancercancerEpothilone B
LargestLargest bacterialbacterial genomegenome sequencedsequenced toto date : date : more more thanthan 13 13 MbMb, 17 , 17 secondarysecondary metabolitemetabolite lociloci
ChemicalChemical DiversityDiversity::
More than 100 different basic structure and 500 structural variants.
Many secondary metabolites have novelmechanism of action
BiosyntheticBiosynthetic diversitydiversity: :
Exceptional combination of PKS and NRPS
WenzelWenzel and and MMüüllerller, , NaturalNatural ProductProduct ReportsReports 20072007
Samples *) Method of isolation
Number of isolates†)
(total=190)
Method of purification
Number of pure
cultures (total =100)
Number of pure cultures per isolation
method
Efficiency of purification methods per
isolation method (%)
Soils (45)
Dung pellets 62 (32.6%)
Second baiting technique 35
51 82.3Direct
purification 8
Sonication 7
Antibiotics 1
E.colibaiting 48 (25.3%)
Second baiting technique 7
15 31.2Direct
purification 2
Sonication 5
Antibiotics 1
Filter paper 24 (12.6%)
Second baiting technique 0
2 8.3Direct
purification 0
Sonication 1
Antibiotics 1
Barks(22) Moist chamber 56 (29.5%)
Second baiting technique 7
32 57.1Direct
purification 20
Sonication 4
Antibiotics 1
GaspariGaspari etet al.al. JAM, 2005JAM, 2005
Isolation of myxobacteria from Israel
Suborder Family Genus Isolated PurifiedCystobacterineae Myxococcaceae Myxococcus 78 65
Corallococcus 34 17
Angiococcus 1 0Cystobacteraceae Archangium 16 10
Cystobacter 8 0Melittangium 4 0
Stigmatella 5 3Sorangineae Polyangiaceae Sorangium 20 0
Polyangium 2 0Chondromyces 2 1Haploangium 0 0
Nannocystaceae Nannocystis 11 0Non identified 9 4
Total strains 190 100
GaspariGaspari etet al.al. JAM, 2005JAM, 2005
Difficult isolation, purification and cultivation
Extremely low yieldsNone scaling-up
Need to develop heterologous expression
developed epothilone heterologous hosts: S. venezuelae, S. coelicolor, M. xanthus, and E. coli
still the industrail producer strain is the mutagenized homologousproducer Sorangium cellulosum
Criteria to be fulfilled
- Be able to produce secondary metabolites
- Possess diversified pathways for secondarymetabolites
- Present significant genetic diversity
- Be retrievable in large numbers
- Be amenable to scale-up for large volumes
Busti et al. Microbiology 2006
Prospecting Microbial Diversity means alsoHigh throughput cultivation and screening
Selective Media
Microbial Sources
Pure Strain
Fermentation
Sample treatment
Extract LibraryStrain Collection
OSMAC*: one strain, many compounds
LongLong--debateddebated questionquestion in microbial in microbial screening: screening:
shouldshould more relative more relative efforteffort bebe appliedapplied totoscreening more screening more phylogeneticphylogenetic diversitydiversity or or
more more physiologicalphysiological parametersparameters??
*Bode*Bode etet alal.20.200202
Fermentation & Extract Generation Process in Screening
Supernatant
Mycelium
“Replicas”
“Replicas”
[Solid phase]
[Solvent]
In In generalgeneral threethree toto fivefivefermentationfermentation//enviromentalenviromental conditionsconditionsper per eacheach strainstrain in in industrialindustrial screening screening
programprogram
Miniaturized parallel fermentations in microplates
"Miniaturise, without compromise !“Enzyscreen
www.enzyscreen.com
System Duetz:
a set of a set of toolstools developeddeveloped at the ETH, at the ETH, ZurichZurich forfor the the parallelparallel preservationpreservationand and cultivationcultivation in 96in 96--or 24or 24--well well microplatesmicroplates
Bacteria, , Duetz et al. Appl Env Microbiol2000
Streptomycetes, Minas et al. Antonievan Leeuwenhoek, 2001
Fungi, Bills et al. J App Microbiol 2008
Actinoplanes teichomyceticus, Taurino et al. Microb Cell Factories, 2011
High throughput cultivation
Inoculum
Fermentative medium
Supernatant
30 ml
myceliumFiltration
Ethanol 20 ml
50 ml
replicas
200 µl-well
Resin
Strain collec.
Other actinomycetes
48%
Myxobacteria 0.4%
Fungi 35%Streptomyces 15%
Other bacteria 1%
An example of industrial microbial collection:
70.000 strains
RecentRecent developmentsdevelopments in in analyticalanalytical techniquestechniques(LC(LC--MS, UHPLCMS, UHPLC--DAD, NMR DAD, NMR etcetc))
BiologicalBiological activityactivity--basedbased screeeningscreeening
versusversus
chemistrychemistry--basedbased screening ?screening ?
High throughput assays for anti-infectives
Antibacterial projects
Cell wall inhibitors
Protein synthesis inhibitors
RNA polymerase inhibitors
Antimicrobial whole cells
(Gram -, MR Gram+, specific strains)
Antifungal projects
Protein synthesis inhibitors
Cell wall inhibitors
Antimicrobial whole cells
(yeasts, filamentous, specific strains)
Current trends
Screening of chemical, semisynthetic and natural product libraries by made available bybig pharma donations
Mining old targets validated by usefulantibiotics
Re-evaluation of “negletected molecules”(es.mannopeptimycins,ramoplanin,fidaxomicin)
Introduction of cell lines and animal model in the initial phases of screening (es.nematodeCaenorhabditis elegans)
Genomics-metagenomics, genetic/functional/bioinformatic screening and heterologous expression