Transcript TIM barrels
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 • Finding the right markers (M) Metagenomics ”finding Enzymes” Protein Engineering ”making enzymes” Bioprocess Development ”using enzymes” • 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 Metagenomics • Parallelization Protein Engineering • High Throughput approaches ”finding Enzymes” ”making enzymes” Bioprocess Development ”using enzymes” • Modelling • Bioinformatics Biocatalysis at our Facilities Where three key components meet... Biocatalysts A Thermostability example molecules phosphorylases Ligands Substrates Used for validation and process optimization Process Development 0.100 ml 10 000 ml B TIM barrels versatile platform for isomerisation Inhibitors Used to find ideal starting biomolecules for directed evolution 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 A Thermostability example molecules phosphorylases Ligands Substrates Used for validation and process optimization Process Development 0.100 ml 10 000 ml B TIM barrels versatile platform for isomerisation Inhibitors Used to find ideal starting biomolecules for directed evolution 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 Enzymes... Biocatalysts A Thermostability example molecules phosphorylases 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. B TIM barrels versatile platform for isomerisation • 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 A. Thermostability example molecules phosphorylases Evolutionary Significance of Thermophilic MO Structure-stability Relationships Strategies for improved Protein stability Basic research Simple and cheap Purification Thermostable Phosphorylases Higher general Stability Suitability for specific industrial Processes Industrial application Biocatalysis at our Facilities Enzymes... Biocatalysts A Thermostability example molecules phosphorylases 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 B TIM barrels versatile platform for isomerisation • 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 R α-hydroxy keton R α-hydroxy aldehyde B. TIM barrels versatile platform for isomerisation Directed evolution Potential enzyme libraries Structure-function Relationships Strategies with altered Substrate specificity Basic research Chirally pure α-hydroxy aldehydes Novel enzymes Kealases Xylose isomerase without cofactors Ribose sugers for modified nucleosides Industrial application Wild Type Dimer 4000 s-1 (!!!) Wild type TIM monoTIM ml1 TIM ml8b TIM A-TIM variants Wild Type Dimer 4000 s-1 (!!!) Wild type TIM monoTIM ml1 TIM ml8b TIM A-TIM variants Loop 3 deletion monoTIM Monomer 5 s-1 (!) Wild type TIM monoTIM ml1 TIM ml8b TIM A-TIM variants Loop 1 rigdify Ml1 TIM Monomer 5 s-1 (!) Wild type TIM monoTIM ml1 TIM Loop 8 deletion ml8b TIM A-TIM variants Ml8b TIM Monomer Not Active Wild type TIM monoTIM ml1 TIM Point mutation V233A ml8b TIM A-TIM variants ATIM Monomer Active site = ok Perfect start for Directed Evolution Wild type TIM monoTIM ml1 TIM ml8b TIM A-TIM variants Biocatalysis at our Facilities Enzymes... Biocatalysts A Thermostability example molecules phosphorylases B TIM barrels versatile platform for isomerisation Monomeric TIM is a very suitable protein for biocatalysis: • 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 Random mutagenesis Mutant Libraries Screening Structural changes DNA Sequence NMR Chemistry Growth Automated Proof-Of-Principle studies Characterization of monomeric TIMs Binding studies NMR/Mass Spectrometry Chemical synthesis X-ray/docking A-TIM A-TIM-A178L A-TIM-S96P A-TIM-I245A Start Active enzymes Active enzymes A-TIM-X* Directed Evolution A-TIM-Y** Screening *RpiA/B activity *AraA activity *XylA activity NMR **new activity Enzyme based Chemical based Growth based Automated Biocatalysis at our Facilities Directed evolution... Biocatalysts Lead Enzyme Improved ATIM Variants Mutagenesis Screening A) fully random in vivo A Thermostability example molecules phosphorylases B TIM barrels versatile platform for isomerisation B) targeted random Random mutagenesis Mutant Libraries Screening Structural changes DNA Sequence NMR Chemistry Growth Automated The libraries – selection of good targets Loop 6 Loop 4 A178L Lead enzyme S96P ATIM Rational Design: Site-directed mutagenesis creates four starting points for the directed evolution approach Loop 8 I245A Starting points (4) 4 Starting points ATIM (A) ATIM-S96P (ASP) - ATIM (A) ATIM-A178L (AAL) - ATIM-S96P (ASP) ATIM-I245A (AIA) - ATIM-A178L (AAL) - ATIM-I245A (AIA) Mutagenesis Mutagenesis I) fully random II) targeted random GeneMorph II Random Mutagenesis Kit Megaprimer PCR (WuWu et al. 2005) error prone PCR: ATIM The libraries – selection of good targets Loop 4 Loop 7 Mutagenesis II) targeted random W100 V214/ N215 Rational Design: Megaprimer PCR creates different libraries of ATIM mutants Regions (3) Targeted mutagenesis (megaprimer method ) 3 Regions - W100 (W) - V214/N215 (VN) - A233/G234/K239/E241 (AGKE) W100 (W) Loop 8 A233/G234/ K239/E241 V214/N215 (VN) A233/G234/K239/E2 41 (AGKE) The libraries – creating the experimental space 16 libraries of A-TIM variants Fully randomized mutagenesis Starting points (4) - ATIM (A) Error rate 0.3-0.6 % amino acid change (Fu) - ATIM-S96P (ASP) - ATIM-A178L (AAL) - ATIM-I245A (AIA) Active enzymes A-TIM-X* *RpiA/B activity *AraA activity *XylA activity Targeted mutagenesis (megaprimer method ) Results Regions (3) Libraries (16) - W100 (W) - A (Fu,W,VN,AGKE) - V214/N215 (VN) - ASP (Fu,W,VN,AGKE) - A233/G234/K239/E241 (AGKE) - AAL(Fu,W,VN,AGKE) - AIA (Fu,W,VN,AGKE) • All methods are verified and introduced mutations into the A-TIM sequence. • Screening based on Growth of Knock-out Knock-out strains strains on selective media is ongoing. • Screening methods for High Throughput approaches are under development The libraries – creating the experimental space 16 libraries of A-TIM variants Fully randomized mutagenesis Starting points (4) - ATIM (A) Error rate 0.3-0.6 % amino acid change (Fu) - ATIM-S96P (ASP) - ATIM-A178L (AAL) - ATIM-I245A (AIA) Targeted mutagenesis (megaprimer method ) Results Regions (3) Libraries (16) - W100 (W) - A (Fu,W,VN,AGKE) - V214/N215 (VN) - ASP (Fu,W,VN,AGKE) - A233/G234/K239/E241 (AGKE) - AAL(Fu,W,VN,AGKE) Pool I W100 43 Every screening 4 plates à 2000 colonies = 8000 Pool II V214/N215 A233/G234… 46 412 Pool III epPCR 3x 109 to 3x1015 Every screening 4 plates à 2000 colonies = 8000 4 screenings every day Every screening 4 plates à 2000 colonies = 8000 4 screenings every day - AIA (Fu,W,VN,AGKE) 1 screening required 525 days of screening required 93750 to 9.4x1010 days of screening required Biocatalysts - Solutions • Miniaturization Metagenomics • Parallelization Protein Engineering • High Throughput approaches Bioprocess Development • Modelling • Bioinformatics Biocatalysis at our Facilities The right Tools for the Right Methods... Tools High Throughput * Hamilton pipetting station Examples AThermostability expression of themophilic phosphorylases (diploma work – end 2008) Kathleen Zseker Parallelization * Small scale cultivation technology (EnBase) * Parallel cloning library Miniaturization * Cultivations * Parallel cloning library BTIM 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) 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 Metagenomics B Protein Engineering Biocatalysis at our Facilities C Bioprocess Development Where three key components meet... Biocatalysts Ligands Thermostability Substrates example molecules phosphorylases Used for validation and process optimization Process Development 0.100 ml 10 000 ml TIM barrels versatile platform for isomerisation Inhibitors Used to find ideal starting biomolecules for directed evolution 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 The chemistry of interactions... Ligands Substrates Used for validation and process optimization Matti Weismaa O O S R Nano Alho (NMR) OH O Inhibitors Used to find ideal starting biomolecules for directed evolution Cl O O S R OH HO O O O HO Detailed understanding Of interactions Biocatalysis at our Facilities Where three key components meet... Biocatalysts A Thermostability example molecules phosphorylases Ligands Substrates Used for validation and process optimization Process Development 0.100 ml 10 000 ml B TIM barrels versatile platform for isomerisation Inhibitors Used to find ideal starting biomolecules for directed evolution 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 More is less... Process Development 0.100 ml Cell density EnBase Fed batch 10 000 ml Small scale High Throughput is scaleable to Production Traditional Cultivation time Modelling In solico design of future experiments d (Vy) = Fi yi + Qiyg,i - F0 δy – Q0yg,0 + Vry dt Kathleen Szeker (formula for mass balance [kg h-1] 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 Wild type TIM Wild type studies X-Ray Crystallography NMR Mass Spec. High Throughput methods Binding Studies monoTIM ml1 TIM Chemical compounds ml8b TIM Pool of enzymes Process development Screen for activity Input Applications Input A-TIM variants ICM docking Technology Random mutagenesis /shuffling Selection of best mutants iterative directed evolution Output Kealases The search continues... Methods What was done: A) Usually done, when structure of enzyme is not well or not at all known error prone PCR: Mutagenesis GeneMorph II Random Mutagenesis Kit A) fully random B) targeted random B) Structure of TIM is well known: Rational Design Megaprimer PCR: according to Wu et al. 2005 B) Why? A) Why? 1. Combination of two polymerases lowers bias of single bases 2. includes a cloning kit 1.Simple primers with Wobbles (Ns) within the targeted areas 2.one-step PCR, no interference necessary