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];
A239P;S426N)TAI: 4.7-fold
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
5GMutagenic PCR+in vivo shuffling
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
4GMutagenic PCR+in vivo shuffling
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
Evolutionary 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
Miguel Alcalde (ICP)Miguel Alcalde
Directed
Enzyme
Evolution
Laboratory, ICP, CSIC
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
Miguel Alcalde (ICP)Miguel Alcalde
Directed
Enzyme
Evolution
Laboratory, ICP, CSIC
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
MUTAGÉNESIS DIRIGIDA
Miguel Alcalde (ICP)Miguel Alcalde
Directed
Enzyme
Evolution
Laboratory, ICP, CSIC
1st
Generation 4th
Generation
3rd
Generation2nd
Generation
Charles Darwin (1809-1882)
Millions
of
yearsNatural evolution
In vitro evolution Weeks
or
monthsTEMPORAL SCALE
Miguel Alcalde (ICP)Miguel Alcalde
Directed
Enzyme
Evolution
Laboratory, ICP, CSIC
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
Miguel Alcalde (ICP)Miguel Alcalde
Directed
Enzyme
Evolution
Laboratory, ICP, CSIC
3rd generation2nd generation1st generationParent
NATURAL EVOLUTION DIRECTED MOLECULAR EVOLUTION
ThermostabilityNovel activitiesFunctional expression levelsStability in non-conventional mediaRegio and stereo- selectivities…
FITNESS OR SURVIVAL
Miguel Alcalde (ICP)Miguel Alcalde
Directed
Enzyme
Evolution
Laboratory, ICP, CSIC
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
Miguel Alcalde (ICP)Miguel Alcalde
Directed
Enzyme
Evolution
Laboratory, ICP, CSIC
“CRECIMIENTO CIENTÍFICO E INDUSTRIAL ”
DE LA EVOLUCIÓN DIRIGIDA*
PNAS, 2009, 106: 9995-10000Current Opinion in Biotechnology 2004, 15:305-313
Miguel Alcalde (ICP)Miguel Alcalde
Directed
Enzyme
Evolution
Laboratory, ICP, CSIC
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
Detergente para lavadoras
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
Miguel Alcalde (ICP)Miguel Alcalde
Directed
Enzyme
Evolution
Laboratory, ICP, CSIC
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
SCREENING
“YOU GET WHAT YOU SCREEN FOR”
Miguel Alcalde (ICP)Miguel Alcalde
Directed
Enzyme
Evolution
Laboratory, ICP, CSIC
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”
Miguel Alcalde (ICP)Miguel Alcalde
Directed
Enzyme
Evolution
Laboratory, ICP, CSIC
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
Miguel Alcalde (ICP)Miguel Alcalde
Directed
Enzyme
Evolution
Laboratory, ICP, CSIC
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
SCREENING
Miguel Alcalde (ICP)Miguel Alcalde
Directed
Enzyme
Evolution
Laboratory, ICP, CSIC
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
Miguel Alcalde (ICP)Miguel Alcalde
Directed
Enzyme
Evolution
Laboratory, ICP, CSIC
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)
Miguel Alcalde (ICP)Miguel Alcalde
Directed
Enzyme
Evolution
Laboratory, ICP, CSIC
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”
Miguel Alcalde (ICP)Miguel Alcalde
Directed
Enzyme
Evolution
Laboratory, ICP, CSIC
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
Miguel Alcalde (ICP)Miguel Alcalde
Directed
Enzyme
Evolution
Laboratory, ICP, CSIC
Saccharomyces cerevisiae as a DNA-recombination toolbox to generate diversity
Miguel Alcalde (ICP)Miguel Alcalde
Directed
Enzyme
Evolution
Laboratory, ICP, CSIC
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
MUTAGÉNESIS DIRIGIDA
PRODUCCIÓNDEL NUEVO BIOCATALIZADOR
DISEÑO RACIONAL: MUTAGÉNESIS DIRIGIDA
Diseño combinatorio y computacional: estudios semi-racionales
Miguel Alcalde (ICP)Miguel Alcalde
Directed
Enzyme
Evolution
Laboratory, ICP, CSIC
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)
Miguel Alcalde (ICP)Miguel Alcalde
Directed
Enzyme
Evolution
Laboratory, ICP, CSIC
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.
Miguel Alcalde (ICP)Miguel Alcalde
Directed
Enzyme
Evolution
Laboratory, ICP, CSIC
Terra
Nova
Bovine
rumen microflora
Guts
from
Lumbricus terrestris
Miguel Alcalde (ICP)Miguel Alcalde
Directed
Enzyme
Evolution
Laboratory, ICP, CSIC
PALEOENZIMOLOGIA O COMO RESUCITAR ENZIMAS EN EL LABORATORIO
Miguel Alcalde (ICP)Miguel Alcalde
Directed
Enzyme
Evolution
Laboratory, ICP, CSIC
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
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];
A239P;S426N)TAI: 4.7-fold
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
5GMutagenic PCR+in vivo shuffling
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
4GMutagenic PCR+in vivo shuffling
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
Evolutionary approach
OB‐1 mutant
Algunos ejemplos prácticos…
P450s
Galactose oxidase
Laccases
Versatile Peroxidase
Directed evolution for:
Substrate specificityFunctional expressionThermal stabilityStability in co-solvents
Miguel Alcalde (ICP)Applied
Biocatalysis
Group
Directed
Enzyme
Evolution
Laboratory, ICP, CSIC
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.
Miguel Alcalde (ICP)Miguel Alcalde
Directed
Enzyme
Evolution
Laboratory, ICP, CSIC
Could we evolve the 139-3 mutant towards olefins epoxidation?
EPOXIDES APPLICATIONS intermediates in organic synthesis (chiral compounds)
pharmaceuticals, surfactants, fumigants, industrial sterilants, cosmetics
Miguel Alcalde (ICP)Miguel Alcalde
Directed
Enzyme
Evolution
Laboratory, ICP, CSIC
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
Miguel Alcalde (ICP)Miguel Alcalde
Directed
Enzyme
Evolution
Laboratory, ICP, CSIC
Miguel Alcalde (ICP)Miguel Alcalde
Directed
Enzyme
Evolution
Laboratory, ICP, CSIC
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
Miguel Alcalde (ICP)Miguel Alcalde
Directed
Enzyme
Evolution
Laboratory, ICP, CSIC
Miguel Alcalde (ICP)Miguel Alcalde
Directed
Enzyme
Evolution
Laboratory, ICP, CSIC
•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
DIRECTED EVOLUTION OF MTLT2 FOR ORGANIC COSOLVENT TOLERANCE
Chemistry and Biology (2007)14: 1052-1064.
Miguel Alcalde (ICP)Miguel Alcalde
Directed
Enzyme
Evolution
Laboratory, ICP, CSIC
DIRECTED EVOLUTION OF MTLT2 FOR ORGANIC COSOLVENT TOLERANCE
Structural reinforcement
Miguel Alcalde (ICP)Miguel Alcalde
Directed
Enzyme
Evolution
Laboratory, ICP, CSIC
SECONDARY WALL OF A NON-WOODY PLANT
Miguel Alcalde (ICP)Miguel Alcalde
Directed
Enzyme
Evolution
Laboratory, ICP, CSIC
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.
Miguel Alcalde (ICP)Miguel Alcalde
Directed
Enzyme
Evolution
Laboratory, ICP, CSIC
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
Miguel Alcalde (ICP)Miguel Alcalde
Directed
Enzyme
Evolution
Laboratory, ICP, CSIC
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
Evolutionary approach
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
3GMutagenic PCR+in vivo shuffling
5GMutagenic PCR+in vivo shuffling
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
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];
A239P;S426N)TAI: 4.7-fold
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
5GMutagenic PCR+in vivo shuffling
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
4GMutagenic PCR+in vivo shuffling
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
MUTATIONALEXCHANGE
WITH A RELATEDEVOLVED LACCASE
(77 % IDENTITY)
Lab evolution of high-redox potential laccases
Miguel Alcalde (ICP)Miguel Alcalde
Directed
Enzyme
Evolution
Laboratory, ICP, CSIC
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
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];
A239P;S426N)TAI: 4.7-fold
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
5GMutagenic PCR+in vivo shuffling
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
4GMutagenic PCR+in vivo shuffling
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
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
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··
Miguel Alcalde (ICP)Applied
Biocatalysis
Group
Directed
Enzyme
Evolution
Laboratory, ICP, CSIC
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
Deletion of N-terminal tail
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
Miguel Alcalde (ICP)Miguel Alcalde
Directed
Enzyme
Evolution
Laboratory, ICP, CSIC
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
Miguel Alcalde (ICP)Miguel Alcalde
Directed
Enzyme
Evolution
Laboratory, ICP, CSIC
Miguel Alcalde (ICP)Miguel Alcalde
Directed
Enzyme
Evolution
Laboratory, ICP, CSIC
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
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).