Marinus G. Casteleijn February, Helsinki KETJU meeting Modular Biocatalyst Platform for Chiral Synthesis of Chemical Compounds by Structure-based Directed Evolution the BIOCAT project

Marinus G. Casteleijn February, Helsinki KETJU meeting Biocatalysis at our Facilities Where three key components meet... Ligands Substrates Used for validation and process optimization Inhibitors Used to find starting ideal biomolecules for directed evolution Process Development ml ml Small scale High Throughput is scaleable to Production Prof. Peter Neubauer Directed evolution Molecular biology Enzymology Prof. Rik Wierenga Structural studies Ph.D Mari Ylianttila Ph.D.Markus Alahuhta Marco Casteleijn Mikko Salin Mirja Krause Prof. Marja Lajunen Organic chemistry Ph.D. Sampo Mattila NMR Matti Vaismaa Nanna Alho Prof. Peter Neubauer Process Development Ph.D Tomi Hillukkala Jaakko Soini Johanna Panula-Perälä Narendar Kumar Khatri Biocatalysts TIM barrels versatile platform for isomerisation

Marinus G. Casteleijn February, Helsinki KETJU meeting Biocatalysis The Project BIOCAT: New enzymes for the chiral* synthesis of new 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 Type Kealases α-hydroxy keton R R α-hydroxy aldehyde Biocatalysts TIM barrels versatile platform for isomerisation * *

Marinus G. Casteleijn February, Helsinki KETJU meeting Proof-Of-Principle studies A-TIM A-TIM-A178L A-TIM-S96P A-TIM-I245A Characterization of monomeric TIMs Binding studies NMR/Mass Spectrometry Chemical synthesis X-ray/docking Start A-TIM-X* *RpiA/B activity**new activity A-TIM-Y** Directed Evolution Screening Active enzymes Selection Directed Evolution *AraA activity *XylA activity Added based on the previous KETJU meeting

Marinus G. Casteleijn February, Helsinki KETJU 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 S96P Lead enzyme ATIM 4 Starting points -ATIM (A) -ATIM-S96P (ASP) -ATIM-A178L (AAL) -ATIM-I245A (AIA)

Marinus G. Casteleijn February, Helsinki KETJU meeting Rational Design: Megaprimer PCR creates different libraries of ATIM mutants Regions (3) W100 (W) V214/N215 (VN) A233/G234/K239/E2 41 (AGKE) V214/ N215 A233/G234/ K239/E241 W100 Mutagenesis 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. Casteleijn February, Helsinki KETJU meeting Fully randomized mutagenesis 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. Casteleijn February, Helsinki KETJU 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 o C 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 space Utilizing ribose sugars Materials and protocols were a kind gift from: Prof. R. Sterner Dr. J. Claren University of Regensburg Knock out strains W3110 F- λ- IN (rrnD-rrnE)1 (Datsenko and Warren PNAS 2000)

Marinus G. Casteleijn February, Helsinki KETJU meeting Selection – the use of the experimental space Replacing 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 Selection Cell Plate Knock Out E. coli Strain Plasmid Rondom gene from library Plate 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. Casteleijn February, Helsinki KETJU meeting Biocatalysis at our Facilities The 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 46

Marinus G. Casteleijn February, Helsinki KETJU meeting Summary Current 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. Casteleijn February, Helsinki KETJU meeting BIOCAT The project * A-TIM libraries * knock-out strains A-TIM * Selection assays * New libraries * New knock-out strains Quantitative structural Enzymological studies: * X-Ray * surface plasmon resonance * CD * Docking, biocomputing * Mass spectroscopy * Fluorescence * Enzyme kinetics Kealases New methods Wild Type Kealases α-hydroxy keton R R α-hydroxy aldehyde * *

Marinus G. Casteleijn February, Helsinki KETJU meeting BIOCAT - Network summary Analytical tools ml8b TIM monoTIM Kealases iterative directed evolution Pool of enzymes Random mutage- nesis /shuffling Selection of best mutants Screen for activity Chemical compounds Input Output Process development ICM docking Technology Applications Wild type TIM ml1 TIM Input Wild type studies X-Ray Crystallography NMR Mass Spectrometry Binding Studies A-TIM variants