Metabolic Engineering:
A Survey of the Fundamentals
Lekan Wang
CS374 Spring 2009
Overview
Standard Bioengineering Techniques
Metabolic Engineering Strategies
Case 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
1
Lee, 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
Overview
Standard Bioengineering Techniques
Metabolic Engineering Strategies
Case 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
Overview
Standard Bioengineering Techniques
Metabolic Engineering Strategies
Case 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)
Overview
Standard Bioengineering Techniques
Metabolic Engineering Strategies
Case Study 1:
Biofuels
Case Study 2:
Artemisinic
Acid
Important Factors
Cost
Relatively
Common
Lower
Specificity
Image Credits: AP, SciELO
The Major Players Today
•
Ethanol
•
Biodiesel
•
Cellulosic Fuels?
Image from The Score
Gasoline Properties
•
C
4
–
C
12
with antiknock additives
•
Octane
•
Energy content
•
Transportability
Gasoline Alternatives
•
Ethanol
•
Butanol
•
Pentanol
Diesel
•
C
9
–
C
23
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
Overview
Standard Bioengineering Techniques
Metabolic Engineering Strategies
Case 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 P450
BM3
interacting with amorphadiene substrate
•
Phe87 causing steric hindrances!
•
But the fix caused more problems since the
P450
BM3
G1 now oxidizes lots of things
•
Repeat process with other interactions, to
produce P450
BM3
G3 and P450
BM3
G4.
Dietrich, J.A.
et al
Sources
Papers
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 Notes
Wang, Cliff. ENGR25 Lecture Notes. Stanford University.
Altman, Russ. CS274 Lecture Notes. Stanford University.
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