Towards new biocatalytic activity of ATIM by structure based directed evolution Projects, Bottlenecks and Where to Go Next?

Biocatalysts - trends Davenport, R. VOL. 4 NO. 1 March 2008 INDUSTRIAL BIOTECHNOLOGY

Biocatalysts - Bottlenecks Metagenomics ”finding Enzymes” Protein Engineering ”making enzymes” Bioprocess Development ”using enzymes” Finding the right markers (M) Vast amount of genetic data (M, PE)  amount of DNA, processing many clones and sequences, library strategies Vast amount of gene products (M, PE, BD)  purity, activity, selectivity Effective screening of activity (M, PE, BD)  data mining, gene isolation, product isolation Adjustable product-gene expression, inteference-free operation (BD)

Biocatalysts - Solutions Miniaturization Parallelization High Throughput approaches Modelling Bioinformatics Metagenomics ”finding Enzymes” Protein Engineering ”making enzymes” Bioprocess Development ”using enzymes”

Biocatalysis at our Facilities Where three key components meet... Biocatalysts Thermostability example molecules phosphorylases TIM barrels versatile platform for isomerisation A B Ligands Substrates Used for validation and process optimization Inhibitors Used to find ideal starting biomolecules for directed evolution Process Development ml ml Small scale High Throughput is scaleable to Production Modelling In solico design of future experiments 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/ Kathleen Szeker 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

Biocatalysis at our Facilities Where three key components meet... Biocatalysts Thermostability example molecules phosphorylases TIM barrels versatile platform for isomerisation A B Process Development ml ml Small scale High Throughput is scaleable to Production Modelling In solico design of future experiments 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/ Kathleen Szeker 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 Ligands Substrates Used for validation and process optimization Inhibitors Used to find ideal starting biomolecules for directed evolution

Biocatalysis at our Facilities Enzymes... Biocatalysts Thermostability example molecules phosphorylases TIM barrels versatile platform for isomerisation A B BIOCAT-HT: Production of active thermostable phosphorylases based on High Throughput strategies Parallel transformations and expressions of phosphorylases isolated from thermophilic organisms by using a fusion-partner plasmid library. High quantity approach: automated, fed-batch small scale cultivations, on-line evaluation of proper folding Starting points Novel thermostable phosphorylases Development of High Throughput methods 45 gene cultivation product High Throughput parallel optimization

Thermostable Phosphorylases Simple and cheap Purification Higher general Stability Strategies for improved Protein stability Suitability for specific industrial Processes Structure- stability Relationships Evolutionary Significance of Thermophilic MO A. Thermostability example molecules phosphorylases Basic research Industrial application

Biocatalysis at our Facilities Enzymes... Biocatalysts Thermostability example molecules phosphorylases TIM barrels versatile platform for isomerisation A B 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

Novel enzymes Kealases Chirally pure α-hydroxy aldehydes Xylose isomerase without cofactors Strategies with altered Substrate specificity Ribose sugers for modified nucleosides Structure- function Relationships Directed evolution Potential enzyme libraries B. TIM barrels versatile platform for isomerisation Basic research Industrial application

Wild Type Dimer 4000 s -1 (!!!) ml8b TIM monoTIM Wild type TIM ml1 TIM A-TIM variants

Wild Type Loop 3 deletion Dimer 4000 s -1 (!!!) ml8b TIM monoTIM Wild type TIM ml1 TIM A-TIM variants

monoTIM Monomer Loop 1 rigdify 5 s -1 (!) ml8b TIM monoTIM Wild type TIM ml1 TIM A-TIM variants

Ml1 TIM Monomer Loop 8 deletion 5 s -1 (!) ml8b TIM monoTIM Wild type TIM ml1 TIM A-TIM variants

Ml8b TIM Monomer Point mutation V233A Not Active ml8b TIM monoTIM Wild type TIM ml1 TIM A-TIM variants

ATIM Monomer Active site = ok Perfect start for Directed Evolution ml8b TIM monoTIM Wild type TIM ml1 TIM A-TIM variants

Biocatalysis at our Facilities Enzymes... Biocatalysts Thermostability example molecules phosphorylases TIM barrels versatile platform for isomerisation A B WT-TIM is very active and very well studied Small size: easy to crystallize, suitable for NMR, suitable for biocomputational studies Easily actively expressed in high amounts in E. coli Stable Monomeric protein No cofactors needed Monomeric TIM is a very suitable protein for biocatalysis: Mutant Libraries Random mutagenesis DNA Sequence Structural changes Screening NMR Chemistry Growth Automated

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 Proof-Of-Principle studies A-TIM-X* *RpiA/B activity**new activity A-TIM-Y** Directed Evolution Screening  NMR  Enzyme based  Chemical based  Growth based  Automated *AraA activity *XylA activity Active enzymes

Biocatalysis at our Facilities Directed evolution... Biocatalysts Thermostability example molecules phosphorylases TIM barrels versatile platform for isomerisation A B Lead Enzyme ATIM Improved Variants Mutagenesis A) fully random B) targeted random Screening in vivo Mutant Libraries Random mutagenesis DNA Sequence Structural changes Screening NMR Chemistry Growth Automated

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) Loop 6 Loop 8 Loop 4

error prone PCR: GeneMorph II Random Mutagenesis Kit ATIM Mutagenesis I) fully random Mutagenesis II) targeted random Megaprimer PCR (WuWu et al. 2005)

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 II) targeted random The libraries – selection of good targets Targeted mutagenesis (megaprimer method ) 3 Regions -W100 (W) -V214/N215 (VN) -A233/G234/K239/E241 (AGKE) Loop 7 Loop 8 Loop 4

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 % 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 All methods are verified and introduced mutations into the A-TIM sequence. Screening based on Growth of Knock-out strains on selective media is ongoing. Screening methods for High Throughput approaches are under development A-TIM-X* *RpiA/B activity *AraA activity *XylA activity Active enzymes Knock-out strains

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 % 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 to 9.4x10 10 days of screening required Every screening 4 plates à 2000 colonies = screenings every day 3x 10 9 to 3x10 15 Pool III epPCR 525 days of screening required Every screening 4 plates à 2000 colonies = screenings every day Pool II V214/N215 A233/G234… 1 screening required Every screening 4 plates à 2000 colonies = Pool I W100

Biocatalysts - Solutions Miniaturization Parallelization High Throughput approaches Modelling Bioinformatics Metagenomics Protein Engineering Bioprocess Development

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 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 A B Examples Thermostability expression of themophilic phosphorylases (diploma work – end 2008) TIM barrels Parallel optimization of expression of a known active, instable monomer (project work – end 2008) High Throughput production of monomeric TIM crystals for Crystallography (diploma work – feb 2009) Kathleen Zseker

Biocatalysis at our Facilities Where three key components meet... Biocatalysts Thermostability example molecules phosphorylases TIM barrels versatile platform for isomerisation Metagenomics Protein Engineering Bioprocess Development A B C Ligands Substrates Used for validation and process optimization Inhibitors Used to find ideal starting biomolecules for directed evolution Process Development ml ml Small scale High Throughput is scaleable to Production Modelling In solico design of future experiments 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 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/ Kathleen Szeker

Ligands Substrates Used for validation and process optimization Inhibitors Used to find ideal starting biomolecules for directed evolution Biocatalysis at our Facilities The chemistry of interactions... Detailed understanding Of interactions Matti Weismaa Nano Alho (NMR)

Biocatalysis at our Facilities Where three key components meet... Biocatalysts Thermostability example molecules phosphorylases TIM barrels versatile platform for isomerisation A B Process Development ml ml Small scale High Throughput is scaleable to Production Modelling In solico design of future experiments 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 Ligands Substrates Used for validation and process optimization Inhibitors Used to find ideal starting biomolecules for directed evolution 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/ Kathleen Szeker

Process Development ml ml Small scale High Throughput is scaleable to Production Modelling In solico design of future experiments Biocatalysis at our Facilities More is less... d (Vy) dt = F i y i + Q i y g,i - F 0 δy – Q 0 y g,0 + Vr y (formula for mass balance [kg h -1 ] Kathleen Szeker

Biocatalysis at our Facilities Presentations... BIOCAT: New enzymes for the chiral synthesis of new chemical compounds by structure based directed evolution Towards new biocatalytic activity of ATIM by structure based directed evolution Marco Casteleijn High throughput methods for the production of thermostable enzymes Kathleen Szeker The design, synthesis and evaluation of new substrate candidates based on Triosephosphate isomerase. Matti Vaismaa Utilization of NMR and MS techniques in biocatalysis research Nanna Alho Protein crystallographic characterization of the A-TIM binding properties Mikko Salin

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 Spec. High Throughput methods Binding Studies A-TIM variants

The search continues...

Methods What was done: error prone PCR: GeneMorph II Random Mutagenesis Kit A) Why? 1.Combination of two polymerases lowers bias of single bases 2. includes a cloning kit A) Usually done, when structure of enzyme is not well or not at all known Mutagenesis A) fully random B) targeted random B) Why? 1.Simple primers with Wobbles (Ns) within the targeted areas 2.one-step PCR, no interference necessary B) Structure of TIM is well known: Rational Design Megaprimer PCR: according to Wu et al. 2005

What was done: Megaprimer PCR (WuWu et al. 2005) Methods Mutagenesis B) targeted random 1st step: Only mutagenic primer (high Tm)can anneal Flanking primers cannot anneal 2nd step: Generation of Megaprimer via one flanking primer reverse (here F2)and smDNA; wt- template is NOT amplified, restores ds status, Tra is used to make sure target is reannealed. 3rd step: repetition of 1 will generate an entire mutagenic ss template DNA (high Temp) Repetition of 2 will obtain desired mutagenic DNA by using the low Tm flanking primer forward(here R1)