1. Im Rahmen des K plus Programms gefördert durch: Pichia pastoris: Protein Production and More Franz Hartner 1, Liu Zhibin 1, Beate Pscheidt 1, Bettina Janesch 1, Roland Weis 1,2, Sandra Abad 1, Karl Gruber 1 and Anton Glieder 1 1 Institute of Molecular Biotechnology, Research Centre Applied Biocatalysis, Petersgasse 14, A-8010, Graz, AUSTRIA, anton.glieder@a-b.at 2 VTU Engineering GmbH, Grambach, Austria Innovation from Nature Cofactor Product Substrate Byproduct Cosubstrate

2. © Angewandte Biokatalyse-Kompetenzzentrum GmbH Identification, Isolation Expression Adaptation Production (Expression) The Making of Industrial Biocatalysts „Bio“Contributions Organic, Food, Polymer Chemistry Bioprocess Engineering Organic, Food, Polymer Chemistry Bioprocess Engineering

3. © Angewandte Biokatalyse-Kompetenzzentrum GmbH * Most industrial enzymes isolated from microorganisms * Produced by microorganisms Is Simple & fast * Enzymes from higher eukaryotes * Additional unique diversity * Produced by microorganisms Should be Simple & fast Natural Sources for Biocatalysts

4. © Angewandte Biokatalyse-Kompetenzzentrum GmbH Excellent folding capabilities Eukaryotic post-translational modifications High Cell density, cheap media High purity of secreted proteins High variations in screening (copy effects) Pichia pastoris – methylotrophic yeast Plasmid cloning in E. coli before Pichia transformation Slower than E. coli and labour intensive

5. © Angewandte Biokatalyse-Kompetenzzentrum GmbH Reliable 96 well plate screening Rational design of enzyme variants Plasmid/ E. coli independent library generation Directed Evolution Pichia pastoris: Expression, Engineering, Biotransformation Enhanced 2 nd generation expression system (vectors/platform strains) Whole Cell Biocatalysis

6. © Angewandte Biokatalyse-Kompetenzzentrum GmbH Pichia - Reliable Micro Cultivation methanol inducible system OD595 after 60 h = time of induction 0,2123 Glucose concentration / % enzyme activity after 75 h of induction Glucose concentration / % 0,2123 123 Weis, R., R. Luiten, W. Skranc, H. Schwab, M. Wubbolts, and A. Glieder. 2004. FEMS Yeast Research 5:179-189.

7. © Angewandte Biokatalyse-Kompetenzzentrum GmbH Apoptosis/Necrosis Reporter for media/screening optimization Weis, R., R. Luiten, W. Skranc, H. Schwab, M. Wubbolts, and A. Glieder. 2004. FEMS Yeast Research 5:179-189. OD595 after 60 h = time of induction 0,2123 Glucose concentration / % enzyme activity after 75 h of induction Glucose concentration / % 0.2% D 1% D2% D 3% D negative control H2O2- induced Tunel assay Necrosis assay

8. © Angewandte Biokatalyse-Kompetenzzentrum GmbH 300 µL 4000 L HRP Hb HNL 4000 L Weis, R., R. Luiten, W. Skranc, H. Schwab, M. Wubbolts, and A. Glieder. 2004. FEMS Yeast Research 5:179-189.

9. © Angewandte Biokatalyse-Kompetenzzentrum GmbH (R)-2-chloro mandelonitrile key intermediate for (R)-2-chloro-mandelic acid for production of cardiovascular drug (R)-2-hydroxy-4-phenylbutyronitrile Intermediate for „-prils“ Glieder et al., Angew. Chem. Int. Ed. (2003) 42; 4815-4818 Weis et al., Angew. Chem. Int. Ed. (2005) 44 (30), 4700-4704 Structural Design HydroxyNitrileLyase HNL

10. © Angewandte Biokatalyse-Kompetenzzentrum GmbH 2-chloro mandelonitrile in model of active centre of PaHNL5 ee > 95 % Glieder, A., et al (2003) Angew Chem, Int Ed, 42, 4815-4818 Synthesis of (R)-2-Chloro mandelonitrile 6-7 fold increase in activity Designed for High Activity

11. © Angewandte Biokatalyse-Kompetenzzentrum GmbH Synthesis of precursors of ACE (Angiotensin Converting Enzyme) inhibitors Designed Stereoselectivity Low Stereoselectivity with substrates with CH 2 spacers between C=O and aromatic ring 3-phenylpropenal 3-phenylpropanal R. Weis, R. Gaisberger, W. Skranc, K. Gruber, A.Glieder (2005), Angewandte Chemie Int. Ed., 44, 4700-4704

12. © Angewandte Biokatalyse-Kompetenzzentrum GmbH Anticoagulant ACE inhibitors (R)-Hydroxynitrile lyases New R-HNLs for API Production structure guided design Directed evolution Further increase in ee and activity Glieder, A., et al (2003) Angew Chem, Int Ed, 42, 4815-4818 R. Weis, R. Gaisberger, W. Skranc, K. Gruber, A.Glieder (2005) Angew. Chemie Int. Ed., 44, 4700-4704 Liu, Z., Pscheidt, B., et al (2007) ChemBioChem, in press

13. © Angewandte Biokatalyse-Kompetenzzentrum GmbH Linear Expression Cassette by OE-PCR: Random Mutagenesis – epPCR Site-Saturation Mutagenesis New Strategy for Library Generation Random & Site Directed mutagenesis Partial GAP promotor Alpha-factor sequence Partial gene Mutated geneZeocin cassette Overlap 2 Overlap 1 ╳ Partial GAP promotor Alpha-factor sequence Mutated geneZeocin cassette Overlap 2 ╳╳╳╳ Overlap 1

14. © Angewandte Biokatalyse-Kompetenzzentrum GmbH Single gene recombination: combining favorable mutations HNL variants of 1st round as templates Pool of fragments generated by PCR In vitro recombination Linear Expression Cassette by OE-PCR: promotor Mutated geneSelection marker Overlap 2 ╳╳╳╳ Overlap 1 linker New Strategy for Library Generation Recombination

15. © Angewandte Biokatalyse-Kompetenzzentrum GmbH Pichia pastoris a host for screening and production Direct transformation of linear integration cassette  A280  t -1 /10 -3 min -1

16. © Angewandte Biokatalyse-Kompetenzzentrum GmbH Synthesis of Cl-, Br- and F- substituted ( R ) -mandelonitriles 0 10 20 30 40 50 60 70 80 90 100 0,5 h 1 h2 h4 h Conversion% A111G4x A111G muteins Higher activity and higher ee by Directed Evolution in Pichia pastoris Liu, Z., Pscheidt, B., et al (2008) ChemBioChem,

17. © Angewandte Biokatalyse-Kompetenzzentrum GmbH Linear Expression Cassette by OE-PCR: Random Mutagenesis – epPCR Site-Saturation Mutagenesis New Strategy for Library Generation Random & Site Directed mutagenesis Partial GAP promotor Alpha-factor sequence Partial gene Mutated geneZeocin cassette Overlap 2 Overlap 1 ╳ Partial GAP promotor Alpha-factor sequence Mutated geneZeocin cassette Overlap 2 ╳╳╳╳ Overlap 1

18. © Angewandte Biokatalyse-Kompetenzzentrum GmbH Site Saturation Approach (sterically hindered aliphatic aldehydes) Saturation of hydrophobic sites in substrate binding pocket –~200 transformants per site screened for improved conversion ~10 fold improved TOF value enantiomeric excess ≥ 95% possible - reasonable amount of enzyme For pivalaldehyde and hydroxypivalaldehyde conversion R-HNL/NaCN (R)-Hydroxypivalaldehyde cyanohydrin Hydroxypivalaldehyde 3M Citrate- phosphate- buffer (pH 2.4) precursor for panthotenic acid All these enzymes produced by Pichia pastoris

19. © Angewandte Biokatalyse-Kompetenzzentrum GmbH Engineered Promoter Libraries Transcription factor binding sites: e.g. HSF......heat shock factor HAP…..O 2 and glucose regulation STRE…..stress response element GCR….glucose repressor  Deletions on many positions  Short deletions (5-60 bp), covering putative transcription factor binding sites Hundreds of different variants AOX1 promoter 953 bp delta1 delta2 delta3 delta4 delta5 delta6 delta7 delta8 delta9 HSF GCR1 HAP234 ADR1 RAP1 (TUF1) HAP1 HAP234 STRE HSF QA-1F MAT1MC ABAA Transcription Start

20. © Angewandte Biokatalyse-Kompetenzzentrum GmbH First Deletions Library d2 d2d6 d6* d1  Strength  Regulatory features  Promoter length 0% 20% 40% 60% 80% 100% 120% 140% 160% relative GFP fluorescence after 72h of induction WT (single copy clones) Next generation system commercialized by VTU Technology

21. © Angewandte Biokatalyse-Kompetenzzentrum GmbH Basal (short) promoters  Basal promoters were cut 5’ of the TATA box  Different fractions of the promoter were added  Variants with small putative cis-acting elements added to the basal promoters ScLeu2 basal or AOX1 basal promoter fragment P(AOX1) P(AOX1) basal TATA box Transcription Initiation Site

22. © Angewandte Biokatalyse-Kompetenzzentrum GmbH Schematic time course of GFP expression  Different promoters, different regulatory features, different levels of GFP expression (growth, derepression, methanol phase) Growth phase Derepression phase Methanol induction phase

23. © Angewandte Biokatalyse-Kompetenzzentrum GmbH Platform Strains & Combinatorial Expression expression enhancers Models: Horseradish Peroxidase (Isoenzyme C) Candida antarctica lipase B (CALB)

24. © Angewandte Biokatalyse-Kompetenzzentrum GmbH HRP Overexpression HRP single copy strain HRP multi copy strain 1.6x 30x 21x 11.6x 51x 18x 28x GAP: constitutive ∆6* : derepression, medium induction AOX1: strong induction by methanol

25. © Angewandte Biokatalyse-Kompetenzzentrum GmbH CALB Overexpression low and high copy number CALB-AOX∆6* low copy strain CALB-AOX∆6* high copy strain 1.4x 1.6x 0.1x 1.6x 2.4x 3.3x A combinatorial problem !!!

26. Introduction Alternative Oxidases Providing metabolic flexibility –Developmental conditions (senescence, fruit ripening) –Environmental fluctuations – stress answer, fungicide resistance Minimize generation of ROS Maintain TCA cycle –citric acid production (Aspergillus niger) Antimycin A Cyanide, azide Hydroxamic acids, alkyl gallates

27. Structure AOD structure Nuclear encoded Plants: dimer, fungi and yeasts: monomer 2 highly conserved E-X-X-H motifs → di-iron di-carboxylate proteins Model for C-terminal, membrane embedded part → four helix bundle matrix Inner mitochondrial membrane Structural model, Berthold, 2003

28. Expression system Expression host: Pichia pastoris WT Enzymesplant and fungal AODs Expression constructs: –AOD-linker-Streptag II –AOD-EK-GFP-Streptag II PCR based linear expression cassettes Structural genomics (membrane proteins)

29. Purification Cells   Mitochondria Cell debris   Matrix (3) Mitochondrial membranes (2)   Sol. Proteins (5) Residual membranes(4)   Unbound Proteins Strep-tagged proteins (6-9)(12-14) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 MW [kDa] 191 97 64 51 39 28 19 14 AOD verified by MALDI analysis (S. Deller) MW [kDa] 191 97 64 51 39 28 19 14 purified membrane enzyme = active CD spectra

30. © Angewandte Biokatalyse-Kompetenzzentrum GmbH Metabolic Engineering

31. © Angewandte Biokatalyse-Kompetenzzentrum GmbH Engineering Central Metabolic Pathways of P. pastoris Bottlenecks in NAD + regeneration and ATP feedback inhibitions limit flux through glycolysis and TCA cycle Uncoupling of NAD + regeneration from ATP production by overexpressing AOD higher growth rate higher substrate uptake rate  lower final biomass yield → higher flux through central carbon metabolism → improved NAD + regeneration Glucose Pyruvate AcetylCoA ADP, 2 NAD + 2 CO 2 TCA cycle 6 NAD +, 2 FAD 2 NAD + +PFK overexpression

32. © Angewandte Biokatalyse-Kompetenzzentrum GmbH The lucky 4….. methanol induces expression of the biocatalysts Pichia pastoris is designed to tolerate methanol and methanol can act as a solvent for the substrate Methanol is a nutrient and Cosubstrate for the Cofactorregeneration Methylotrophic Yeasts for Whole Cell Biocatalysis Expression of endogenous cofactor regeneration system induced by methanol Back to the cell…..

33. © Angewandte Biokatalyse-Kompetenzzentrum GmbH Co-factor Regeneration !using the whole pathway! Toxic intermediates 2 NADH per MeOH CO 2 as byproduct  irreversible reaction q S,max 10-17 mmol g -1 h -1 q S,max 15 mmol g -1 h -1 S. cerevisiae on glucose P. pastoris on methanol Blank et al., 2004 Jahic et al., 2002

34. © Angewandte Biokatalyse-Kompetenzzentrum GmbH Growth of DAS Knockout Strains wild type  aox1  das1  das2  das1  das2

35. © Angewandte Biokatalyse-Kompetenzzentrum GmbH Butanediol DH catalysed Biotransformtion Increased cell density (60 g/L) 25 g/L substrate concentration 6% methanol Shake flasks

36. © Angewandte Biokatalyse-Kompetenzzentrum GmbH Synthetic ADH3 and ADH4 genes ADH3_WT ADH3_synthetic_high expression ADH3ADH4 ADH3, ADH4 are NADPH dependent (BASF)

37. © Angewandte Biokatalyse-Kompetenzzentrum GmbH Yields in Shake flasks shake flask cultures, 1 g/L substrate

38. © Angewandte Biokatalyse-Kompetenzzentrum GmbH Pichia pastoris based Synthetic biotechnology Optimized synthetic genes Engineered enzymes laboratory evolved & designed

39. © Angewandte Biokatalyse-Kompetenzzentrum GmbH Synthetic Biotechnology PCR based linear expression cassettes d2 d2d6 d6* d1 0% 20% 40% 60% 80% 100% 120% 140% 160% WT Synthetic (modular) promoter libraries promotor Mutated geneSelection marker Overlap 2 ╳╳╳╳ Overlap 1 Engineered platform hosts License free and advanced expression system

40. © Angewandte Biokatalyse-Kompetenzzentrum GmbH Synthetic Biotechnology in Whole Cell Biocatalysis Minimal Cell for NAD(P)H dependent catalysis Classic: Growing Cells By-product: biomass Classic: Resting Cells No catalyst regeneration Engineered methylotrophic Yeast

41. © Angewandte Biokatalyse-Kompetenzzentrum GmbH Glieder group Pichia Team (Graz) GLIEDER GROUP July 2007 DSM, BASF, VTU Roland Weis Hannelore Mandl Beate Pscheidt (Karl Gruber) Liu Zhibin Franz Hartner Sandra Abad Claudia Ruth Ulrike Schreiner Kerstin Kitz Bettina Janesch Manuel Peter Astrid Hoermann Maria Freigassner Andrea Mellitzer &

42. © Angewandte Biokatalyse-Kompetenzzentrum GmbH Project Ideas Strain engineering by promoter replacements Design of fully synthetic promoters for different yeast starins