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Metabolic Engineering: A Survey of the Fundamentals

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?.

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Metabolic Engineering: A Survey of the Fundamentals

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  1. Metabolic Engineering:A Survey of the Fundamentals Lekan Wang CS374 Spring 2009

  2. Overview Standard Bioengineering Techniques Metabolic Engineering Strategies Case Study 1: Biofuels Case Study 2: Artemisinic Acid

  3. What Is It? Image Credits: Genentech, Portland State University, Uni-Graz

  4. 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”

  5. Why? • Control • Chemical Factors • Cost • Yield and Efficiency

  6. What things can it make? • Drugs • Chemical precursors • Increasingly, biofuels

  7. Overview Standard Bioengineering Techniques Metabolic Engineering Strategies Case Study 1: Biofuels Case Study 2: Artemisinic Acid

  8. 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!

  9. Choosing a Host • Compatibility • Cost • Speed • Safety Adapted from Cliff Wang’s Bioengineering Lecture Notes

  10. Obtain some DNA Introns Exons Splicing! What we want!

  11. Inserting DNA into a Vector

  12. Inserting DNA into a Vector • PCR to get more of desired DNA • Tools for insertion: • Restriction Enzymes • Ligase • Recombinases

  13. Delivering the Vector • Combine the plasmid and host cell • Hope for the best

  14. Isolating the Good Cells • Kill off cells with antibiotics • Cells with resistance survive • Culture surviving cells • Agar plate • Bioreactor

  15. Overview Standard Bioengineering Techniques Metabolic Engineering Strategies Case Study 1: Biofuels Case Study 2: Artemisinic Acid

  16. Lee, et al

  17. Host Strain Selection • Natural metabolic capabilities • Current tools for organism • Available genomic and metabolic information

  18. Computational Analysis • Omics techniques • Simulation of complex pathways (“Genetic Circuits”) • Metabolic Flux Analysis (aka Flux Balance Analysis, Constraints-Based Flux Analysis, etc)

  19. Overview Standard Bioengineering Techniques Metabolic Engineering Strategies Case Study 1: Biofuels Case Study 2: Artemisinic Acid

  20. Important Factors Relatively Common Lower Specificity Cost Image Credits: AP, SciELO

  21. The Major Players Today • Ethanol • Biodiesel • Cellulosic Fuels? Image from The Score

  22. Gasoline Properties • C4 – C12with antiknock additives • Octane • Energy content • Transportability

  23. Gasoline Alternatives • Ethanol • Butanol • Pentanol

  24. Diesel • C9 – C23with antifreeze • Cetane • Freezing temperature • Vapor pressure

  25. Diesel Alternatives • FAMEs (Fatty Acid Methyl Esters) • Isoprenoids

  26. Jet Fuel Properties • Very low freezing temperatures • Density • Net heat of combustion

  27. Jet Fuel Alternatives • Biodiesel • Alkanes • Isoprenoids

  28. Outlook • In silico models to utilize alternative substrates • Cellulose • Xylose • Discarded biomass • Upstream optimizations • Synthetic Biology

  29. Overview Standard Bioengineering Techniques Metabolic Engineering Strategies Case Study 1: Biofuels Case Study 2: Artemisinic Acid

  30. Artemisinin • Antimalarial • $$ Expensive $$ • Difficulty 1: Amorphadiene • Difficulty 2: Redox to Dihydroartemisinic acid

  31. Biological Solution? • Previous E. coli and S. cerevisiae usage • Try genes expressing native enzymes? • Uh oh…

  32. To a Solution First, some good biochemistry Dietrich, J.A. et al

  33. To a Solution First, some good biochemistry Dietrich, J.A. et al

  34. ROSETTA Image from Rosetta@Home

  35. Molecular Dynamics (MD) • Simulation • See whiteboard

  36. 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.

  37. Dietrich, J.A. et al

  38. 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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