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 TimeCostGlycosylation E. coli30 minLowNone S. cerevisiae1-2 hoursLowYes, but often incompatible with human Mammalian (CHO/BHK) ~ dayVery HighYes, 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

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

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: /cb900006h Lee, S.Y. et al. (2009). Metabolic engineering of microorganisms: general strategies and drug production. Drug Discovery Today 14, 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, 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, Lectures and Notes Wang, Cliff. ENGR25 Lecture Notes. Stanford University. Altman, Russ. CS274 Lecture Notes. Stanford University.