Bacterias para el medio ambiente: Bacterias para el medio ambiente:

de la Bioremediación de la Bioremediación

a la Biología Sintéticaa la Biología Sintética

Víctor de LorenzoVíctor de Lorenzo

Centro Nacional de Biotecnología Centro Nacional de Biotecnología

Madrid (Madrid (SpainSpain))

La Cloaca Maxima (Roma, s. V adC)

Los tres dominiosdel árbol de la vida

LUCA

Microbial properties of environmental interest

• Biodegradative pathways for recalcitrant compounds

• mineralization of toxic pollutants

• removal or organic sulphur from soil

• biosensors

• green chemistry

• Tolerance to heavy ions

• volatilization

• bioavailability/toxicity assays

• bioaccumulation:removal of toxic ions

• biotransformation and re-speciation

• Surfactant production: bioremediation of oil spills

BIO-REMEDIACION

ATóxico

BIO-REMEDIACION

A BTóxico Inocuo

BIO-REMEDIACION

A BTóxico Inocuo

Catalizador biológico

BIO-REMEDIACION

A BTóxico Inocuo

Catalizador biológico

BIO-REMEDIACION

B

CO2 + H2Omineralización

C transformación

B inmovilización

• Natural attenuation

• Bioaugmentation

(incl. genetic engineer.)

• Biostimulation

(Eco-engineering)

Strategies in Bioremediation

Our Our experimental experimental systemsystem……

CH3

Cl

Cl

Cl

Cl

Cl

NO2

CH3

(CH)n

CH3

OH

CH3

Cl

CH3

NO2

NH2

CH3

OH

Enrichment cultures

Accessing the diversity of soil microbes

Cepa (Plásmido) Contaminante

P. putida mt-2 (pWW0) Tolueno

Pseudomonas sp, CF600 (pVI150) Fenol

Burkholderia sp. RP007 Fenantreno

P. putida TMB Trimetilbenceno

P. putida NAH (NAH7) Naftaleno

Acinetobacter sp. ADP1 Aril esteres

R. eutropha (pJP4) 2,4 D

Pseudomonas sp. (pP51) Triclorobenceno

P. putida UCC22 (pTDN1) Anilina

P. oleovorans (pOCT) n-alcanos

Burkholderia cepacia LB400 Bifenilo, PCBs

Pseudomonas sp. VLB120 Estireno

CH3 COOH OH

OH TCA

Xyl UWCMABN

Upperoperon Xyl XYZL

toluatedioxygenase

Xyl TEGFJQKIH

C2,3O et alLower (meta)

operon

El sistema TOL de Pseudomonas putida

CH3 COOH

Xyl UWCMABN

Upperoperon

CH3 CH2OH

De la biodegradación a la química verde

COH COOH

xylA xylB xylC

CH3 CH2OH

De la biodegradación a la química verde

xylA

CH CH2CH=CH2

xylA

O

estireno epóxido

microorganismos

(156 k)

ecosistemas

Diversidad conocida

plant

(270 k)

animal

(1.230 k)

microorganisms

200 k

ecosystems

plants(250k)

animal1500k

animalsplants

microbes

human

known existing

fungi

80 k

protista

100 k

known

eubacteria & archaea

existing

nonculturable

culturable

10 k

Exploring new worlds…

Metagenomics

16S RNA

DGGE analysis of 16 s RNAs of lindane-polluted soils

enriched

Acceso al metagenoma microbiano

Extraction of intact bacterial fraction from soil

Fractionation of metagenomicsoil DNA for cloning in lambda vectors

Pero… ¿podemos mejorar

a la Naturaleza?

¡Si! pero utilizando sus

mismos mecanismos

Declorination activity of linA-like clones

0,000

0,200

0,400

0,600

0,800

1,000

1,200

1,400

5F0

9

10

H0

8F0

5

5H

09

8A

0

3C

04

1E

07

2A

0

8E

12

4D

0

3G

11

5G

04

9A

0

8C

09

5G

11

clone

Ac

tiv

ity WT

Act

LinA geneMutagenic

PCR

ScreenActivity assay

DataAnalysis

Cys -> Arg

Study ofImprovedVariants

Next round

DIRECTED EVOLUTION OF Lindane Dechlorinase (LinA)

plac

Reintroducing linA-in the

biodegradation network with

transgenic plants

José Eduardo González-Pastor, Carolina González de Figueras y

Víctor de Lorenzo

Centro de Astrobiología (INTA-CSIC) (Madrid)

Centro Nacional de Biotecnología (Madrid)

pCAMBIA3500, a binary vector to transform

Arabidopsis thaliana

• Replication origins for Escherichia coli and for Agrobacterium tumefaciens

• T-DNA from Agrobacterium (DNA that is excised and integrated in the plant) was

engineered to contain:

– a 35S promoter from cauliflower mosaic virus (CaMV35S) driving a gene encoding

resistance to phosphinothricin (herbicide) for selection of the plant transformants

– Two CaMV35S promoters driving expression of the transgenes, and a CaMV polyA

(modified by Carlos Alonso-CNB)

T-border

(left)

T-border

(right)

CaMV

polyA

CaMV

polyA

CaMV

35S

2x

CaMV

35S

linA

phosphinothricin

linA-transgenic Arabidopsis thaliana growing in thepresence of lindane (10 m M)

linA control linA (C-His) control

70s

80s

90s

2Ks

Widening of Biotechnologies

Health

Food

Environment

ChemistryNanotech

?

Biología Sintética

Rediseñar los sistemas vivos

The big leap forward: from GeneticEngineeringto SyntheticBiology

Catalog of parts

Bacterial promoters as logic gates (I)

Bacterial promoters as logic gates (II)

xylUWCMABNPu Pm xylXYZLTEGFJQKIH xylS Ps Pr xylR

Upper operon meta operon

benzoate

m, p-xylene

The regulation of the TOL system

toluateCH3

R

COOH

R

Regulation of the TOL system as a whole of logic gates

What can be done with allthis?

Engineering developmental patterns in bacterial layers

by designing genetic circuitswith Quorum-Sensing components

R. Weiss

Programming anartificial genetic circuit with parts ofthe bacterialQuorum Sensingsystem

Designing E. coli strains withLight-responsive properties

Voigt

Chimaeric light receptor

• Phytochrome Cph1 from Synechocystis• EnvZ• Phycocyanobilin (PCB) ho1, pcyA

Light-dependent lacZ expression

Mercury vapor lamp

Projected image

Double Guass

focusable lens

632nm

bandpass fi lter35mm slide

Only games?

H H H H H H

H

H H H H H

H

H

H

H

H

H

H

H

Cl Cl

Cl Cl

Cl Cl

H H H H H

H

H H H H

H

H

H

+ H+ + Cl-

+ H+ + Cl-

+ H+ + Cl-

[e- don or] ox

[e- don or]red Pd(0)cell

Pd(II) sol

H

Pd(0)cell

Pd(0)cell /H2 [e- don or]red

[e- don or]ox

S. oneidensi s a

b

c

Dechlorinating PCBs with Bio/inorganic catalysts

G6P

Glucose

PYRAcCoA

Acetate

Isocitrate

-Ketoglutarate

SuccinateGlyoxylate

Fumarate

Malate

Oxaloacetate

LactateEthanol

GlucoseG6P

PEP

PYR

ATP ADP

CO2

Formate

Formate

(Secreted) H2

NAD

H

NAD

Citrate

Pentose

Phosphate

Pathways

Building abetter H2

producerBuilding a new

chromosome based

on genome

sequencesMaximizing

conversion

to H2

Maximizing

renewable

resource

utilization

Towards a an H2 super-producer (?)

2006 2008 2010 2012 2014

Synthetic

microbial core Functional

genomics core

Computational

microbial design lab Protein

complex core

Genetic circuit

repository

Microbial Fuel Cell

R

I

Transcriptional regulators à la carte?

Antb reporter

Epitope presentation

Enzymatic activity

Optical output

R

Circuitdesign

P

Area Reduction:THE DISTRIBUTED & SPECIALIZED SENSOR

The MMDS provides a distributed & specialized sensor thatdetects explosive traces and reacts providing an enhanced EMevidence.

A second sensor (a mobile one) easily detects the new EMevidence, which is the translation and amplification of theoriginal explosive one.

No DNT 2,4-DNT Salicylate

Dnt

RX

ylR

5

CH3

O2NNO2

CH3

CH3

XylR5

luxCDABE

wt XylR XylR5

Spreading sensor bacteria on a soil microcosm

Revealing underground 2,4 DNT in a soil microcosm