Metabolic Engineering - Stanford AI Lab - Stanford University

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.