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.