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Metabolic Engineering:A Survey of the Fundamentals
Lekan WangCS374 Spring 2009
OverviewStandard Bioengineering Techniques
Metabolic Engineering StrategiesCase Study 1: Biofuels
Case Study 2: Artemisinic Acid
What Is It?
Image Credits: Genentech, Portland State University, Uni-Graz
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”
Why?
• Control• Chemical Factors• Cost• Yield and Efficiency
What things can it make?
• Drugs• Chemical precursors• Increasingly, biofuels
OverviewStandard Bioengineering Techniques
Metabolic Engineering StrategiesCase Study 1: Biofuels
Case Study 2: Artemisinic Acid
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!
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
Obtain some DNA
Introns Exons
Splicing!
What we want!
Inserting DNA into a Vector
Inserting DNA into a Vector
• PCR to get more of desired DNA• Tools for insertion:– Restriction Enzymes– Ligase– Recombinases
Delivering the Vector
• Combine the plasmid and host cell• Hope for the best
Isolating the Good Cells
• Kill off cells with antibiotics• Cells with resistance survive• Culture surviving cells– Agar plate– Bioreactor
OverviewStandard Bioengineering Techniques
Metabolic Engineering StrategiesCase Study 1: Biofuels
Case Study 2: Artemisinic Acid
Lee, et al
Host Strain Selection
• Natural metabolic capabilities• Current tools for organism• Available genomic and metabolic information
Computational Analysis
• Omics techniques• Simulation of complex pathways (“Genetic
Circuits”)– Metabolic Flux Analysis (aka Flux Balance Analysis,
Constraints-Based Flux Analysis, etc)
OverviewStandard Bioengineering Techniques
Metabolic Engineering StrategiesCase Study 1: Biofuels
Case Study 2: Artemisinic Acid
Important Factors
CostRelativelyCommon
LowerSpecificity
Image Credits: AP, SciELO
The Major Players Today
• Ethanol• Biodiesel• Cellulosic Fuels?
Image from The Score
Gasoline Properties
• C4 – C12 with antiknock additives
• Octane• Energy content• Transportability
Gasoline Alternatives
• Ethanol• Butanol• Pentanol
Diesel
• C9 – C23 with antifreeze
• Cetane• Freezing temperature• Vapor pressure
Diesel Alternatives
• FAMEs (Fatty Acid Methyl Esters)• Isoprenoids
Jet Fuel Properties
• Very low freezing temperatures• Density• Net heat of combustion
Jet Fuel Alternatives
• Biodiesel• Alkanes• Isoprenoids
Outlook
• In silico models to utilize alternative substrates– Cellulose– Xylose– Discarded biomass
• Upstream optimizations• Synthetic Biology
OverviewStandard Bioengineering Techniques
Metabolic Engineering StrategiesCase Study 1: Biofuels
Case Study 2: Artemisinic Acid
Artemisinin
• Antimalarial• $$ Expensive $$
• Difficulty 1: Amorphadiene• Difficulty 2: Redox to
Dihydroartemisinic acid
Biological Solution?
• Previous E. coli and S. cerevisiae usage• Try genes expressing native enzymes?• Uh oh…
To a Solution
First, some good biochemistry
Dietrich, J.A. et al
To a Solution
First, some good biochemistry
Dietrich, J.A. et al
ROSETTA
Image from Rosetta@Home
Molecular Dynamics (MD)
• Simulation• See whiteboard
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.
Dietrich, J.A. et al
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.