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

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
 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
• Compatibility
• Cost
• Speed
• Safety

Doubling Time Cost Glycosylation

E. coli 30 min Low None
S. cerevisiae 1-2 hours Low Yes, but often
incompatible with
human
Mammalian ~ day Very High Yes, and more
(CHO/BHK) similar with human

Adapted from Cliff Wang’s Bioengineering Lecture Notes

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
 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

Relatively Lower
Cost
Common Specificity
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
 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 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
 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.