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.
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