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Biocatalysis at our Facilities Where three key components meet...

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

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Biocatalysis at our Facilities Where three key components meet...

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  1. Modular Biocatalyst Platform for Chiral Synthesis of ChemicalCompounds by Structure-based Directed Evolutionthe BIOCAT project

  2. Biocatalysis at our FacilitiesWhere three key components meet... Biocatalysts TIM barrelsversatile platform for isomerisation Ligands SubstratesUsed for validation and process optimizationInhibitorsUsed to find starting ideal biomolecules for directed evolution ProcessDevelopment 0.100 ml 10 000 mlSmall scale High Throughput is scaleable to Production 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 CasteleijnMikko SalinMirja Krause Prof. Marja Lajunen Organic chemistryPh.D. Sampo Mattila NMR Matti VaismaaNanna Alho

  3. Biocatalysis The Project Biocatalysts TIM barrelsversatile platform for isomerisation 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 Type * Kealases R R α-hydroxy aldehyde α-hydroxy keton

  4. *AraA activity *XylA activity Added based on the previous KETJU meeting 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** Directed Evolution Directed Evolution Screening Selection Start *RpiA/B activity **new activity

  5. The libraries – selection of good targets A178L Lead enzyme ATIM S96P Rational Design: Site-directed mutagenesis creates four starting points for the directed evolution approach 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)

  6. The libraries – selection of good targets W100 Mutagenesis 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 ) A233/G234/ K239/E241 3 Regions • W100 (W) • V214/N215 (VN) • A233/G234/K239/E241 (AGKE)

  7. 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)

  8. 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 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 Knock out strainsW3110 F- λ- IN (rrnD-rrnE)1 (Datsenko and Warren PNAS 2000) Materials and protocols were a kind gift from:Prof. R. SternerDr. J. ClarenUniversity of Regensburg

  9. Knockout strains RPIA-/B-: Collaboration XylA-: Created own strain based on E. coli K12:W3110 AraA-: based on E. coli K12:W3110  ongoing Selection – the use of the experimental space Replacing known isomerase activity Libraries (16) • A (Fu,W,VN,AGKE) • ASP (Fu,W,VN,AGKE) • AAL(Fu,W,VN,AGKE) • AIA (Fu,W,VN,AGKE) Selection Loop 8 Cell Plate Neg. control Neg. control Pos. control 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 No Hits Two Hits Knock Out E. coli Strain Rondom gene from library Positive controle gene Plate with selective media (i.e. One (1) carbon source) Plasmid Colony utilizing selective sugars

  10. 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 46 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

  11. SummaryCurrent results... Knockout strains RPIA-/B-: Collaboration XylA-: Created own strain based on E. coli K12:W3110 AraA-: based on E. coli K12:W3110  ongoing Loop 8 Libraries (16) • A (Fu,W,VN,AGKE) • ASP (Fu,W,VN,AGKE) • AAL(Fu,W,VN,AGKE) • AIA (Fu,W,VN,AGKE) 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 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

  12. BIOCAT The project New methods * * Wild Type Kealases R R α-hydroxy keton α-hydroxy aldehyde A-TIM * New libraries* New knock-out strains * A-TIM libraries* knock-out strains Quantitative structuralEnzymological studies:* X-Ray* surface plasmon resonance* CD* Docking, biocomputing* Mass spectroscopy* Fluorescence* Enzyme kinetics * Selection assays Kealases

  13. BIOCAT - Network summary Wild type TIM Analytical tools Wild typestudies X-RayCrystallography NMR Mass Spectrometry 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

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