High Throughput Expression of Thermostable Phosphorylases: Technology & Solutions

1. Parallel high throughput expression of Thermostable PhosphorylasesFocus on technology

2. Linnanmaa campus Rovaniemi University of Oulu OULU Vaasa Kuopio Joensuu Jyväskylä Tampere Lappeenranta Turku Helsinki Biocatalyst platform Faculty of Science: Chemistry (3 groups)Biochemistry (1 group) Faculty of Technology: Bioprocess Engineering (1 group)

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

4. 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”

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

6. Biocatalysis at our FacilitiesEnzymes... Biocatalysts Thermostabilityexample moleculesphosphorylasesTIM barrelsversatile platform for isomerisation 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 scalecultivations, on-line evaluation of proper folding • Starting points • Novel thermostable phosphorylases • Development of High Throughput methods 45 High Throughput parallel optimization gene cultivation product

7. TechnologyEnBaseTM 96 well plates 1 [Johanna Panula-Perälä et al. 2008] (University of Oulu, BPEL)

8. Recombinant protein expression TechnologyCytoplasmic expression library Expression vector library for Cytoplasmic Protein Expression & Optimization from Uwe Horn2 45 different expression vectors • 3 promoters • 3 Ribosomal binding sites (SD) • 5 different fusion tags Ribosome-biding Site Promotor 2 [Kraft et al. 2007]

9. Generation of expression plasmids Transformation HT-cloning Gateway System Robotic Systems Evaluation Protein expression Online monitoring of protein aggregation1 LucA reporter plasmid TechnologyHigh Throughput strategies High cell density Cultivation Enbase Sampling, OD determination Transformations: ~ 500/dayProtein expression: 60 x 4 x 2 (T1&T2) = 480/experimentOnline: OD490 / Aggregation/ Sampling

10. Case study 1: The Phosphorylase 1 family Deinococcus geothermalis • Gram (+) Bacterium • Thermophilic radiophile • Optimal growth at 47°C • Isolated in hot springs in Italy and Portugal [Ferreira et al., 1997] Dgeo 1497 - PNP * APE 2105.1 - UP Aeropyrum pernix -Aerobic Archaeon - Optimal growth: 95°C - Isolated from hydrothermal vents in Japan [Sako at al., 1996] - Genome: 67% GC content sequenced in 1999 [Kawarabayasi et.al, 1999] reannoted in 2006 [Yamazaki et al., 2006] APE 0993.1 - MTAP * Poster:Parallel high throughput expression of Thermostable Phosphorylases

11. Case study 1: The Phosphorylase 1 family Basic research Industrial application

12. Case study 1: The Phosphorylase 1 family Activity assayby NMR(E. Coli UP in Cell Lysate) Protein expression

13. Case study 2: EnBaseTM 24-DWP replaces traditional Shake Flasks Challenge: Saving time and manpower by scaling down the expression format of 4 x 72 SF (288 SF) per week to EnBaseTM 24 DWP Test of 15 random clones….. … • Outcome: • Average final cell density over OD600=25 • Protein yield was best with MSM + booster (blue lines), cells are still in exponential phase • Preliminary results look promising that 6 shakes flasks can be replaced by one EnBaseTM24DWP Partners: J.Ottosson, H. Tegel, M. Hjelmare; M. Uhlen KTH Stockholm (Human Protein Atlas)

14. Case study 2: New developments: EnBaseTM Flo Challenge: High cell density cultivation on complex medium EnBase CM EnBase MSM + booster EnBase MSM +glucose +booster Low ---------- pH ---------- high NH4+ glucose Solution: Controlled growth on EnBase with MSM and boosting at time of induction Example: E. coli B21(DE3) and human PDI, 30°C, 180 rpm, EnBase mini shake flask system

15. 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  ongoingEnBaseTM Flo

16. Acknowledgements Academic Partners Industry Partners Oulu University Bioprocess Engineering Laboratory Prof. Peter NeubauerPh.D Mari Ylianttila Kathleen SzekérJohanna Panula-Perälä Chemistry Department Ph.D. Sampo Mattila Silja PelttariNanna Alho Biochemistry Department Prof. Rik Wierenga Ph.D. Andre JufferNiko Pursiainen Biosilta (FI) High Throuput expression system Metkinen (FI) Modified Nucleosides Fermentas (LV) Methodology, Scale-up: Juozas Šiurkus Technical University Berlin (GE) Prof. Peter Neubauer University of Kuopio (FI) Prof. Seppo LapinjokiProf. Igor Mikhailopulo Jarkko Roivainen Leibniz-Institute for Natural Product Research and Infection Biology – HKI (GE) Dr. Uwe HörnMario Kraft