Metabolic Engineering: A Survey of the Fundamentals Lekan Wang CS374 Spring 2009.
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Transcript of Metabolic Engineering: A Survey of the Fundamentals Lekan Wang CS374 Spring 2009.
![Page 1: Metabolic Engineering: A Survey of the Fundamentals Lekan Wang CS374 Spring 2009.](https://reader031.fdocuments.net/reader031/viewer/2022032723/56649d175503460f949ed668/html5/thumbnails/1.jpg)
Metabolic Engineering:A Survey of the Fundamentals
Lekan WangCS374 Spring 2009
![Page 2: Metabolic Engineering: A Survey of the Fundamentals Lekan Wang CS374 Spring 2009.](https://reader031.fdocuments.net/reader031/viewer/2022032723/56649d175503460f949ed668/html5/thumbnails/2.jpg)
OverviewStandard Bioengineering Techniques
Metabolic Engineering StrategiesCase Study 1: Biofuels
Case Study 2: Artemisinic Acid
![Page 3: Metabolic Engineering: A Survey of the Fundamentals Lekan Wang CS374 Spring 2009.](https://reader031.fdocuments.net/reader031/viewer/2022032723/56649d175503460f949ed668/html5/thumbnails/3.jpg)
What Is It?
Image Credits: Genentech, Portland State University, Uni-Graz
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What is it?
Holistic genetic engineering
“Metabolic engineering considers metabolic and cellular system as an entirety and accordingly allows manipulation of the system with consideration of the efficiency of overall bioprocess, which distinguishes
itself from simple genetic engineering.”1
1Lee, S.Y., et al., “Metabolic engineering of microorganisms”
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Why?
• Control• Chemical Factors• Cost• Yield and Efficiency
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What things can it make?
• Drugs• Chemical precursors• Increasingly, biofuels
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OverviewStandard Bioengineering Techniques
Metabolic Engineering StrategiesCase Study 1: Biofuels
Case Study 2: Artemisinic Acid
![Page 8: Metabolic Engineering: A Survey of the Fundamentals Lekan Wang CS374 Spring 2009.](https://reader031.fdocuments.net/reader031/viewer/2022032723/56649d175503460f949ed668/html5/thumbnails/8.jpg)
Bioengineering 101
• Choose host cell• Create or obtain DNA that expresses desired
phenotypes• Insert DNA into a DNA vector• Deliver vector to host cell• Isolate only cells that received the vectors• Profit!
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Choosing a Host
Doubling Time Cost Glycosylation
E. coli 30 min Low None
S. cerevisiae 1-2 hours Low Yes, but often incompatible with human
Mammalian (CHO/BHK)
~ day Very High Yes, and more similar with human
Adapted from Cliff Wang’s Bioengineering Lecture Notes
• Compatibility• Cost• Speed• Safety
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Obtain some DNA
Introns Exons
Splicing!
What we want!
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Inserting DNA into a Vector
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Inserting DNA into a Vector
• PCR to get more of desired DNA• Tools for insertion:– Restriction Enzymes– Ligase– Recombinases
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Delivering the Vector
• Combine the plasmid and host cell• Hope for the best
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Isolating the Good Cells
• Kill off cells with antibiotics• Cells with resistance survive• Culture surviving cells– Agar plate– Bioreactor
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OverviewStandard Bioengineering Techniques
Metabolic Engineering StrategiesCase Study 1: Biofuels
Case Study 2: Artemisinic Acid
![Page 16: Metabolic Engineering: A Survey of the Fundamentals Lekan Wang CS374 Spring 2009.](https://reader031.fdocuments.net/reader031/viewer/2022032723/56649d175503460f949ed668/html5/thumbnails/16.jpg)
Lee, et al
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Host Strain Selection
• Natural metabolic capabilities• Current tools for organism• Available genomic and metabolic information
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Computational Analysis
• Omics techniques• Simulation of complex pathways (“Genetic
Circuits”)– Metabolic Flux Analysis (aka Flux Balance Analysis,
Constraints-Based Flux Analysis, etc)
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OverviewStandard Bioengineering Techniques
Metabolic Engineering StrategiesCase Study 1: Biofuels
Case Study 2: Artemisinic Acid
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Important Factors
CostRelativelyCommon
LowerSpecificity
Image Credits: AP, SciELO
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The Major Players Today
• Ethanol• Biodiesel• Cellulosic Fuels?
Image from The Score
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Gasoline Properties
• C4 – C12 with antiknock additives
• Octane• Energy content• Transportability
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Gasoline Alternatives
• Ethanol• Butanol• Pentanol
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Diesel
• C9 – C23 with antifreeze
• Cetane• Freezing temperature• Vapor pressure
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Diesel Alternatives
• FAMEs (Fatty Acid Methyl Esters)• Isoprenoids
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Jet Fuel Properties
• Very low freezing temperatures• Density• Net heat of combustion
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Jet Fuel Alternatives
• Biodiesel• Alkanes• Isoprenoids
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Outlook
• In silico models to utilize alternative substrates– Cellulose– Xylose– Discarded biomass
• Upstream optimizations• Synthetic Biology
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OverviewStandard Bioengineering Techniques
Metabolic Engineering StrategiesCase Study 1: Biofuels
Case Study 2: Artemisinic Acid
![Page 30: Metabolic Engineering: A Survey of the Fundamentals Lekan Wang CS374 Spring 2009.](https://reader031.fdocuments.net/reader031/viewer/2022032723/56649d175503460f949ed668/html5/thumbnails/30.jpg)
Artemisinin
• Antimalarial• $$ Expensive $$
• Difficulty 1: Amorphadiene• Difficulty 2: Redox to
Dihydroartemisinic acid
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Biological Solution?
• Previous E. coli and S. cerevisiae usage• Try genes expressing native enzymes?• Uh oh…
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To a Solution
First, some good biochemistry
Dietrich, J.A. et al
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To a Solution
First, some good biochemistry
Dietrich, J.A. et al
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ROSETTA
Image from Rosetta@Home
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Molecular Dynamics (MD)
• Simulation• See whiteboard
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To a Solution
• ROSETTA-based simulation of P450BM3 interacting with amorphadiene substrate
• Phe87 causing steric hindrances!• But the fix caused more problems since the
P450BM3 G1 now oxidizes lots of things
• Repeat process with other interactions, to produce P450BM3 G3 and P450BM3 G4.
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Dietrich, J.A. et al
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SourcesPapers
Dietrich, J.A., et al. (2009). A novel semi-biosynthetic route for artemisinin production using engineered substrate-promiscuous P450. ACS Chemical Biology Letters. DOI:10.1021/cb900006h
Lee, S.Y. et al. (2009). Metabolic engineering of microorganisms: general strategies and drug production. Drug Discovery Today 14, 78-88.
Lee, S.K. et al. (2008). Metabolic engineering of microorganisms for biofuels production: from bugs to synthetic biology to fuels. Current Opinion in Biotechnology 19, 556-563.
Edwards, J.S, Ibarra, R.U., Palsson, B.O. (2001). In silico predictions of Escherichia coli metabolic capabilities are consistent with experimental data, Supplementary Appendix 1. Nature Biotechnology 19, 125-130.
Lectures and NotesWang, Cliff. ENGR25 Lecture Notes. Stanford University.Altman, Russ. CS274 Lecture Notes. Stanford University.