University College London. Scale-up Issues for Whole-cell Biocatalytic Oxidation. John M Woodley

Transcription

1 University College London Scale-up Issues for Whole-cell Biocatalytic Oxidation John M Woodley

2 Scale-up Issues Productivity Production capacity Scale Lab equipment Plant equipment

3 Scale-up and Implementation Exquisite (chiral) chemistry, under mild conditions but.. availability of biocatalysts integration with chemistry productivity limitations

4 Productivity Limitations Substrate and product instability Substrate and product inhibition / toxicity Biocatalyst instability Non-natural substrate access into whole cells and low rates of reaction Aqueous media Integration with neighbouring operations

5 Potential Solutions Auxiliary phase biocatalysis two-liquid phase resin Feed and bleed Catalyst immobilisation Genetic engineering Protein engineering

6 BVMO

7 Model Baeyer-Villiger Reaction bicyclo[3.2.0]hept-2-en-6-one (-) 1(S), 5(R) 2- oxabicyclo[3.3.0]oct-6-en-3-one H H O O O O + O O 2, NADPH, H + H 2 O, NADP + H (-) 1(R), 5(S) 3- oxabicyclo[3.3.0]oct-6-en-2-one H Alphand et al 1989 Tet Lett 30, 3663; Alphand and Furstoss 1992 J Org Chem 57, 1306

8 BVMO Challenges Wild type host is pathogenic, contains contaminating activity and is difficult to grow CHMO is susceptible to oxidation and therefore unstable Stoichiometric quantities of NADPH are required Many reactions suffer from low intrinsic reaction rates and inhibition Reactions require molecular oxygen

9 Recombinant CHMO (pqr239) Antibiotic Ampicillin Inducer - Arabinose

10 BVMO Fermentation Arabinose induction DOT (%), CER, OUR Agitation speed (rpm) OD 670nm Time (h)

11 1. Choice of Catalyst Form

12 Catalyst selection software Database of enzymes with known reactions and e.e. Is a R-WC available Is the pathogenicity of the WT-WC acceptable Free enzyme Low enough contaminating activity Low enough contaminating activity Low enough contaminating activity Purification Does the R-WC have high enough activity Is the low activity due to the cell barrier Does the WT-WC have high enough activity Is the low activity due to the cell barrier Does the homogenate have high enough activity Protein engineering Permeabilise Permeabilise Is homogenate stability high enough Does protease inhibition improve stability Protein engineering Loss of activity on immobilisation Does the R-WC have high enough stability Is the enzyme the cause of the low stability Does the WT-WC have high enough stability Is the enzyme the cause of the low stability Does the R-WC suffer reactant or product toxicity Is the enzyme the cause of the inhibition Does the WT-WC suffer reactant or product toxicity Is the enzyme the cause of the inhibition Does free enzyme suffer reactant or product inhibition Protein engineering Reactant or product inhibition R-WC WT-WC Free enzyme Immobilised enzyme

13 Process options air Fermentation + Biocatalysis Purification Product substrate air air Fermentation Biocatalysis Purification Product substrate air Fermentation Biocatalyst preparation Biocatalysis Purification Product substrate

14 Isolated enzyme issues Isolation procedure and losses Cost of immobilisation support Number of recycles achievable Diffusional limitations (Thiele modulus / Damkohler number) Cofactor recycle

15 Process Options air Fermentation + Biocatalysis Purification Product substrate air air Fermentation Biocatalysis Purification Product substrate Clean media for improved DSP Optimise production and use of catalyst Catalyst concentration independent of fermentation

16 Whole cell catalyst issues Effects on downstream process Side reactions and over metabolism Access of substrate to the enzyme Need for molecular oxygen Toxic effects on the host cell

17 Oxidation activities of different ketones Ketone Enzyme/Cell Bicyclo[3.2.0]hept-2-en-6-one methylcyclohexanone hexylcyclopentanone 5.0 Enzyme Microb Technol (2003)

18 Oxidation of bicyclo[3.2.0]hept-2-en-6-one Catalyst %X %ee g/l/h g/l g/gdcw Cell Enzyme Trade off between downstream process (g/l) and fermentation (g/gdcw) Potential to overcome fermentation limit by recycle of immobilised enzyme

19 Scale-up issues Process intensity Feed-rates of reactant / media Rates of product removal Robustness to cope with heterogeneity Oxygen uptake

20 2. Oxygen Supply

21 Evaluation of Biocatalyst Kinetics and Stability 70 Volumetric activity (U.l -1 ) H H O BVMO catalysed lactone synthesis = 250 µl scale = 1 L scale Ketone concentration (g.l -1 ) [Doig et al (2002) Biotech. Bioeng., 80, 41]

22 Product formation during reaction 3.5 Product Lactone (g/l) Time (min)

23 Oxygen Supply Reaction Rate Oxygen supply [Catalyst]

24 75L scale (10 g/l dcw) biomass CHMO reaction Ketone / Lactone (g/l) DOT (%) Agitation speed (rpm) OD 670nm Time (h)

25 75L scale (5 g/l dcw) biomass CHMO reaction Ketone / Lactone (g/l) DOT (%) Agitation speed (rpm) OD 670nm Time (h)

26 Process limitations 6.0 max specific activity (0.65g/g.h) Initial Production rate (g/l.h) SF/1.5L 75L SF 1.5L 75L SF 1.5L Biomass (g/l)

27 Enriched air supply 6.0 Initial Production rate (g/l.h) % Oxygen in air

28 Process limitations 6.0 max specific activity (0.65g/g.h) 1.5L 60%O 2 Initial Production rate (g/l.h) SF/1.5L 75L SF 1.5L 1.5L 40%O 2 75L SF 1.5L 21%O 2 1.5L 10%O 2 Increase in Oxygen Biomass (g/l)

29 Whole Cell Reaction Model Metabolism + Reaction Oxygen demand O 2 supply O 2 limitation Metabolism O 2 limitation Biomass concentration

30 Productivity Limitations Oxygen limitation (Rate) Biocatalyst lifespan (Time) Product Lactone (g/l) Product Inhibition (concentratio 0 Time (min)

31 Biocatalyst concentration Product Lactone (g/l) g/l 5.0 g/l 3.0 g/l 1.0 g/l 0.2 g/l [Cell] Time (min)

32 3. Product Concentration

33 Resin Based Reactor Concept O O O

34 Recycle Reactor with Fixed Bed fixed bed of adsorbent. V CYCLE entry fermenter ph-sensor O -sensor 2 glycerol 1M H PO, 4M KOH 3 4 biocatalyst/cell exchange air outlet fermenter

35 Oxidation of bicyclo[3.2.0]hept-2-en-6-one Catalyst %X %ee g/l/h g/l g/gdcw Cell Enzyme Cell* Integration with ISPR is critical

36 4. Process Integration

37 Effect of cell concentration O 2 Stability Product concentration [Cell] Bioconversion time [Product]

38 Catalyst Concentration Determines interaction with fermentation Determines what is limiting productivity in the reactor catalyst preparation conversion downstream processing

39 Catalyst Preparation oncentration Fermentation Dewater Biocatalysis Dilution Fermentation Dilution Biocatalysis Direct Fermentation Biocatalysis

40 Catalyst Concentration Map Expression Direct [Catalyst] in fermentation ineffective Dilution Concentration ineffective B ineffective A A Rate limited B Product / Catalyst stability limited [Catalyst] in reaction

41 Process Drivers Catalyst Production Conversion Downstream Processing

42 Reaction profiles [Product] A B C Time

43 Process Metrics Metric Cost g/g g/l/h g/l catalyst production conversion downstream process

44 Regime analysis A B C Metric g/g g/l/hr g/l [Catalyst] Stability Product Rate limited limited limited

45 Process Mapping Trans I Chem E C (2002) 80 51

46 CHMO Available.

47 Products available.

48 Productivity Targets 100 g product /g biomass Optimise [Cell] and oxygen + ISPR Optimise [Cell] and oxygen ISPR g product /L

49 Conclusions Recombinant Escherichia coli containing CHMO 300 L scale Conversion using CHMO 200 L and 50 L scale / 1 Kg Oxygen supply modelled and understood limitations Product inhibition modelled and implementation of ISPR Scalable process using whole cell CHMO

50 Future Rapid methods of removing product Adequate means of oxygen supply Modelling for process analysis Isolation of product Improving stability of whole cells

51 Acknowledgements Jenny Littlechild, Exeter, UK John Ward, UCL, UK Dick Janssen, Groningen, NL Marcel Wubbolts, DSM, NL Giacomo Carrea, CNR Milan, IT Roland Wohlgemuth, Sigma-Aldrich Chemie, CH Roger Cripps, Consultant, UK Roland Furstoss, CNRS Marseille, FR European Commission BIO4-CT QLK3-CT

52 EC BVMO Programme