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

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Simplifying Biocatalytic Process can Mitigate Scale-up and Separation Issues

Chemical & Biochemical Engineering; Center for Biocatalysis & Bioprocessing (CBB), University of Iowa 4th Annual Next Generation Bio-based Chemicals: Bio-manufacturing & Scale-up Jan 28, 2013

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Broader Research Philosophy in Subramanian Laboratory: Bio-feedstock to (Fine) Chemicals via Microorganisms/Enzymes

Microbial Metabolism Applications: Chemicals, Dignostics, Biofuels, Environmental Assessment (New) Pathways/Enzymes/Genes Enzyme Mechanisms

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• Over-Arching Technology
• *Niche (Redox) Application

One Process

One Enzyme Several Products Many (Redox) Enzymes* *Many (Redox) Products *No NAD(P)/H input *No co-substrate

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Spray Dried Catalyst

**

**WO 2009/064277

One Process: Spray Dried Cells For Single or Multi-Enzyme-Based Catalysis

• Simplifies Unit Operation: Fermentation

to Biocatalysis

• Small molecules in/out. Proteins IN
• No loss of activity like upon immobilization
• No support needed
• Solid phase catalysis: Just add the powder!
• Highly stable enzyme prep.
• No leaching of enzymes
• Easily recyclable
• Readily scalable
• Robust
• Patent pending

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Buchi B-190 Mini Spray-dryer

Air Path Hea+ng Element Drying Chamber Cyclone Inlet Temp Probe Outlet Temp Probe Collec+on Vessel Feed Spray Nozzle

(Not Shown) (Not Shown)

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Spray-Dried Pichia GO Strain

12 Microns; 3-5% Moisture

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• Over-arching Technology
• *Niche (Redox) Application

One Process

One Enzyme - Glycolate Oxidase (+ Catalase) Several Products:

• Pyruvate, glyoxylate
• Other keto acids, R-OH acids

Many (Redox) Enzymes *Many (Redox) Products *No NAD(P)/H input *No co-substrate WO 2009/064277

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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.

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Redox reaction with spray- dried cells, recovery of products

L-Lactate Pyruvate Production of High-Cell Density Biocatalyst = 20 Kg/100 L

Spray Dry Catalyst

**

• 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

Production of Pyruvic Acid by SD rPichia

WO 2009/064277

MVS 1/13 20 40 60 80 100 120 24 48 72 96 120 144 168 192 216 240

pO2.PV [%DO] Runtime [h]

15+2g/L A 15+2g/L B 12+2g/L A 12+2g/L B

+2 g/L cells +3 g/L cells

Batch-wise Recycling of GO for Pyruvate Production

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Biologically Produced Pyruvate from 8L Bioreactor - Freeze dried Sample

Bottle contains 152 g

• f total 1 Kg pyruvate

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Dynamic Resolution of RS-2-Hydroxy acids using GO

• One pot reac+on
• >95% yield of R‐HBA or D‐Lac+c acid
• Reac+on in “dead‐cell” format, hence no issue with NaBH4

OH O R OH OH O R O O H O R OH Glycolate Oxidase 2 + (R)-1 R = Different aliphatic and aromatic side chain Reduction (NaBH4) OH O R OH (S)-1 + (R)-1 1 = 2-Hydroxy Acids 2 = 2-Keto Acids + Catalase

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D-Lactate from L-Lactate by GO & Catalase

D‐Lactate Produced From 1.0 L of 250 mM L‐Lactate – in One Pot Reac+on Mixture

0.00 50.00 100.00 150.00 200.00 250.00 1 2 3 4 5 6 Concentration (mM) Reaction Coordinate (h) D-LA L-LA BH PA

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• Over-arching Technology
• *Niche Application

One Process

One Enzyme - Glycolate Oxidase Several Products:

• Pyruvate, glyoxylate
• Other keto acids, R-OH acids

Many (Redox) Enzymes *Many (Redox) Products *No NAD(P)/H input *No co-substrate

Provisional 2/10 Utility 2/3/11 – US Serial #13/020,588

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Redox reaction with spray- dried cells, recovery of products

Crude Hemicellulose hydrolysate Xylitol Production of High-Cell Density Biocatalyst* = 20 Kg/100 L

Spray Dry Catalyst

**

• 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.

Production of Bioxylitol by SD rPichia

Agricultural residue: Eg: Corn stover

Dilute acid

Provisional 2/10, Utility 2/3/11 – USSerial #13/020,588

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Biocatalysis Progress: C-5 Hydrolysate Reaction Mixture to Xylitol Crystals

C5-Hydrolysate Post-Reaction Mixture 1-Step SMB* Purified Xylitol Xylitol Crystals

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After Warming @ 50°C Water bath for 20 min & Allowing to cool to Room Temp Xylitol

SMB Purified Concentrated Sample was Diluted 1/100 times And Injected into HPLC HPLC Conditions Column: Biorad Aminex HPX-87H (300 x 7.8 mm2 i.d.) Flow rate: 0.6 mL/min Detector: RID Mobile Phase: 5mM Sulfuric acid

Purity: 98.72%

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Xylitol Crystals on the Side Glass Surface After keeping few hours @ 4°C

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HPLC Profile of Xylitol Crystal (50 mM)

Purity: 99.35%

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

Xylitol Process Needs Further Optimization

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Utilizing Native or Engineered Enzymes, in Combination OR Metabolically Engineered in Spray Dried (Dead) Powder* to Generate Products

SDP: A Different Approach to Metabolic Engineering!

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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

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Metabolic Engineering

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Metabolic Engineering of 3-HP producer

Glucose NADH ATP NAD+ Pyruvate Lactate Acetyl-CoA Formate Ethanol Acetyl-P

2

PEP OAA Succinate Acetate NAD+ NAD+ ATP

3-HP

NAD+

β-Ala

Find Enzymes Assemble Enzymes Improve Enzymes Find Enzymes Assemble Enzymes Improve Enzymes Regulate & Optimize “Carbon”-flow to the Desired Product

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Utilizing Native or Engineered Enzymes, in Combination OR Metabolically Engineered in Spray Dried Powder* to Generate Products

SDP – A Different Approach to Metabolic Engineering?

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

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Subramanian Research Group 6/11