Metabolic Engineering A Survey of the Fundamentals by bjb17276

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									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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