Timothy M. Vogel

www.GenomEnviron.org

Mining Metagenomes for Novel Enzymes

Extraction

ADN

Culture

in vitro

Cloning

Transformation

vector

Clone Library

PCR

Cloning

and/or

sequencing

RISA, T-RFLP

DGGE,

Phylochip

Molecular

screening

Chemical

screening

Biological

activity

OMe CH3

O

O

CH3

CH3

O

OHOH

OMe

OMe

Lombard et al., 2006

Metagenomic approach:

454 Life science Technology

MS NAT CSA MR

Number of sequences

182 710 163 581 181 022 162 290

Total Length ofSequences (Mb)

46 41 43 38

Average Length of Sequences (bp)

251 247 235 234

Sequence annotation : MG-RAST server (Meyer et al, 2008 )

MS NAT CSA MR

Number of coding sequences

82 504 80 828 121 381 100 524

Number of subsystems

566 583 562 555

Feature in subsystems (%)

55 46 52 51

Hypothetical proteins (%)

20 27 20 20

41.62%

31.59%

PCA comparing general functional subsystems

distributions among 32 metagenomes

41.62%

31.59%

Amino Acids and Derivatives

Carbohydrates

Cell Division and Cell Cycle

Cell Wall and Capsule Clustering.based subsystems

Cofactors. Vitamins. Prosthetic

Groups. Pigments

DNA Metabolism

Dormancy and

Sporulation

Fatty Acids and LipidsMacromolecular Synthesis

Membrane Transport

Metabolism of Aromatic

Compounds

Miscellaneous

Motility and Chemotaxis

Nitrogen Metabolism

Nucleosides and

Nucleotides

Phosphorus Metabolism

Photosynthesis

Potassium metabolism

Prophage

Protein Metabolism

Regulation and Cell signaling

Respiration

RNA Metabolism

Secondary Metabolism

Stress Response

Sulfur Metabolism

Unclassified

Virulence

PCA comparing general functional subsystems

distributions among 32 metagenomes

104

107

4 x 1010

4 x 1013

106

109

“Species”/g soil Number of bp Number of clones

109 4 x 10151011

Number of bp Number of clonesTotal b&a/g soil

40 kb inserts; average genome size 4 x 106

Scientific Committee on Problems of the Environment

SCOPE

http://www.icsu-scope.org/

SCOPE program on Microbial Environmental Genomics

MicroEnGen III

Soil Metagenome International Consortium

METASTED

TERRAGENOME

http://www.terragenome.org/

The long-term experimental site in the

UK: Rothamsted http://www.rothamsted.ac.uk/

Extensive metadata

From 50 to 140 years

of controlled

experiments

Comparison of the relative distribution of ARGD in the

different metagenomes by principal component analysis

d = 0.5

Aac2I

Aac2Ib

Aac2Ic Aac2Id

Aac3Ia

Aac3IIa

Aac3III

Aac3IV

Aac3IX

Aac3VI

Aac3VII

Aac3VIII

Aac3X

Aac6I Aac6Ia

Aac6Ib

Aac6Ic

Aac6Ie

Aac6If

Aac6Ig Aac6IIa

Aac6IIb

aad9Ib

AadD

AcrA

AcrB

AdeA

AdeB

AdeC

AmrA

AmrB

Ant2Ia

Ant2Ib Ant3Ia Ant4IIa

Ant6Ia

Aph33Ib

Aph3Ia

Aph3Ic

Aph3IIIa

Aph3IVa

Aph3Va Aph3Vb Aph3VIa Aph4Ib

Aph6Ia

Aph6Ib

Aph6Ic

Aph6Id

arnA

BacA

Bcr

BcrA

BcrC

BL1_acc

BL1_ampc

BL1_asba

BL1_ceps

BL1_cmy2

BL1_ec

BL1_fox

BL1_mox

BL1_och

BL1_pao

BL1_pse

BL1_sm

BL2_ges

BL2_veb

BL2a_1

BL2a_III

BL2a_III2

BL2be_per

BL2d_lcr1

BL2d_oxa1

BL2d_oxa10

BL2d_oxa2

BL2d_oxa5

BL2d_oxa9

BL2d_r39

BL2e_cbla

BL2e_cepa

BL2e_cfxa

BL2e_fpm BL2e_y56

BL3_ccra

BL3_cit

BL3_cphA

BL3_gim

BL3_imp

BL3_l

BL3_shw

BL3_sim

BL3_vim

Ble

Blt

Bmr

CarA

catA1

catA11

catA13

catA14

catA15

catA16 catA2

catA3

catA4

catA6

catA8

catB1

catB2

catB3

catB4

catB5

CeoA

CeoB

cml_e1

cml_e3

cml_e4

cml_e6

cml_e7

cml_e8

dfrA1

dfrA12 dfrA13

dfrA14

dfrA15 dfrA17 dfrA19

dfrA20

dfrA21

dfrA22

dfrA24

dfrA26

dfrB1

EmeA

EmrD

EmrE

EreB

ErmA

ErmB

ErmC

ErmD

ErmE ErmF

ErmG

ErmH

ErmO

ErmQ

ErmR

ErmT

ErmU

ErmV

ErmW

ErmX

ErmY

FosA

FosB

FosC

FosX

fusH

KsgA

LmrA

LmrB

LmrP LnuA

LnuB

lsa

MacB

MdfA

Mdr

mdtE

mdtF mdtG

MdtH

MdtK

MdtL

MdtM

MdtN

MdtO

MdtP

mecA

MecR1

MefA

MepA

MexA

MexB

MexC

MexD

MexE

MexF

MexH

MexI

MexW

MexX

MexY

MfpA

MphA

MphC

MsrA

NorA

norM

OleB

OpcM

OpmD

OprA

OprJ

OprM

OprN

otrA

otrB

pac

pbp1a

pbp1b

pbp2 pbp2b

pbp2x

PmrA

pur8

Qac

QacA

QacB

QnrA

QnrB

QnrS

RosA

RosB

SmeA

SmeB

SmeC

SmeD

SmeE

SmeF

SrmB

sta

str

Sul1

Sul2

Sul3

tcmA

tcr3

tet

tet30

tet31

tet32

tet33

tet34

tet36

tet37

tet38

tet39

tet40

tet41

tetA

tetB

tetC

tetD tetE

tetG

tetH

tetK

tetL

tetM

tetO

tetPA

tetPB

tetQ

tetS

tetT

tetV

tetW

tetX

tetZ

TlrC

TmrB

TolC

tsnr

VanA

VanB

VanC

VanD

VanE

VanG

VanHA VanHB

VanHD VanRA VanRB

VanRC

VanRD

VanRE

VanRG

VanSA VanSB

VanSC

VanSD

VanSE

VanSG

VanT

VanTE VanTG

VanUG

VanWB

VanWG

VanXA VanXB

VanXD

VanXYC

VanXYE VanXYG

VanYA

VanYB

VanYD

VanYG

VanZ

VatA

VatB

VatC

VatD VatE

VgaA VgaB

VgbA VgbB

YkkC YkkD

d = 1 d = 1

Antarctic

Chicken

Cow

Deep_Ocean

Human_Feces

Mouse

Ocean Sediment

Sludge

Rotham

PC1 (29.1%)

PC

2 (1

8.2

%)

Metagenomes ARGD

Pick soil sample

Separate

bacterial

cells

Lyse cells, extract and

purify DNA

indirect

direct

The soil metagenome

Cut DNA with restriction

enzymes

Clone DNA fragments into the

appropriate vector

Transform preferred host (s):

soil metagenomic library

Screen transformants

Functional: identify

metabolites

Genetic: mine DNA

sequences

Deep soil sequencing

Annotate and assemble

sequence data

Deep soil sequencing

High throughput

shotgun sequencing

technology aiding

metagenomics

Whole-insert sequencing,

Characterization of ORFs

Composite or single samples,

Sample size and suitability,

Enrichments, SIP

Multiple expression hosts in

functional screenings

Clone pooling, Microarrays, SIGEX-

FACS and other high throughput

methods

Screening efficiency for bacterial genes /

metabolites

Soil particles

Nycodenz

cushion

Vector choice: insert and library size, host

range, selection markers, GFP labeling

DNA molecular weight, yield, purity

and integrity

Calibration of DNA fragment

size

Metagenomics

stepsCritical issues

van Elsas et al

Preserved sampling during winter

Diversity differences

and distribution

MIX

Plot 1Plot 2

Plot 3

Plot 5Plot 4

PFGE

:

1 EXTRACTION5 EXTRACTIONS

Montrond (MR)

Châteaurenard (CR)

Cell

extraction

Soil

microbes

Cell lysis

Cell

separation

DNA

purification

Nycodenz CsCl

SOIL

Bertrand et al

Cell separation by Nycodenz density gradient

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

DM SN CSA MR

autres euk

Metazoaires

Plantes

CHAMP

Virus

ARCH

BACT

DIRECT

INDIRECT

454 FLX Data

d = 10

Beadbeatingd1

BBd2

BBd3 BBd4

BBd5

BBd6

BBd7

BBP2 BBP2.1

BBc9

BBc1

BBc2

BBc5

BBtop

BBtop.1

BBtop.2

BBtop.3

Mobio1

M2

M3

M4 M5

M6 M7 M7.1

Abot

Atop

A3 A1A2

Gc9

Gc9

Gc2

Gtop

Gtop

Gtop

Gtop

Gtop Gtop

Gtop

Gtop Gtop Gtop

Gtop

Ttop

Ttop

Ttop Ttop Ttop Ttop

Ttop

Tc

Tc

A4

A5

Aw1 Aw2

Aw3

Bead beating

UltraClean Mobio

Epicentre Gram+

Nucleospin Tissue

AGAROSE PLUG

PCA (Rothamsted soil, axes 1 and 2):

02

46

81

01

21

4

14.6%

13.6%

Direct

Cell ring

Hunting for Nickel resistance

100 m

Ultramafic rock

2-3% of Ni

1-1,5% of Ni

Mine spoils (20g Ni/ kg)

Natural ecosystem (4g Ni/kg)

High levels

of heavy

metalsNi

Co Fe

Essential

element

deficiencies

C, N…

Metagenomic approach

Analysis by sequencing : to determine the bacterial

community structureAnalysis by screening based on activity:

to select nickel resistant clones

Ligation

1. 16S rDNA library

Transformation

Vector

Soil or mine spoils samples

2. Total plasmidic DNA library

Transformation

Ligation

E. Coli

Plasmid pUC19

Direct DNA extraction

16S rDNA amplification

Enzymatic restriction

DNA fragments 2-9kb

Bacterial extraction by gradient of density

DNA extraction (50kb)

Ligation Cosmid pWEB

Encapsidation and transformation in E. coli

3. Total cosmidic DNA library

Bacterial community structure analysisBased on 16S rRNA library sequences

Mine spoils (134 sequences)

Natural soil (116 sequences)

0

5

10

15

20

25

30

35

40

45

50

Prop

ortion

s (%

)

Hery et al

Metagenomic insert identification

DME3

Sequencing

Gene inactivation

Transposon insertion(EZ::Tn5<Kan 2>)

2 sensible clones on nickel

Sub-cloning

Without nickel Nickel (4 mM)

ORF2 nreB Hyp. prot

BamHI BamHI

Tn5-1;8

nreA

Tn5-2;3;6Tn5-4

Tn5-7

Fall et al

Numbers of producers vs numbers of resistors

Production diversity vs resistance diversity Gene transfer and gene diversity Hunt for new molecules

The structure of polyketides produced by PKSI is related to the specific linear organization in domains and modules

Type I PKS (PKSI)

Antibiotic Production Diversity

Ginolhac et al

* A ketosynthase domain (KS) for decarboxylativecondensation of the extender unit onto the growing chain

* An acyltransferasedomain (AT) for substrate selection and transfer

* An acyl carrier protein (ACP) which loads the growing chain

A minimal extender module is composed with at least three domains:

Ginolhac et al

Metagenomic clone

library: 16,000 clones

77 000 clones

Functional:

antagonism against

R. solani AG3

Molecular:

screening for PKS1

biosynthetic genes

Clone selection

Slipstream inhibition of mycelial growth

Hybridization with PKS1 probe

Ginolhac et al

van Elsas et al

Functional and molecular screening

Demanèche et al

Sequenced

clones and

original DNA

by 454 FLX

Ginolhac et al

Identification of active fractionsBioguided Purification

1 11 21 31 41 51 61 71 81 91 101

111

121

131

141

151

161

171

0

20

40

60

80

100

Gro

wth

inh

ibit

ion

(%

)

Fractions

Negative Clone Producing Clone

Clone producteur

Clone Témoin

PRODUCTION D'EXTRAITS

FRACTIONNEMENT

Test des fractions sur Staph. aureus

Gro

wth

inhib

itio

n (

%)

Fractions

Active fraction

Analyse HPLC-MS des fractions activesMax. 1,7e4 mAU.

0 1 2 3 45

6 7 8 910

11 12 1314Time, min

-8000,0

-6000,0

-4000,0

-2000,0

0,0

2000,0

4000,0

6000,0

8000,0

1,0e4

1,2e4

1,4e4

1,6e4

1,8e4

Absorbance

, mA

U

0,98

14,54

5,42

2,35

2,15

3,382,853,64 8,557,01 11,738,024,680,90 11,346,08

Active pic ?

Libragen S.A.

CONCLUSIONS

Metagenomics provides access to « novel » genes

1. Sequencing helps target appropriate environments and

possible gene families

• Sequence interpretation is heavily dependent on

genome sequences in database – need more genomes

sequenced

2. Clone librairies provide phenotypic and genotypic screening

• Phenotypic screening is dependant on host cell, etc, but

can be used to produce targeted enzyme – compound

purification

www.genomenviron.org

Environmental Microbial Genomics Group