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Biocatalysts - trends

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)

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Biocatalysts - trends

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  1. Towards new biocatalytic activity of ATIM by structure based directed evolutionProjects, Bottlenecks and Where to Go Next?

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

  3. Biocatalysts - Bottlenecks • 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) Metagenomics”finding Enzymes” Protein Engineering”making enzymes” Bioprocess Development”using enzymes”

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

  5. Biocatalysis at our FacilitiesWhere three key components meet... Ligands SubstratesUsed for validation and process optimizationInhibitorsUsed to find ideal starting biomolecules for directed evolution Biocatalysts Thermostabilityexample moleculesphosphorylasesTIM barrelsversatile platform for isomerisation A ProcessDevelopment 0.100 ml 10 000 mlSmall scale High Throughput is scaleable to ProductionModellingIn solico design of future experiments B Prof. Peter NeubauerProcess Development Ph.D Tomi HillukkalaJaakko SoiniJohanna Panula-PeräläNarendar Kumar Khatri Prof. Peter NeubauerDirected evolution Molecular biologyEnzymologyProf. Rik Wierenga Structural studies Ph.D Mari YlianttilaPh.D.Markus AlahuhtaMarco Casteleijn / Mikko SalinMirja Krause/ Kathleen Szeker Prof. Marja LajunenOrganic chemistryPh.D. Sampo Mattila NMR Matti VaismaaNanna Alho

  6. Biocatalysis at our FacilitiesWhere three key components meet... Ligands SubstratesUsed for validation and process optimizationInhibitorsUsed to find ideal starting biomolecules for directed evolution Biocatalysts Thermostabilityexample moleculesphosphorylasesTIM barrelsversatile platform for isomerisation A ProcessDevelopment 0.100 ml 10 000 mlSmall scale High Throughput is scaleable to ProductionModellingIn solico design of future experiments B Prof. Peter NeubauerProcess Development Ph.D Tomi HillukkalaJaakko SoiniJohanna Panula-PeräläNarendar Kumar Khatri Prof. Peter NeubauerDirected evolution Molecular biologyEnzymologyProf. Rik Wierenga Structural studies Ph.D Mari YlianttilaPh.D.Markus AlahuhtaMarco Casteleijn / Mikko SalinMirja Krause/ Kathleen Szeker Prof. Marja Lajunen Organic chemistryPh.D. Sampo Mattila NMR Matti VaismaaNanna Alho

  7. Biocatalysis at our FacilitiesEnzymes... Biocatalysts Thermostabilityexample moleculesphosphorylasesTIM barrelsversatile platform for isomerisation BIOCAT-HT: Production of active thermostable phosphorylases based on High Throughput strategies A Parallel transformations and expressions of phosphorylases isolated from thermophilic organisms by using a fusion-partner plasmid library. • High quantity approach: automated, fed-batch small scalecultivations, on-line evaluation of proper folding • Starting points • Novel thermostable phosphorylases • Development of High Throughput methods B 45 High Throughput parallel optimization gene cultivation product

  8. A. Thermostabilityexample moleculesphosphorylases Basic research Industrial application

  9. Biocatalysis at our FacilitiesEnzymes... Wild Type Kealases R R α-hydroxy aldehyde α-hydroxy keton Biocatalysts Thermostabilityexample moleculesphosphorylasesTIM barrelsversatile platform for isomerisation BIOCAT: New enzymes for the chiral synthesis ofnew chemical compounds by structure based directed evolution A 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 B

  10. B. TIM barrelsversatile platform for isomerisation Basic research Industrial application

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

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

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

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

  15. Wild type TIM Ml8b TIM Monomer monoTIM Not Active ml1 TIM Point mutationV233A ml8b TIM A-TIM variants

  16. Wild type TIM ATIM Monomer monoTIM Active site = okPerfect startfor Directed Evolution ml1 TIM ml8b TIM A-TIM variants

  17. Biocatalysis at our FacilitiesEnzymes... Random mutagenesis Mutant Libraries Screening NMRChemistryGrowthAutomated DNA Sequence Structural changes Biocatalysts Thermostabilityexample moleculesphosphorylasesTIM barrelsversatile platform for isomerisation Monomeric TIM is a very suitable protein for biocatalysis: A • 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 B

  18. Proof-Of-Principle studies Characterization of monomeric TIMs Binding studiesNMR/Mass SpectrometryChemical synthesis X-ray/docking Active enzymes Active enzymes A-TIM A-TIM-A178L A-TIM-S96P A-TIM-I245A A-TIM-X* A-TIM-Y** DirectedEvolution Screening Start *RpiA/B activity **new activity • NMR • Enzyme based • Chemical based • Growth based • Automated *AraA activity *XylA activity

  19. Biocatalysis at our FacilitiesDirected evolution... Random mutagenesis Mutant Libraries Screening NMRChemistryGrowthAutomated DNA Sequence Structural changes Biocatalysts Thermostabilityexample moleculesphosphorylasesTIM barrelsversatile platform for isomerisation Lead Enzyme ATIM Improved Variants A Screening in vivo Mutagenesis A) fully random B) targeted random B

  20. The libraries – selection of good targets Loop 6 A178L Loop 4 Lead enzyme ATIM S96P Rational Design: Site-directed mutagenesis creates four starting points for the directed evolution approach Loop 8 I245A Starting points (4) ATIM (A) ATIM-S96P (ASP) ATIM-A178L (AAL) ATIM-I245A (AIA) 4 Starting points • ATIM (A) • ATIM-S96P (ASP) • ATIM-A178L (AAL) • ATIM-I245A (AIA)

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

  22. The libraries – selection of good targets Loop 4 Loop 7 W100 Mutagenesis II) targeted random V214/ N215 Rational Design: Megaprimer PCR creates different libraries of ATIM mutants Regions (3) W100 (W) V214/N215 (VN) A233/G234/K239/E241 (AGKE) Targeted mutagenesis(megaprimer method ) Loop 8 3 Regions • W100 (W) • V214/N215 (VN) • A233/G234/K239/E241 (AGKE) A233/G234/ K239/E241

  23. Active enzymes • 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 Knock-out strains A-TIM-X* *RpiA/B activity *AraA activity *XylA activity The libraries – creating the experimental space 16 libraries of A-TIM variants Fully randomizedmutagenesis Targeted mutagenesis(megaprimer method ) Results Starting points (4) • ATIM (A) • ATIM-S96P (ASP) • ATIM-A178L (AAL) • ATIM-I245A (AIA) Error rate 0.3-0.6 %amino acid change(Fu) Regions (3) • W100 (W) • V214/N215 (VN) • A233/G234/K239/E241 (AGKE) Libraries (16) • A (Fu,W,VN,AGKE) • ASP (Fu,W,VN,AGKE) • AAL(Fu,W,VN,AGKE) • AIA (Fu,W,VN,AGKE)

  24. Pool I W100 43 Every screening 4 plates à 2000 colonies = 8000 1 screening required Pool II V214/N215 A233/G234… 46 412 Every screening 4 plates à 2000 colonies = 8000 4 screenings every day 525 days of screening required Pool III epPCR 3x 109 to 3x1015 Every screening 4 plates à 2000 colonies = 8000 4 screenings every day 93750 to 9.4x1010 days of screening required The libraries – creating the experimental space 16 libraries of A-TIM variants Fully randomizedmutagenesis Targeted mutagenesis(megaprimer method ) Results Starting points (4) • ATIM (A) • ATIM-S96P (ASP) • ATIM-A178L (AAL) • ATIM-I245A (AIA) Error rate 0.3-0.6 %amino acid change(Fu) Regions (3) • W100 (W) • V214/N215 (VN) • A233/G234/K239/E241 (AGKE) Libraries (16) • A (Fu,W,VN,AGKE) • ASP (Fu,W,VN,AGKE) • AAL(Fu,W,VN,AGKE) • AIA (Fu,W,VN,AGKE)

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

  26. Biocatalysis at our FacilitiesThe right Tools for the Right Methods... Tools High Throughput* Hamilton pipetting stationParallelization* Small scale cultivation technology (EnBase)* Parallel cloning library Miniaturization* Cultivations* Parallel cloning library Examples Thermostabilityexpression of themophilicphosphorylases(diploma work – end 2008)TIM barrelsParallel 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 B Kathleen Zseker

  27. Biocatalysis at our FacilitiesWhere three key components meet... Ligands SubstratesUsed for validation and process optimizationInhibitorsUsed to find ideal starting biomolecules for directed evolution Biocatalysts Thermostabilityexample moleculesphosphorylasesTIM barrelsversatile platform for isomerisation ProcessDevelopment 0.100 ml 10 000 mlSmall scale High Throughput is scaleable to ProductionModellingIn solico design of future experiments Metagenomics Protein Engineering Bioprocess Development A B C Prof. Peter NeubauerProcess Development Ph.D Tomi HillukkalaJaakko SoiniJohanna Panula-PeräläNarendar Kumar Khatri Prof. Peter NeubauerDirected evolution Molecular biologyEnzymologyProf. Rik Wierenga Structural studies Ph.D Mari YlianttilaPh.D.Markus AlahuhtaMarco Casteleijn / Mikko SalinMirja Krause/ Kathleen Szeker Prof. Marja Lajunen Organic chemistryPh.D. Sampo Mattila NMR Matti VaismaaNanna Alho

  28. Biocatalysis at our FacilitiesThe chemistry of interactions... Ligands SubstratesUsed for validation and process optimizationInhibitorsUsed to find ideal starting biomolecules for directed evolution Matti Weismaa Nano Alho (NMR) Detailed understanding Of interactions

  29. Biocatalysis at our FacilitiesWhere three key components meet... Ligands SubstratesUsed for validation and process optimizationInhibitorsUsed to find ideal starting biomolecules for directed evolution Biocatalysts Thermostabilityexample moleculesphosphorylasesTIM barrelsversatile platform for isomerisation A ProcessDevelopment 0.100 ml 10 000 mlSmall scale High Throughput is scaleable to ProductionModellingIn solico design of future experiments B Prof. Peter NeubauerProcess Development Ph.D Tomi HillukkalaJaakko SoiniJohanna Panula-PeräläNarendar Kumar Khatri Prof. Peter NeubauerDirected evolution Molecular biologyEnzymologyProf. Rik Wierenga Structural studies Ph.D Mari YlianttilaPh.D.Markus AlahuhtaMarco Casteleijn / Mikko SalinMirja Krause/ Kathleen Szeker Prof. Marja Lajunen Organic chemistryPh.D. Sampo Mattila NMR Matti VaismaaNanna Alho

  30. Biocatalysis at our FacilitiesMore is less... ProcessDevelopment 0.100 ml 10 000 mlSmall scale High Throughput is scaleable to ProductionModellingIn solico design of future experiments d (Vy) = Fiyi + Qiyg,i - F0δy – Q0yg,0 + Vry (formula for mass balance [kg h-1] dt Kathleen Szeker

  31. Biocatalysis at our FacilitiesPresentations... 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

  32. BIOCAT - Network summary Wild type TIM Analytical tools Wild typestudies X-RayCrystallography NMR Mass Spec. High Throughput methods Binding Studies monoTIM Chemical compounds Process development ml1 TIM Screen for activity Pool of enzymes ml8b TIM Input Applications Kealases ICM docking Technology Input Output Random mutage-nesis /shuffling Selection of best mutants • A-TIM • variants iterative directed evolution

  33. The search continues...

  34. Methods What was done: A) Usually done, when structure of enzyme is not well or not at all known error prone PCR: GeneMorph II Random Mutagenesis Kit Mutagenesis 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? • Simple primers with Wobbles (Ns) within the targeted areas • one-step PCR, no interference necessary A) Why? • Combination of two polymerases lowers bias of single bases • includes a cloning kit

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