The Marine Biodiscovery PipelineMarcel JasparsMarine Biodiscovery CentreUniversity of AberdeenScotland, UKm.jaspars@abdn.ac.uk

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Professor Marcel Jaspars

• BA Hons (Cantab) in Natural Sciences (Chemistry)• PhD in Synthetic Organic Chemistry• Post-doc with Prof Phil Crews, University of California, Santa Cruz on

Marine Natural Products• Professor of Organic Chemistry & Director of Marine Biodiscovery

Centre, University of Aberdeen• Project Leader, PharmaSea Consortium• Co-Author of ESF Marine Board Position Paper “Marine Biotechnology

– A New Vision and Strategy for Europe”• Visiting Professor (20%) at University of Tromsø• Scientific advisor to MabCent, Tromsø• Member of the Industrial Biotechnology Sector Group of the

Biosciences Knowledge Transfer Network• Unpaid advisor to Aquapharm Biodiscovery Ltd and Glycomar Ltd, UK.

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Why Use Marine Bioresources?

Offers advantage over comparable terrestrial resource:Superior performanceBetter economics

Unprecedented activity in particular application:Enzymes: new reactivity/new biotransformationSmall molecules: new mechanism of actionMaterials: new properties

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Biodiscovery is the discovery of compounds and associated ideas from natural sources to develop novel biomedicines.Biodiscovery generates

chemical diversity that is used to find initial biological activity in disease focused screensBiodiscovery also includes

the development of biomedical research tools, antifoulants, catalysts, nutraceuticals and cosmeceuticals.

The Marine Biodiscovery Process

Development

Sampling

Curation

Biomass

Extraction

Assay

Purification

Active NCE

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Why Marine?

Diversity of Habitat

Jørgensen Nat Rev Microbiology, 2007, 5, 770

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Extreme Marine Environments

Deep Oceans95 % > 1000 m deep50 % > 3000 m deepAverage depth = 3790 m1-3% trench ecosystems

Cold Oceans

Thermal Vents

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Marine Animal Biodiversity

0

1

2

3

4

5

Porife

ra

Placo

zoa

Cnida

ria

Cten

opho

ra

Platyh

elmint

hes

Gnath

ostom

ulida

Neme

rtea

Nema

toda

Rotife

ra

Gastr

otrich

a

Kinorh

ynch

a

Loric

ifera

Tardi

grada

Priap

ula

Mollu

sca

Kamp

tozoa

Pogo

noph

ora

Sipun

cula

Echiu

ra

Anne

lida

Onyc

hoph

ora

Crus

tacea

Cheli

cerat

a

Unira

mia

Chae

togna

tha

Phoro

nida

Brac

hiopo

da

Bryo

zoa

Echin

oderm

ata

Hemi

chord

ata

Uroc

horda

ta

Ceph

aloch

ordata

Verte

brata

Phylum

Spec

ies

estim

ate

(Log

) MarineTerrestrial

Chart1

PoriferaPorifera

PlacozoaPlacozoa

CnidariaCnidaria

CtenophoraCtenophora

PlatyhelminthesPlatyhelminthes

GnathostomulidaGnathostomulida

NemerteaNemertea

NematodaNematoda

RotiferaRotifera

GastrotrichaGastrotricha

KinorhynchaKinorhyncha

LoriciferaLoricifera

TardigradaTardigrada

PriapulaPriapula

MolluscaMollusca

KamptozoaKamptozoa

PogonophoraPogonophora

SipunculaSipuncula

EchiuraEchiura

AnnelidaAnnelida

OnychophoraOnychophora

CrustaceaCrustacea

ChelicerataChelicerata

UniramiaUniramia

ChaetognathaChaetognatha

PhoronidaPhoronida

BrachiopodaBrachiopoda

BryozoaBryozoa

EchinodermataEchinodermata

HemichordataHemichordata

UrochordataUrochordata

CephalochordataCephalochordata

VertebrataVertebrata

Marine

Terrestrial

Phylum/Subphylum

Species estimate (Log10)

3

1

3

1

3

2

2

2

1

3

3

1

1

2

2

1

1

1

1

5

3

1

2

2

1

2

4

3

1

4

2

2

4

1

5

1

1

2

3

3

1

3

1

3

3

Chart2

PoriferaPorifera

PlacozoaPlacozoa

CnidariaCnidaria

CtenophoraCtenophora

PlatyhelminthesPlatyhelminthes

GnathostomulidaGnathostomulida

NemerteaNemertea

NematodaNematoda

RotiferaRotifera

GastrotrichaGastrotricha

KinorhynchaKinorhyncha

LoriciferaLoricifera

TardigradaTardigrada

PriapulaPriapula

MolluscaMollusca

KamptozoaKamptozoa

PogonophoraPogonophora

SipunculaSipuncula

EchiuraEchiura

AnnelidaAnnelida

OnychophoraOnychophora

CrustaceaCrustacea

ChelicerataChelicerata

UniramiaUniramia

ChaetognathaChaetognatha

PhoronidaPhoronida

BrachiopodaBrachiopoda

BryozoaBryozoa

EchinodermataEchinodermata

HemichordataHemichordata

UrochordataUrochordata

CephalochordataCephalochordata

VertebrataVertebrata

Marine

Terrestrial

Phylum

Species estimate (Log)

3

1

3

1

3

2

2

2

1

3

3

1

1

2

2

1

1

1

1

5

3

1

2

2

1

2

4

3

1

4

2

2

4

1

5

1

1

2

3

3

1

3

1

3

3

Phyla Figure

PHYLA AND DISTRIBUTION IN THE ANIMAL KINGDOM

Habitat

PhylumMarineFreshwaterTerrestrialSymbiotic

Porifera311

Placozoa1

Cnidaria32111

Ctenophora11

Platyhelminthes313214

Gnathostomulida2

Nemertea21111

Nematoda31313133

Rotifera1122111

Gastrotricha22

Kinorhyncha2

Loricifera1

Tardigrada121

Priapula1

Mollusca5133111

Kamptozoa111

Pogonophora2

Sipuncula21

Echiura2

Annelida41232

Onychophora1

Crustacea4332222

Chelicerata21224321

Uniramia11325322

Chaetognatha11

Phoronida1

Brachiopoda2

Bryozoa31

Echinodermata31

Hemichordata1

Urochordata31

Cephalochordata1

Vertebrata33233311

BPBPMXEcEn

Numbers indicate the approximate number of recorded living species:

1 = 1-102; 2 = 102-103; 3 = 103-104; 4 = 104-105; 5 = 105 or more. (Chris these are exponents)

B = benthic; P = pelagic; M = moist; X = xeric; Ec = ecto; En = endo.

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Marine Microbial Diversity

Streptoverticilum

Taxonomic ‘space’Marine and terrestrial speciesclearly separated.

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Biological Diversity = Chemical Diversity

H

NNH H

Br N

O

N

OH

BrO

Br

N

N

O

HO

Br

O

OH

OH

COOH

OO

OH

O

O

O

OH

OOH

O

OH

HOOH

OH

OH

O

OH

HOOH

N

HO

H

HH

HO

NN

NOH

N+H

H

H

OH

H3C

O

OH

O

OHO

O

O

HOH

H H

H

OHHO O

HO

O

H

H

OHOH

HHO

O

O

NHN

S

HN

SN

NH

O

O

ON

N

O

O

O

O

HO

H

OHOOH

OH

O

HN

N

N

O

OO

N

HN

O

N

O NH

HN

O

HN

O

HN

N

HN

N

OO

OO

HN

O

HN

NH

NNH

OO

O

OH

ON

OMe

BrBr

HN

OO

Br

Br

HO

R

HN

N

O

OHMeO

Br

Br

O R

Br

Small Molecules Biomolecules

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Marine and Terrestrial Chemical Diversity are Different

• 71% of scaffolds are exclusively marine

• These cover only 30% of marine natural products

• Many marine natural products scaffolds appear only once

Kong, Drug Discovery Today, 2010, 15, 884

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Marine Natural Products on the Market

Vent Polymerase w-3 polyunsaturated fatty acidsfor heart disease

Prialt for pain Halaven for cancer

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The Marine Biodiscovery Process

Development

Sampling

Curation

Biomass

Extraction

Assay

Purification

Active NCE

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Little Sampling Done Beyond 3000 m

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Research Vessels

• High daily rate• Limited number of vessels globally• Access competitive• Long time between bid for time and actual cruise• Many different types of science accommodated may lead to compromises• Shared resources and bartering systems operate to optimise usage

RRS Discovery (UK) Chikyu (Japan)

http://www.marineinsight.com/wp-content/uploads/2013/03/RRS-Discovery.jpghttp://www.marineinsight.com/wp-content/uploads/2013/03/RRS-Discovery.jpg

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Submersibles

ROV Isis (UK)(6500 m)

Shinkai (Japan)(6500 m)

ROV Nereus (US)(11000 m)

Deepsea challenger (US)(11000 m)

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Data Logging for Research Cruises

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Sampling Devices

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The Marine Biodiscovery Process

Development

Sampling

Curation

Biomass

Extraction

Assay

Purification

Active NCE

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Metadata may includeLocationDepthTemperatureSalinitypHOxygen contentSeafloor conditions

Sample Data and Storage

Sample storageAmbient temperatureCooler (4oC)Freezer (-20oC)-80oC FreezerLiquid nitrogen (-196oC)FormaldehydeEthanolDNA/RNA preservation liquids

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Are Current Repositories Sufficient?

Further sampling is essential for the following reasons:• Origin may be difficult to ascertain (eg location, depth, collector,

date, ownership etc). A minimal data set is imperative. • IP status not clear• May not have been collected in a way consistent with proposed use• May not have been stored correctly to ensure sufficient quality for

proposed use.• The amount of material may not be sufficient for proposed multiple

uses. • Very few locations have been sampled so repository may not be

representative of ABNJs.

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If previous points can be addressed then such a repository might be viable• The rules for terrestrial biorepositories may not apply for

their marine equivalent. • Much information on biorepositories is based on situation

with respect to plants where samples can be propagated. • A deepsea core sample or a marine macroorganism

collected on one sampling expedition is finite. • Microbes can be cultured but again culturing a microbe

from a hydrothermal vent is quite different from culturing a microbe from a terrestrial habitat.

An ABNJ Sample Biorepository?

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The Marine Biodiscovery Process

Development

Sampling

Curation

Biomass

Extraction

Assay

Purification

Active NCE

23

Biomass

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The Marine Biodiscovery Process

Development

Sampling

Curation

Biomass

Extraction

Assay

Purification

Active NCE

25

Extraction and Purification

Solvent-solventpartition

Size-exclusion chromatography High performance

Liquid chromatography

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The Marine Biodiscovery Process

Development

Sampling

Curation

Biomass

Extraction

Assay

Purification

Active NCE

27

Assay

S1Inactive

S2Inactive

S3Inactive

S4Inactive

S6F1Inactive

S6F2Inactive

S6F3Inactive

S6F4H1Inactive

S6F4H2Inactive

Pure CompoundActive

S6F4Active

S5Active

S6Inactive

Crude extractActive

Cell based Enzyme based

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The Marine Biodiscovery Process

Development

Sampling

Curation

Biomass

Extraction

Assay

Purification

Active NCE

29

Structure Determination

ppm (t2) 1.0 2.0 3.0 4.0 5.0 6.0 7.0

50

100

150

ppm (t1)

Spectroscopic data

NH SON N

NH

ON

NH

S

NNH

O

O

O

O 2D Structure

3D Structure

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Metagenomic Approach

Prochloron gDNA

VTACITFCTACITFCVACITFCVTCITFCVTAITFCVTACTFCVTACIFCVTACITCVTACITF

Genomesequencing

Search for allpermutations

50000bp

50000bp

50000bp

Shotguncloning

E. coliclone library

Identificationof biosynthetic

pathway

1 2 3 4 5 6 7 8 9 10 11 12

A

B

C

D

E

F

G

H

Positive plate1 2 3 4 5 6 7 8 9 10 11 12

A

B

C

D

E

F

G

H

Positive column

1 2 3 4 5 6 7 8 9 10 11 12

A

B

C

D

E

F

G

H

Positive well

LC-MS deconvolutionof clone library

50000bp

50000bp

50000bp

Cloning and heterologous

expressionof pathway

50000bp

Identification ofproductive clones

Sequencing

gDNA

NH

SO

N N

HN

ON

NH

S

N HN

R1

O

O

O

R4

O

R2

R3

Product

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Bioinformatic Databases and the Metagenomic Approach

• Who acquired/deposited the data and with what authority?

• Who has access to the data?• Is genome and metagenome information

sufficient?• Many genes found in marine species are

not in the current bioinformatic databases• The function of many of these genes

cannot be determined without laboratory work

• Difficulty in cloning genes of marine origin• Lack of suitable tools (vectors/hosts)

0

200

100300

400

50

150250

350

EhV-86407,339 bp

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Gene Synthesis

GATTACAGGACGCTTATTTTTCGACGATGCTTGGGGAAATGCAAAGATTCAGCTAAAGTC

Gene sequence

DNA

ProteinMolecule

The Marine Biodiscovery PipelineProfessor Marcel JasparsWhy Use Marine Bioresources?The Marine Biodiscovery ProcessWhy Marine?Extreme Marine EnvironmentsMarine Animal BiodiversityMarine Microbial DiversityBiological Diversity = Chemical DiversityMarine and Terrestrial Chemical Diversity are DifferentMarine Natural Products on the MarketThe Marine Biodiscovery ProcessLittle Sampling Done Beyond 3000 mResearch VesselsSubmersiblesData Logging for Research CruisesSampling DevicesThe Marine Biodiscovery ProcessSample Data and StorageAre Current Repositories Sufficient?An ABNJ Sample Biorepository?The Marine Biodiscovery ProcessBiomassThe Marine Biodiscovery ProcessExtraction and PurificationThe Marine Biodiscovery ProcessAssayThe Marine Biodiscovery ProcessStructure DeterminationMetagenomic ApproachBioinformatic Databases and the Metagenomic ApproachGene SynthesisSlide Number 33