Simplifying Biocatalytic Process can Mitigate Scale-up and - PowerPoint PPT Presentation

Simplifying Biocatalytic Process can Mitigate Scale-up and Separation Issues Chemical & Biochemical Engineering; Center for Biocatalysis & Bioprocessing (CBB), University of Iowa 4 th Annual Next Generation Bio-based Chemicals: Bio-manufacturing & Scale-up Jan 28, 2013 MVS 1/13

Broader Research Philosophy in Subramanian Laboratory: Bio-feedstock to (Fine) Chemicals via Microorganisms/Enzymes Microbial Metabolism (New) Pathways/Enzymes/Genes Applications: Chemicals, Dignostics, Biofuels, Enzyme Mechanisms Environmental Assessment MVS 1/13

• Over-Arching Technology • *Niche (Redox) Application One Enzyme Many (Redox) Enzymes * One Process *Many (Redox) Products *No NAD(P)/H input Several Products *No co-substrate MVS 1/13

One Process: Spray Dried Cells For Single or Multi-Enzyme-Based Catalysis **WO 2009/064277 • Simplifies Unit Operation: Fermentation ** to Biocatalysis • Small molecules in/out. Proteins IN Spray • No loss of activity like upon immobilization Dried • No support needed Catalyst • Solid phase catalysis: Just add the powder! • Highly stable enzyme prep. • No leaching of enzymes • Easily recyclable • Readily scalable • Robust • Patent pending MVS 1/13

Buchi B-190 Mini Spray-dryer Air Path Inlet Temp Probe (Not Shown) Hea+ng Element (Not Shown) Spray Nozzle Feed Drying Chamber Cyclone Outlet Temp Probe Collec+on Vessel MVS 1/13

Spray-Dried Pichia GO Strain 12 Microns; 3-5% Moisture MVS 1/13

• Over-arching Technology • *Niche (Redox) Application One Enzyme - Glycolate Oxidase Many (Redox) Enzymes (+ Catalase) One Process WO 2009/064277 *Many (Redox) Products Several Products: *No NAD(P)/H input *No co-substrate • Pyruvate, glyoxylate • Other keto acids, R -OH acids 7 MVS 1/13

Glycolate Oxidase: One Enzyme, One Process, Several Products • L-Lactate to pyruvic acid • Other α –hydroxy acid to keto acids • R,S- Hydroxy acids to R -hydroxy acid in a dynamic process (>95% yield) • L-Amino acids to R-hydroxy acids in a dynamic process (on-going) S. Das, J. Glenn IV and M.V. Subramanian (2010). Enantioselective oxidation of 2-hydroxycarboxylic acids by glycolate oxidase and catalase coexpressed in methylotrophic Pichia pastoris . Biotechnology Progress 26: 607-615. MVS 1/13

Production of Pyruvic Acid by SD rPichia L-Lactate Production of WO 2009/064277 High-Cell Density Biocatalyst = ** 20 Kg/100 L Redox reaction Spray with spray- Dry dried cells, Catalyst recovery of products Pyruvate • Only 2-3 unit operations • Enzyme:Product ratio, 1:45 (5 kg enzyme = 225 kg pyruvate) • Purity >99%. Conversion in water. • *No specific gene IP issue/FTO issues • ** Proprietary technology • Technology licensed to a large company. Another has just signed an Option Agreement MVS 1/13

Batch-wise Recycling of GO for Pyruvate Production 120 100 80 pO2.PV [%DO] 15+2g/L A 60 15+2g/L B 12+2g/L A 12+2g/L B 40 20 0 0 24 48 72 96 120 144 168 192 216 240 Runtime [h] +2 g/L cells +3 g/L cells MVS 1/13

Biologically Produced Pyruvate from 8L Bioreactor - Freeze dried Sample Bottle contains 152 g of total 1 Kg pyruvate MVS 1/13

Dynamic Resolution of RS -2-Hydroxy acids using GO O O O O + + R R R R Glycolate Oxidase OH OH OH O + Catalase H OH OH O OH 2 ( R )-1 ( R )-1 ( S )-1 Reduction (NaBH 4 ) R = Different aliphatic and 1 = 2-Hydroxy Acids aromatic side chain 2 = 2-Keto Acids • One pot reac+on • >95% yield of R‐HBA or D‐Lac+c acid • Reac+on in “dead‐cell” format, hence no issue with NaBH 4 MVS 1/13

D -Lactate from L -Lactate by GO & Catalase 250.00 Concentration (mM) 200.00 D-LA 150.00 L-LA BH 100.00 PA 50.00 0.00 0 1 2 3 4 5 6 Reaction Coordinate (h) D‐Lactate Produced From 1.0 L of 250 mM L‐Lactate – in One Pot Reac+on Mixture MVS 1/13

• Over-arching Technology • *Niche Application One Enzyme - Many (Redox) Enzymes Glycolate Oxidase Provisional 2/10 One Process Utility 2/3/11 – US Serial #13/020,588 *Many (Redox) Products Several Products: *No NAD(P)/H input *No co-substrate • Pyruvate, glyoxylate • Other keto acids, R -OH acids 14 MVS 1/13

Production of Bioxylitol by SD rPichia Crude Dilute acid Agricultural residue: Hemicellulose Eg: Corn stover hydrolysate Production of Provisional 2/10, Utility 2/3/11 – USSerial #13/020,588 High-Cell Density Biocatalyst* = ** 20 Kg/100 L Redox reaction Spray with spray- Dry dried cells, Catalyst recovery of products Xylitol • Only 2-3 unit operations • Bioxylitol produced with crude hemicellulose hydrolysate, 70% conversion in 2 hours! • Various inhibitory components in corn hydrolysate have no impact on the biocatalysis • *No specific gene IP issue/FTO issues (wild type gene without improvement is best!) • ** Proprietary technology • Bio-xylitol sample available. MVS 1/13

Biocatalysis Progress: C-5 Hydrolysate Reaction Mixture to Xylitol Crystals Post-Reaction 1-Step SMB* Xylitol C5-Hydrolysate Mixture Purified Xylitol Crystals MVS 1/13

After Warming @ 50°C Water bath for 20 min & Allowing to cool to Room Temp SMB Purified Concentrated Sample was Diluted 1/100 times Xylitol And Injected into HPLC HPLC Conditions Purity: 98.72% Column: Biorad Aminex HPX-87H (300 x 7.8 mm 2 i.d.) Flow rate: 0.6 mL/min Detector: RID Mobile Phase: 5mM Sulfuric acid MVS 10/06

Xylitol Crystals on the Side Glass Surface After keeping few hours @ 4°C MVS 1/13

HPLC Profile of Xylitol Crystal (50 mM) Purity: 99.35% MVS 1/13

Xylitol Process Needs Further Optimization Incoming C5 stream from corn stover Xylitol - >98% hydrolysis pure in extract Raffinate – Left over xylose + NAD Redox reaction with spray-dried cells, recovery of product Catalyst Recycle Xylitol in stover hydrolysate MVS 1/13

SDP: A Different Approach to Metabolic Engineering! Utilizing Native or Engineered Enzymes, in Combination OR Metabolically Engineered in Spray Dried (Dead) Powder* to Generate Products MVS 1/13

Metabolic Engineering • Find enzymes that have some native ability to catalyze the reaction of interest • Generate diversity • Select for improved variants • Recruit and assemble genes to encode complete pathways • Optimize regulation and flux (of “C”)* *Limited to mostly E. coli & Saccharomyces MVS 10/06

Metabolic Engineering MVS 1/13

Metabolic Engineering of 3-HP producer Glucose Find Enzymes NADH Assemble Enzymes ATP Improve Enzymes PEP Succinate OAA Find Enzymes NAD + Lactate Pyruvate Assemble Enzymes Improve Enzymes β -Ala NAD + Formate NAD + Acetyl-CoA 3-HP 2 Acetyl-P Acetate Ethanol Regulate & Optimize NAD + ATP “Carbon”-flow to the Desired Product MVS 1/13

SDP – A Different Approach to Metabolic Engineering? Utilizing Native or Engineered Enzymes, in Combination OR Metabolically Engineered in Spray Dried Powder* to Generate Products Metabolic Engineering SDP-Technology Live cells Dead Cells Fermentation 3-7 days Fermentation 3 days + Reaction, few hours Multi-gene modifications Three demonstrated Maximize “C” to product Not necessary Glucose/Glycerol needed Not necessary Redox balance Not necessary Product recovery issues Simple recovery, fewer UOP MVS 1/13

Subramanian Research Group 6/11 MVS 1/13