th α Miguel Alcalde - CSIC · DISEÑO RACIONAL DE BIOCATALIZADORES (Mutagénesis dirigida) Asp23....

P393H mutantTAI: 1.5-fold; TI: 0.5-fold

11A2 mutant(V[α10]D;A[α87]T;V162A;

A239P;D281E;S426N;A461T)TAI: 3.4-fold

1D11 mutant(V[α10]D;A[α87]T;H208Y;

A239P;S426N;A461T)TAI: 3.8-fold

9H4 mutant(V[α10]D;L[α63]S;A[α87]T;

A239P;S426N)TAI: 5.9-fold

8G8 mutant(V10D;E[α83]G;A[α87]T;

N181D;S224G;A239P)TAI: 4.7-fold

5G3 mutant(V[α10]D;A[α87]T;A239P;

S426N;A461T)TAI: 10.3-fold

PM1-60 mutant(A239P)

TAI: 12-fold

PARENT-PM1

PM1-1A2 mutant(D281E)

TAI: 4-fold

PM1-30C mutant(S224G)

TAI: 7-fold

3E1 mutant( V[α10]D;A[α87]T;A239P;V286L)

TAI: 11-fold10D2 mutant

(L[α63]S;A239P;S426N)TAI: 6-fold

4C2 mutant(A239P;A461T)

TAI: 2.6-fold

1GMutagenic PCR

4E12 mutant(V[α10]D;A[α87]T;V162A;

A239P;D281E;P486L)TAI: 5.5-fold

4B8 mutant(V[α10]D;A[α87T];


11B3 mutant(V[α10]D;A[α87]T;

A239P;D281E)TAI: 4.2-fold

2GMutagenic PCR

3GMutagenic PCR+in vivo shuffling

7D2 mutant(V[α10]D;L[α63]S;

A239P;S426N)TAI: 6-fold

7F2 mutant(V[α10]D;A[α87]T;N181D;

S224G;A239P;S426N;A461T)TAI: 2.6-fold


6C8 mutant(V[α10]D;N[α23]K;A[α87]T;V162AH208Y;A239P;S426N;F454S;A461T)

TAI: 1.8-fold

A[α9]D mutantTAI: 0.6 fold; TI: 1-fold

Site directed mutagenesis approach

Evolutionary approach


16B10 mutant(V[α10]D;I[α33]T;A[α87]T;V162A;

H208Y;A239P;A361T;S426N;F454S;A461T;S482L)

TAI: 0.51-foldTI: 1.6-fold

6Gin vivo assembly

(IvAM)

S454F (reverted mutant)TAI: 0.5-fold; TI: 2.3-fold

D281E mutantTAI: 1.3-fold; TI: 1-fold

S224G mutantTAI: 1.3-fold; TI: 1-fold

from PcL evolution

OB1 mutantV[α10]D;N[α23]K;A[α87]T;V162A

H208Y;S224G;A239P;D281E;S426N;A461TTAI vs αPM1 parent: 34000-fold

A[α9]D; P393H

S224G; D281E

7H2 mutant(V[α10]D;A[α87]T;V162A;H208Y;

A239P;S426N;F454S;A461T)TAI: 4.8-fold

2G5 mutant(A239P;D281E)

TAI: 3.6-fold

34000‐foldIMPROVEMENTvs parent type

12‐fold

132‐fold

1360‐fold

5170‐fold

Parent type

24290‐fold

43720‐fold

S426N A461T

A461TS426N

‐FACTOR signal sequence MATURE LACCASE

1st Generation

2nd Generation

7th and 8th

Generations

3rd Generation

V10D A87T A239P V286L

V10D A87T A239PV162A

S224G S454F

A239PV10D A87T

A239P

N23K H208Y

D281E

A461T

4th Generation

A239PV10D A87T H208Y S426N

Preleader Proleader

A461T

5th Generation

A239PV10D A87T H208Y S426NV162A

F454S

A461T

6th Generation

A239PV10D A87T H208Y S426NV162A

F454S

N23K

S224G D281EA[9]D

D[10]V A[9]D-D[10]VP394H

S224G-D281ES454F

Site‐directed mutagenesis approach


OB‐1 mutant

EVOLUCIONANDO ENZIMAS EN EL LABORATORIO

Miguel AlcaldeDirected

Enzyme

Evolution

Laboratory,Applied

Biocatalysis

Group.Institute

of

Catalysis,CSIC, Madrid, Spain

genoma

Célula cromosomas

genes los genes contienen instrucciones para hacer proteínas ADN

enzimas

las enzimas actúan en tareas celularesmuy determinadas regulandocomplejas rutas metabólicas

ADN

ARN

PROTEÍNAS

PRIMERA REVOLUCIÓN BIOTECNOLÓGICA: LA PCR

Miguel Alcalde (ICP)Miguel Alcalde

Directed

Enzyme

Evolution

Laboratory, ICP, CSIC


Directed

Enzyme

Evolution


PRIMERA REVOLUCIÓN BIOTECNOLÓGICA: LA PCR

ESTUDIOESTRUCTURAL PREVIO

DISEÑO RACIONAL DE BIOCATALIZADORES (Mutagénesis dirigida)

Asp23GAC

Phe23UUC

Nivel proteicoNivel genético

GAC por UUC

MUTAGÉNESIS DIRIGIDA

PRODUCCIÓNDEL NUEVO BIOCATALIZADOR


Directed

Enzyme

Evolution


DISEÑO RACIONAL DE BIOCATALIZADORES (Mutagénesis dirigida)

Problemas:•Necesidad de estructuras 3D de la proteína

•Debemos considerar las proteínas como moléculas dinámicas

Concepto: re-diseñar una enzima (cuya estructura cristalina está

resuelta)mediante cambios puntuales en residuos específicos.

GAC por UUC



Directed

Enzyme

Evolution


1st

Generation 4th

Generation

3rd

Generation2nd

Generation

Charles Darwin (1809-1882)

Millions

of

yearsNatural evolution

In vitro evolution Weeks

or

monthsTEMPORAL SCALE


Directed

Enzyme

Evolution


SEGUNDA REVOLUCIÓN BIOTECNOLÓGICA: LA EVOLUCIÓN DIRIGIDA

Genes parentales

Mutagénesis aleatoria

Recombinación

CREACION DE DIVERSIDAD

Inserción de la librería de genesen un vector de expresión

Introducción de la librería/vectoren el microorganismo

Crecimiento de las colonias

Transferencia de coloniasa placas multipocillo

Expresión de la enzima

SELECCION DE LA PROPIEDAD DESEADA

Placa maestra

Gen “ganador”

NUEVA GENERACION

12

3

45

6

7

mutación


Directed

Enzyme

Evolution


3rd generation2nd generation1st generationParent

NATURAL EVOLUTION DIRECTED MOLECULAR EVOLUTION

ThermostabilityNovel activitiesFunctional expression levelsStability in non-conventional mediaRegio and stereo- selectivities…

FITNESS OR SURVIVAL


Directed

Enzyme

Evolution


MUTATIONRECOMBINATION

SCREENING/SELECTION

NATURAL EVOLUTION DIRECTED MOLECULAR EVOLUTION

TEST-TUBE EVOLUTION:simplified and guided process

3rd generation2nd generation1st generationParent

Over millions of years Only months of bench work

At cellular level or in whole organisms One gene: one single winner

Spontaneous process Selective pressure controlled by the scientist


Directed

Enzyme

Evolution


“CRECIMIENTO CIENTÍFICO E INDUSTRIAL ”

DE LA EVOLUCIÓN DIRIGIDA*

PNAS, 2009, 106: 9995-10000Current Opinion in Biotechnology 2004, 15:305-313


Directed

Enzyme

Evolution



Directed

Enzyme

Evolution


Miguel Alcalde (ICP)Applied

Biocatalysis

Group

Directed

Enzyme

Evolution


ENZIMA INDUSTRIAL

LIPOLASA

SUBTILISINA

PEROXIDASA

ANTICUERPO VITAXIN

PROTEÍNA DE SANGRE

SUPRESOR DE TUMORES P53

ALFA INTERFERON

RECEPTOR DE CELULAS T

Pilas de combustible

Detergente para lavadoras



medicamento contra el cáncer

Substituto sanguíneo

medicamento contra el cáncer

fármaco antiviral

Medicamento para la artritis

Oficina de Investigación Naval, USA

Novo Nordisk

Maxygen/Novo Nordisk

Novo Nordisk (Novozymes, A/S)

Ixsys

Diversa

Universidad de Texas, Austin

Maxygen

Universidad de Illinois

ENZIMA IMPLANTADA EN MERCADO

APLICACIÓN COMPAÑÍA


Directed

Enzyme

Evolution


Genes parentales


Recombinación








Placa maestra

Gen “ganador”

NUEVA GENERACION

12

3

45

6

7

mutación

SCREENING

“YOU GET WHAT YOU SCREEN FOR”


Directed

Enzyme

Evolution


Degradación de colorantespoliméricos

Transformación de xenobióticos

Actividad peroxidasa

Evolución de rutas metabólicas:Búsqueda de carotenoides

en fase sólida

Colonias con diferentes colores, contienen distintos carotenoides.Para su análisis se empleanprocesadores digitales

Carotenoidesidentificados

Epoxidación

de alquenos

Métodos directos colorimétricosen formato high-throughput

Degradación de la lignina(blanqueado del papel)

OO2N

OO2N

OH

O-O2NH

O

P450NADPH

O2

+H2 O

+

Métodos indirectos:“Surrogate substrates”


Directed

Enzyme

Evolution


HERRAMIENTAS DE ALTA CAPACIDAD (HIGH-THROUGHPUT)EN CATÁLISIS:

ACELERANDO EL DESCUBRIMIENTO DE NUEVOS CATALIZADORES

FILOSOFIA: llevar a cabo cientos o miles de experimentos,de manera rápida y simultánea.

Miniaturización de los ensayos

ROBOTS


Directed

Enzyme

Evolution


1

43

2

NECESIDAD DE AUTOMATIZACIÓN: FORMATO HIGH-THROUGHPUT

Genes parentales


Recombinación








Placa maestra

Gen “ganador”

NUEVA GENERACION

12

3

45

6

7

mutación

SCREENING


Directed

Enzyme

Evolution


Actividad del parentalActividad del parental

EVOLUCIÓN “ASEXUAL”: PCR PROPENSA A ERROR

INDUCCIÓN DE ERRORES DURANTE ELPROCESO DE AMPLIFICACIÓN DEL DNA

(PCR MUTAGÉNICA)

HERRAMIENTAS: DNA polimerasas(ej.

Mutazima

y Taq

polimerasas)

mutación

mutación


Directed

Enzyme

Evolution


EVOLUCIÓN SEXUAL: LA CRÍA MOLECULAR

ANIMALES Y PLANTAS CRÍA MOLECULAR

Dos organismos parentales Uno, dos o muchos “padres”

Unica

y pequeña generación de cada cruce

Ilimitadas combinaciones de generaciones (sólo restringidasPor el método de screening)

Barreras filogenéticas estrictas(mismas especies)

Barreras filogenéticas muy flexibles(especies diferentes)

Ciclos temporales lentos (1 o dos por año)

Ciclos temporales rápidos (1 o dos al mes)


Directed

Enzyme

Evolution


DNA Wizards

StEP (Staggered extension process)

in vitro DNA shuffling

in vivo DNA shuffling

Parental genes

DNase DNA polimerase

PCR

Repeated cycles

Random digestion

Linearized vector

Transformation along withthe linearized plasmid

Parental genes

primers

Priming and extension Denaturation, random annelingand extension (with introductionof point mutations)

Parental genes

DNA-recombinationand cloning repairingby in vivo gap repair

“DNA Sorcerers”


Directed

Enzyme

Evolution


grown

clonesMaster plate

Parents

Random

prone

PCR

Recombination

CREATING DIVERSITY (LIBRARY)

Ligation

of

library

intoexpression

vector

Transformation

Transfer of

colonies

to

96 well

plates

Enzyme

expression

12

3

45

6

“WINNER”

NEXT GENERATION

7

mutation

Saccharomyces cerevisiae


Directed

Enzyme

Evolution


Saccharomyces cerevisiae as a DNA-recombination toolbox to generate diversity


Directed

Enzyme

Evolution


B

A

PCR 2 (c+d)

in vivo recombination in S. cerevisiae

PCR 1 (a+b)

d5´3´

a

b

c

5´3´

3´5´5´ 3´

Linearized vectorPCR 2 product

PCR 1 product

C

Transformation

in S. cerevisiae

MutagenicPCR 1

Taq

MutagenicPCR 2

Mutazyme

Linearizedvector

+Parent

Parents

MutagenicPCR

+Transformation

in S. cerevisiae

Linearizedvector

1a Generación 4a Generación

3a Generación2a Generación

Charles Darwin (1809-1882)Padre de la Teoría Evolutiva

Miles de millones de añosEvolución natural

Evolución dirigida Semanas o mesesESCALA TEMPORAL

DISEÑO NO-RACIONAL: EVOLUCIÓN DIRIGIDA

ESTUDIOESTRUCTURAL PREVIO

GAC por UUC


PRODUCCIÓNDEL NUEVO BIOCATALIZADOR

DISEÑO RACIONAL: MUTAGÉNESIS DIRIGIDA

Diseño combinatorio y computacional: estudios semi-racionales


Directed

Enzyme

Evolution


Diseño combinatorio y computacional: estudios semi-racionalesFinalidad: reducir el espacio de secuencia proteica

Circular Permutation

Site-directed

MutagenesisDirected

Molecular Evolution

Combinatorial

Saturation

Mutagenesis

Current Opinion in Biotechnology 2005, 16:378-384Nature Biotechnology 2006, 24:328-330; Nature 2009, 10:866-876

SCREENINGS IN SILICO:PDA (Protein

design

automation)SCHEMAEvolutionary

Trace MethodProSAR

(Protein

Sequenceactivity

Relationships)


Directed

Enzyme

Evolution


METAGENOMICA:El acceso a hábitats extremos del planeta, favorecen el aislamiento

de nuevos genes-enzimas

Nature 2005. 3: 510-516

Alcalde, M., Ferrer, M., Plou, F.J. and Ballesteros A. (2006). Environmental biocatalysis: from remediation with enzymes to novel green processes. Trends in Biotechnology 24: 281-287.


Directed

Enzyme

Evolution


> 3500 m


Directed

Enzyme

Evolution


Terra

Nova

Bovine

rumen microflora

Guts

from

Lumbricus terrestris


Directed

Enzyme

Evolution


http://www2.uah.es/gifa/fotos/acantilado_hielo_jhonson.jpg

PALEOENZIMOLOGIA O COMO RESUCITAR ENZIMAS EN EL LABORATORIO


Directed

Enzyme

Evolution


Perez-Jimenez

et al. (2011)Single-molecule

paleoenzymology

probes

the

chemistry

of

resurrected

enzymes.Nature

Structural

& Molecular Biology.18,592–596

•La paleoenzimología

permite viajar atrás en el tiempo para reconstruir a nivel molecular proteínas de organismos ya extintos.

•La reconstrucción se basa en algoritmos para el desarrollo de análisis filogenéticos predictivos.

•Se han reconstruido con este enfoque varias enzimas pre-cámbricas.

•El estudio de enzimas resucitadas puede aportar información básica para comprender la extinción o adaptación de las especies













TAI: 12-fold

PARENT-PM1


TAI: 4-fold


TAI: 7-fold





TAI: 2.6-fold

1GMutagenic PCR







2GMutagenic PCR








TAI: 1.8-fold






H208Y;A239P;A361T;S426N;F454S;A461T;S482L)


6Gin vivo assembly

(IvAM)




from PcL evolution



A[α9]D; P393H

S224G; D281E




TAI: 3.6-fold


12‐fold

132‐fold

1360‐fold

5170‐fold

Parent type

24290‐fold

43720‐fold

S426N A461T

A461TS426N


1st Generation

2nd Generation

7th and 8th

Generations

3rd Generation

V10D A87T A239P V286L


S224G S454F

A239PV10D A87T

A239P

N23K H208Y

D281E

A461T

4th Generation

A239PV10D A87T H208Y S426N

Preleader Proleader

A461T

5th Generation

A239PV10D A87T H208Y S426NV162A

F454S

A461T

6th Generation

A239PV10D A87T H208Y S426NV162A

F454S

N23K

S224G D281EA[9]D

D[10]V A[9]D-D[10]VP394H

S224G-D281ES454F



OB‐1 mutant

Algunos ejemplos prácticos…

P450s

Galactose oxidase

Laccases

Versatile Peroxidase

Directed evolution for:

Substrate specificityFunctional expressionThermal stabilityStability in co-solvents


Biocatalysis

Group

Directed

Enzyme

Evolution


Invertases

.

0 1 2 3 4 5 6

Generation

Rel

ativ

e ac

tivity

1

10

20

139-3

wtrandom mutagenesis

recombination

OO2N

OO2N

OH

O-O2NH

O

P450NADPH

O2

+H2 O

+

(2002). Nature Biotech. 20:1135-1139.


Directed

Enzyme

Evolution


Could we evolve the 139-3 mutant towards olefins epoxidation?

EPOXIDES APPLICATIONS intermediates in organic synthesis (chiral compounds)

pharmaceuticals, surfactants, fumigants, industrial sterilants, cosmetics


Directed

Enzyme

Evolution


Prof. Edgardo FarinasUniversity

of

New

Jersey

Maximum turnover rates for wildtype and 139-3

Wt139-3

Max

imum

turn

over

rate

(m

ole

subs

trate

/min

/mol

e en

zym

e)

0

500

1000

1500

2000

2500

1 2 3 4 5 6 7 8

3-H

exen

e

2-H

exen

e

1-H

exen

e

Cyc

lohe

xene

Isop

rene

Ally

lchl

orid

e

Styr

ene

Prop

ylen

e

2000

2500

1000

0

500

1500

Alcalde, Farinas and Arnold (2004) Alkene epoxidation catalyzed by cytochrome P450 BM-3 139-3. Tetrahedron 60:525-528


Directed

Enzyme

Evolution



Directed

Enzyme

Evolution



Directed

Enzyme

Evolution


http://images.google.com/imgres?imgurl=http://www.belt.es/noticias/2003/03_enero/02_03enero/02enero/ave_prestige.jpg&imgrefurl=http://www.belt.es/noticias/2003/03_enero/02_03enero/02enero/02_chipre_prestige.htm&h=315&w=315&sz=20&hl=en&start=25&tbnid=zy0etaxV_aSDaM:&tbnh=117&tbnw=117&prev=/images%3Fq%3Dhundimiento%2Bprestige%26start%3D18%26gbv%3D2%26ndsp%3D18%26svnum%3D10%26hl%3Den%26sa%3DN

FUNGAL LACCASES-Extracellular oxidoreductase(Blue copper oxidases)-4 Cu-ions-Redox potential at T1 from 475 mV to 800 mV -Broad substrate specificity-Laccase mediator system

-Pulp bleaching-Bioremediation-Biofuel cells-Biosensors-Drinks, Foods and Cosmetics

H450

Electronic transfer from the reducing substrate (RS) towards the O2 in fungal laccases

Alcalde (2007) Laccase: biological functions, molecular structure and industrial applications. In: Industrial Enzymes: structure, function andapplications. J. Polaina and A.P. MacCabe, Eds., Springer, New York p459-474Kunamneni, Camarero, García, Plou, Ballesteros, and Alcalde. (2008). Engineering and applications of fungal laccases for organic synthesis.Microb. Cell Fact. 7, 32.

H400

T1T1--CuCu

T3T3--CuCu

T2T2--CuCu

C451

H452 H456

H395

H109H66

H64

H398

H111O2

RST3T3--CuCu


Directed

Enzyme

Evolution



Directed

Enzyme

Evolution


•13,000 clones, 5 rounds of

evolutionDiversity tools

based

in S. cerevisiae machinery:In vivo DNA-shuffling•IVOE•IvAM

•R2 mutant:-Active and

stable

at 50 % (v/v) cosolvents.-Promiscuity

other

organic

cosolvents-Diferent

espectro-electrochemical

features

Relative activity in ethanol 50 % (v/v)

0 1 2 3 4

Rel

ativ

e ac

tivity

in a

ceto

nitri

le 4

0 %

(v/v

)

0

1

2

3

4

DIRECTED EVOLUTION OF MTLT2 FOR ORGANIC COSOLVENT TOLERANCE

Zumarraga, Bulter, Shleev, Polaina, Plou, Ballesteros and Alcalde. (2007). In vitro evolution of a fungal laccase in high concentrations of organic cosolvents. Chemistry and Biology. 14: 1052-1064.

Dr. Miren Zumárraga

R2 mutant

4 mutations in mature protein2 mutations at the C-terminal tail

Val429

Asn280

Lys182

His552

T1

T2/T3

Val429

Asn280

Lys182

His552

T1

T2/T3


Chemistry and Biology (2007)14: 1052-1064.


Directed

Enzyme

Evolution



Structural reinforcement


Directed

Enzyme

Evolution



Biocatalysis

Group

Directed

Enzyme

Evolution


SECONDARY WALL OF A NON-WOODY PLANT


Directed

Enzyme

Evolution


Pulp

Biobleaching

Wood

White-rot

fungiPleurotus ostreatus Trametes versicolor

Alcalde, Ferrer, Plou and Ballesteros. (2006). Environmental biocatalysis: from remediation with enzymes to novel green processes. Trends in Biotechnology 24: 281-287.


Directed

Enzyme

Evolution



Biocatalysis

Group

Directed

Enzyme

Evolution


Laccase PM1 :(donated by Prof. R. Santamaría, University of Salamanca).Isolated from the wastewater of a paper factory.•Eº at T1 site: +790mV.

Laccase from Pycnoporus cinnabarinus:(donated by Prof. Asther, INRA, Marseille).•Eº at T1 site: +790mV.

Lab evolution of high-redox potential laccases

www.biorenew.org


Directed

Enzyme

Evolution


http://www.biorenew.org/index.php


12‐fold

132‐fold

1360‐fold

5170‐fold

Parent type

24290‐fold

43720‐fold

S426N A461T

A461TS426N


1st Generation

2nd Generation

7th and 8th

Generations

3rd Generation

V10D A87T A239P V286L


S224G S454F

A239PV10D A87T

A239P

N23K H208Y

D281E

A461T

4th Generation

A239PV10D A87T H208Y S426N

Preleader Proleader

A461T

5th Generation

A239PV10D A87T H208Y S426NV162A

F454S

A461T

6th Generation

A239PV10D A87T H208Y S426NV162A

F454S

N23K

S224G D281EA[9]D

D[10]V A[9]D-D[10]VP394H

S224G-D281ES454F



OB‐1 mutant

Romero & Arnold (2009). Exploring

protein

fitness

landscapes

by directed

evolution. Nature Reviews: Molecular cell Biology, 10:866-876

PRE-PRO LEADER MATURE PROTEIN

-PM1

-PcL

LAB EVOLUTIONOF -PM1

PARENT-PcL

1E2 Mutant(A[9]D reverted )

(A[9]A, F[48]S, S[58]G, E[86]G, N208S, D341N, P394H)

TAI: 0.0-fold (-)

3G10 Mutant(L46I)

TAI: 2.3-fold3D3 Mutant(R[2]S, S[58]G)

TAI: 7.3-fold

1D12 Mutant(A[9]D)

TAI: 12.6-fold 5F7 Mutant(P394H)

TAI: 4.5-fold1G

Mutagenic PCR(Medium mutation rate)

19C8 Mutant(R[2]S, S[58]G, D341N, P394H)

TAI: 11.7-fold

10A7 Mutant(A[9]D, F[48]S, S[58]G,

E[86]G, P394H)TAI: 17.1-fold

1C9 Mutant(A[9]D, P394H)

TAI: 8.7–fold

20C7 Mutant(R[2]S, L46I, P394H)

TAI: 11.1-fold2D8 Mutant

(R[2]S, S[58]G, P394H)

TAI: 8.7-fold

1F10 Mutant(A[9]D, S135G, P394H)

TAI: 9.5-fold

5D3 Mutant(A[9]D, F[48]S,S[58]G,

E[86]G, L46I, P394H)TAI:1.9-fold

9C3 Mutant(A[9]D, F[48]S, S[58]G,

E[86]G, L46I, P394H)TAI: 1.7-fold

7A9 Mutant(A[9]D, F[48]S, S[58]G,

E[86]G, N208S, D341N, P394H)TAI: 3.7-fold

2GMutagenic PCR+In vivo shuffling

A240P from PM1L evolutionR[α2]S



1H3 Mutant(A[9]D, F[48]S, S[58]G, G[62]R, E[86]G, N208S,

N331D, D341N, P394H)TAI: 3.1-fold

6A10 Mutant(A[9]D, L[44]S, F[48]S, S[58]G, E[86]G, N130D,

N208S, D341N, P394H)TAI: 2.0-fold

7F11 Mutant(A[9]D, T[24]S, F[48]S, S[58]G, E[86]G, N208S,

D341N, P394H)TAI: 2.0-fold

9E2 Mutant(A[9]D, F[48]S, P[54]Q, S[58]G, E[86]G, N208S,

F332S, D341N, P394H)TAI: 1.9-fold

8B9 Mutant(A[9]D, F[48]S, S[58]G,

E[86]G, N208S, D341N, P394H, T428A)

TAI: 1.8-fold

12B4 Mutant(A[9]D, F[48]S, G[58]D,

E[86]G, N208S, K324M, D341N, P394H)

TAI: 1.5-fold

3B7 Mutant(A[9]D, F[48]S, S[58]G, E[83]K, E[86]G, N208S,

D341N, P394H)TAI: 1.8-fold

6A8 Mutant(A[9]D, L[44]S, F[48]S, S[58]G, E[86]G, N208S,

N331D, D341N, P394H)TAI: 1.3-fold

7C9 Mutant(A[9]D, T[24]S, F[48]S, S[58]G, E[86]G, N208S,

N331D, D341N, P394H)TAI: 1.2-fold

2C8 Mutant(A[9]D, F[48]S, S[58]G, G[62]R, E[86]G, N208S,

R280H, N331D, D341N, P394H)

TAI: 2.6-fold

2D12 Mutant(A[9]D, F[48]S, S[58]G, G[62]R,

E[86]G, N208S, D255G, T294I, N331D, D341N,

P394H, N443S)TAI: 2.0-fold

6Gin vivo shuffling

6GMutagenic PCR

2F9 Mutant(A[9]D, F[48]S, S[58]G, G[62]R,

E[86]G, F81S, N208S, N331D, D341N, P394H)

TAI: 2.3-fold

1GMutagenic PCR

(Low mutation rate)

negative mutantsTAI: 0.0-fold (-)

4GSite directed

mutagenesis + IVOE

LAB EVOLUTIONOF -PcL













TAI: 12-fold

PARENT-PM1


TAI: 4-fold


TAI: 7-fold





TAI: 2.6-fold

1GMutagenic PCR







2GMutagenic PCR








TAI: 1.8-fold






H208Y;A239P;A361T;S426N;F454S;A461T;S482L)


6Gin vivo assembly

(IvAM)




from PcL evolution



A[α9]D; P393H

S224G; D281E




TAI: 3.6-fold

MUTATIONALEXCHANGE

WITH A RELATEDEVOLVED LACCASE

(77 % IDENTITY)

Lab evolution of high-redox potential laccases


Directed

Enzyme

Evolution


OB1 mutant harbours 15 mutations: five in the α-factor prepro-leader (two synonymous), and ten in the mature protein (three synonymous).

Diana Maté

Eva García

Dr. Susana Camarero,CT (CIB, CSIC);

Dr. Carlos García-Burgos













TAI: 12-fold

PARENT-PM1


TAI: 4-fold


TAI: 7-fold





TAI: 2.6-fold

1GMutagenic PCR







2GMutagenic PCR








TAI: 1.8-fold






H208Y;A239P;A361T;S426N;F454S;A461T;S482L)


6Gin vivo assembly

(IvAM)




from PcL evolution



A[α9]D; P393H

S224G; D281E




TAI: 3.6-fold

MAIN FEATURES:

•8 rounds of

evolution+rational

approaches.

•50,000 clones explored.

•An

offspring

of

26 mutants

“winners”

characterized

with

improvements

ranging

from

1.3 to

12-fold.

3D-nanobiodevice

FP7 Project http://www.mah.se/3Dnanobiodevice

•We

have

obtained

a high-redox potential

laccase

highly

active, soluble and

stable.

•OB1 MUTANT shows the

highest

functional

expression

level

ever

reported

for

a high-redox potential

laccase

in S. cerevisiae: 1500 U/L, with

a total laccase

activity

improvement

(TAI) of

34000-fold

Mate, Garcia-Burgos, Garcia, Ballesteros, Camarero & Alcalde. (2010). Laboratory evolution of high-redox potential laccases. Chemistry & Biology.17:1030-1041


Biocatalysis

Group

Directed

Enzyme

Evolution


DIRECTED EVOLUTION OF VERSATILE PEROXIDASE FROM Pleurotus eryingii

García, Ruíz, Martínez, Martínez, and Alcalde (2010). High-redox potential peroxidases engineered by directed evolution. Patent PCT/ES2010/070316.

D175

E36

E40

Mn2+

DMPDMPABTSABTS

((lowlow

affinityaffinity))

HH22

OO22

VAVARB5RB5

ABTSABTS((highhigh

affinityaffinity))

W164W164··


Biocatalysis

Group

Directed

Enzyme

Evolution


11H10 + 15G9 + 1 new mutation

11H10 + 4B5 + 1 new mutation

1 new mutation

1 new mutation

3 new mutations

3 new mutations

11H10 + 2 new mutations

11H10 + 4B1

19C2 + 16E12

19C2 + 16E12 + 1 new mutation

19C2 + 13G1 + 3 new mutations

19C2 + 16E12 + 1 new mutation

20D1 + 16E12 + 1 new mutation

10C3 + 13E4 + 6B1

10C3 + 13E4 + 6B1 + 1 new mutation

1 new mutation

2 new mutations

3H9 +1 new mutation

Deletion of N-terminal tail



T284I

A308T

E[α45]K

Evolutionfortherm

ostability

90ºCSelective

pressure60ºC

α-pre α-pro mature VPNTT

1GMutagenic PCR

2GMutagenic PCR + in vivo shuffling

3GMutagenic PCR + in vivo shuffling

4GStEP + in vivo shuffling

5GIn vivo assembly (IvAM)*

6G

Mutational approach/Suggested

crossovers event

TAI(in fold)

T50(ºC)

MUTANT

Evolutionforfunctionalexpression

andactivity

A[α19]A

V160A

S324T

V[α50]A H39R I278I

E[α27]G

D318D

V[α50]A

V160A

I[α6]T H39R

A[α19]A

T184M

T184M

T184M

T184M

T184M

T184M

T184M

T184M

T184M

E37K

E37K

E37K

E37K

E37K

Q202L

Q202L

T184ME37K Q202L

Q202L

Q202L

V160A

P185P

D318D

F[α12]F L207P

L[α11]S

V[α50]AI[α6]T

P[α54]P

H39R

T184ME37K Q202LV160A

T184ME37K Q202LV160A G330R

T184ME37K Q202LV160A G330R

T184ME37K Q202LV160A G330RD213A

H39R D213AT184ME37K Q202LV160A G330R

T184ME37K Q202LV160A

H39R D213AT184ME37K Q202LV160A G330R

9

7.4

6.3

3.6

36

34.2

28.8

41.4

121

129

128

118

97.7

238

138

109

164

87.4

---

---

60.5

n.d.

n.d.

n.d.

60.5

n.d.

n.d.

n.d.

60.5

n.d.

n.d.

n.d.

n.d.

59.4

62.7

63.9

62.4

65.3

65.6

60.3

58.1

Parentα-vpl2

11H10

15G9

4B5

4B1

16E12

19C2

20D1

13G1

10C3

6B1

13E4

6E7

11F3

R4

24E10

3H9

15B4

2-1B

2-1Bt

R4t

Parentα-vpl2t

581

Deletion mutagenesis by IVOE

---

Mutagenic PCR + in vivo DNA shuffling

Secretion levels: 21 mg/L

T184 M184

L202Q202

V160 A160

E37K37

A235

E304

A235

E304

D30 D30

A174A174

I181I181

BA

haem haem

NATIVE-VP LAB-

EVOLVED VP R4 MUTANT


Directed

Enzyme

Evolution


García, Ruíz, Martínez, Martínez, and Alcalde (2010). High-redox potential peroxidases engineered by directed evolution. Patent PCT/ES2010/070316.

Eva García

Temperature (ºC)

30 40 50 60 70

resi

dual

act

ivity

(%)

0

10

20

30

40

50

60

70

80

90

100

11H10 MUTANT; 1st Generation 16E12 MUTANT; 2nd Generation 10C3 MUTANT; 3rd Generation R4 MUTANT (best mutant of activity); 4th Generation 24E10 MUTANT (Thermostability variant);4th Generation VP expressed in E. coli and in vitro refolded 3H9 MUTANT (Thermostability variant); 5th Generation2-1B MUTANT (Thermostability variant); 6th Generation

T50

=60.5ºC11H10 Mutant; 1st G16E12 Mutant; 2nd

G10C3 Mutant; 3rd

G

T50

=62.7ºC, 24E10 Mutant, 4th G

T50

=63.9ºC3H9 Mutant; 5th

G

T50

=37ºCVp

E.coli

T50

=65.3ºC2-1B Mutant

T50

=59.4ºC, R4 Mutant, 4th

G

Evolving

Thermostability

in VP


Directed

Enzyme

Evolution



Directed

Enzyme

Evolution


Before

evolution After

evolution

MUTANTE OB-1Lacasa

mutante de altopotencial redox

termoestable

Obi Wan Kenobi(Maestro Jedi)

MUTANTE R2Lacasa

mutante resistentea disolventes R2-D2

MUTANTE R4Peroxidasa

versátil mutanteexpresada en levadura

R4

2-1BRobot médico

MUTANTE 2-1BPeroxidasa

versátil mutante termoestable

MUTANTE 3POLacasa

alcalófila 3PO

EVOLUCIONANDO ENZIMAS EN EL LABORATORIO

Institute of CatalysisMadrid, SpainDirected Enzyme Evolution LaboratoryApplied Biocatalysis GroupDepartment of BiocatalysisCSIC (www.icp.csic.es/abg)CIB peopleEuropean partnersand NeuronBioPharma

ACKNOWLEDGMENTS

California InstituteOf Technology, USA Frances Arnold laboratory

Special thanks to:Dr. Thomas Buelter (Gevo.com).Prof. L. Sun (University of Massachusetts,Amherst).Prof. E. Farinas (New Jersey´s Scienceand Technology University).

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