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Biocatalysis at our Facilities Where three key components meet...
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Marinus G. Casteleijn 10-11 February, Helsinki Modular Biocatalyst Platform for Chiral Synthesis of Chemical Compounds by Structure-based Directed Evolution the BIOCAT project
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Modular Biocatalyst Platform for Chiral Synthesis of Chemical Compounds by Structure-based Directed Evolution the BIOCAT project. Biocatalysis at our Facilities Where three key components meet. Biocatalysts TIM barrels versatile platform for isomerisation. Ligands - PowerPoint PPT Presentation
Transcript of Biocatalysis at our Facilities Where three key components meet...
Marinus G. Casteleijn10-11 February, HelsinkiKETJU meeting
Modular Biocatalyst Platform for Chiral Synthesis of Chemical
Compounds by Structure-based Directed Evolution
the BIOCAT project
Marinus G. Casteleijn10-11 February, HelsinkiKETJU meeting
Biocatalysis at our FacilitiesWhere three key components meet...
Ligands
SubstratesUsed for validation and process optimization
InhibitorsUsed to find starting ideal biomolecules for directed evolution
ProcessDevelopment
0.100 ml
10 000 mlSmall scale High Throughput is scaleable to Production
Prof. Peter NeubauerDirected evolution Molecular biologyEnzymologyProf. Rik Wierenga Structural studies
Ph.D Mari YlianttilaPh.D.Markus AlahuhtaMarco CasteleijnMikko SalinMirja Krause
Prof. Marja Lajunen Organic chemistryPh.D. Sampo Mattila NMR
Matti VaismaaNanna Alho
Prof. Peter NeubauerProcess Development
Ph.D Tomi HillukkalaJaakko SoiniJohanna Panula-PeräläNarendar Kumar Khatri
Biocatalysts
TIM barrelsversatile platform for isomerisation
Marinus G. Casteleijn10-11 February, HelsinkiKETJU meeting
Biocatalysis The Project
BIOCAT: New enzymes for the chiral* synthesis ofnew chemical compounds by structure based directed evolution
Structure based directed evolution towards new tailormade active enzymes
• Interdisciplinary approach: Structural biochemistry, chemical synthesis, molecular biology, enzymology.
• Starting points
•a superior structural framework
•a highly interesting chemical reaction: chiral hydroxy compounds
Wild TypeKealases
α-hydroxy keton α-hydroxy keton
R R
α-hydroxy aldehyde α-hydroxy aldehyde
Biocatalysts
TIM barrelsversatile platform for isomerisation
**
Marinus G. Casteleijn10-11 February, HelsinkiKETJU meeting
Proof-Of-Principle studies
A-TIMA-TIM-A178LA-TIM-S96PA-TIM-I245A
Characterization of monomeric TIMs
Binding studiesNMR/Mass Spectrometry
Chemical synthesisX-ray/docking
Start
A-TIM-X*
*RpiA/B activity **new activity
A-TIM-Y**Directed Evolution
Screening
Active enzymesActive enzymes
Active enzymesActive enzymes
Selection
Directed Evolution
*AraA activity
*XylA activity
Added based on the previous KETJU meeting
Marinus G. Casteleijn10-11 February, HelsinkiKETJU meeting
Rational Design:
Site-directed mutagenesis creates four starting points
for the directed evolution approach
Starting points (4)
ATIM (A)
ATIM-S96P (ASP)
ATIM-A178L (AAL)
ATIM-I245A (AIA)
The libraries – selection of good targets
A178L
I245A
S96PLead
enzyme
ATIM
4 Starting points
- ATIM (A)
- ATIM-S96P (ASP)
- ATIM-A178L (AAL)
- ATIM-I245A (AIA)
Marinus G. Casteleijn10-11 February, HelsinkiKETJU meeting
Rational Design:
Megaprimer PCR creates different libraries
of ATIM mutants
Regions (3)
W100 (W)
V214/N215 (VN)
A233/G234/K239/E241
(AGKE)
V214/ N215
A233/G234/
K239/E241
W100Mutagenesis
targeted random
The libraries – selection of good targets
Targeted mutagenesis(megaprimer
method )3 Regions
- W100 (W)
- V214/N215 (VN)
- A233/G234/K239/E241 (AGKE)
Marinus G. Casteleijn10-11 February, HelsinkiKETJU meeting
Fully randomizedmutagenesis
Targeted mutagenesis
(megaprimer method )
Starting points (4)
- ATIM (A)
- ATIM-S96P (ASP)
- ATIM-A178L (AAL)
- ATIM-I245A (AIA)
Regions (3)
- W100 (W)
- V214/N215 (VN)
- A233/G234/K239/E241 (AGKE)
Error rate 0.3–0.6 %
amino acid change
(Fu)
Results
Libraries (16)
- A (Fu,W,VN,AGKE)
- ASP (Fu,W,VN,AGKE)
- AAL(Fu,W,VN,AGKE)
- AIA (Fu,W,VN,AGKE)
16 libraries of A-TIM variants
The libraries – creating the experimental space
Marinus G. Casteleijn10-11 February, HelsinkiKETJU meeting
First strains
Problems* Wild type like strains showed unexpected recombination events
* Wild type like strains showed difficulties to isolate plasmids
Solution* Simple protocol by use of pDK43 expressing λ red recombinase and the pCP20 expressing FLP both a 43 oC
Knockout strains
RpiA-/B-: Collaboration
XylA-: Created own strain based on E. coli K12:W3110
AraA-: based on E. coli K12:W3110 ongoing
Knockout strains – creating the experimental spaceUtilizing ribose sugars
Materials and protocols were a kind gift from:
Prof. R. SternerDr. J. ClarenUniversity of Regensburg
Knock out strains W3110 F- λ- IN (rrnD-rrnE)1
(Datsenko and Warren PNAS 2000)
Marinus G. Casteleijn10-11 February, HelsinkiKETJU meeting
Selection – the use of the experimental spaceReplacing known isomerase activity
L-Arabinose Isomerase
Initial hits (4) for characterization.
However screening will be repeated with AraA- E. coli K12:W3110 strain.
D-Xylose Isomerase
Hits (2) for characterization
Loop 8
Libraries (16)
- A (Fu,W,VN,AGKE)
- ASP (Fu,W,VN,AGKE)
- AAL(Fu,W,VN,AGKE)
- AIA (Fu,W,VN,AGKE)
Knockout strains
RPIA-/B-: Collaboration
XylA-: Created own strain based on E. coli K12:W3110
AraA-: based on E. coli K12:W3110 ongoing
SelectionCell Plate
Knock Out E. coli Strain Plasmid
Rondom gene from libraryPlate with selective media (i.e. One (1) carbon source) Colony utilizing selective sugars
Positive controle gene
Neg. control
Pos. control
No Hits
Two Hits
Neg. control
Marinus G. Casteleijn10-11 February, HelsinkiKETJU meeting
Biocatalysis at our FacilitiesThe right Tools for the Right Methods...
Tools
High Throughput* Hamilton pipetting station
Parallelization* Small scale cultivation technology (EnBase)* Parallel cloning library
Miniaturization * Cultivations* Parallel cloning library
New Methods
High Throughput transformation
High Throughput optimization of protein expression
From Small Scale to Large Scale without further optimization
High Throughput production of crystals for Crystallography ongoing
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Marinus G. Casteleijn10-11 February, HelsinkiKETJU meeting
SummaryCurrent results...
Libraries (16)
- A (Fu,W,VN,AGKE)
- ASP (Fu,W,VN,AGKE)
- AAL(Fu,W,VN,AGKE)
- AIA (Fu,W,VN,AGKE) L-Arabinose Isomerase
Initial hits (4) for characterization.
However screening will be repeated with AraA- E. coli K12:W3110 strain.
D-Xylose Isomerase
Hits (2) for characterization
Loop 8
Knockout strains
RPIA-/B-: Collaboration
XylA-: Created own strain based on E. coli K12:W3110
AraA-: based on E. coli K12:W3110 ongoing
New Methods
High Throughput transformation
High Throughput optimization of protein expression
From Small Scale to Large Scale without further optimization
High Throughput production of crystals for Crystallography ongoing
Marinus G. Casteleijn10-11 February, HelsinkiKETJU meeting
BIOCAT The project
* A-TIM libraries* knock-out strains
A-TIM
* Selection assays
* New libraries* New knock-out strains
Quantitative structuralEnzymological studies:
* X-Ray* surface plasmon resonance* CD* Docking, biocomputing* Mass spectroscopy* Fluorescence* Enzyme kinetics
Kealases
New m
ethods
Wild Type Kealases
α-hydroxy keton α-hydroxy keton
R R
α-hydroxy aldehyde α-hydroxy aldehyde
**
Marinus G. Casteleijn10-11 February, HelsinkiKETJU meeting
BIOCAT - Network summary
Analytical tools
ml8b TIMml8b TIM
monoTIMmonoTIM
Kealases
iterative directed evolution
Pool of enzymes
Random mutage-
nesis/shuffling
Selectionof best
mutants
Screen for activity
Chemical compounds
Input Output
Process development
ICM docking Technology
Applications
Wild type TIM
Wild type TIM
ml1 TIMml1 TIM
Input
Wild type studies
X-Ray Crystallograph
yNMR Mass
SpectrometryBindingStudies
A-TIMvariants
A-TIMvariants
