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

Metagenomics

1 g soil contains 2000 - 18000 prokaryotic genomes (~99 % unexplored)

Insights into metagenomics for smallmolecules

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

Our pannel of media for actinomycetes

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 ?

AnAn exampleexample of of extractextract collectioncollection

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

A screening 'case history‘“safe to say that a good established target (as bacterial cell wall) is

better than a new target”

Novel Lantibiotics Acting on Bacterial Cell Wall Synthesis Produced by

Uncommon ActinomycetesF.Castiglione et al. Biochemistry 2007

F.Castiglione et al. Chem&Biol 2008