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Pathway-PonderingMetabolic Engineering Problem
Space
Srebrenka Robic Department of Biology Agnes Scott
College Kam Dahlquist Department of
Biology Loyola Marymount University
June 16, 2007 BioQUEST Summer Workshop
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Classical Text Book Representation of
Glycolysis from Alberts et al. Molecular Biology
of the Cell

• Balancing the check book
• Carbons
• ATP
• NAD/NADH

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The Two Fates of Pyruvate from Alberts et al.
Molecular Biology of the Cell
Fermentation
TCA Cycle
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Struggles with Teaching Metabolism

• Memorizing steps and intermediates
• Getting lost in the details
• Static pictures do not convey the dynamics of
  metabolic flux
• Linking metabolic pathways to each other
• Anabolic and catabolic processes
• Linking metabolic pathways to other cellular
  processes
• Regulation of gene expression

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What are Your Challenges/Goals when Teaching
Metabolism?

• Students do not understand resident molecule idea
  (sources, sinks)
• Plants have mitochondria
• More than glucose metabolism
• Obsessed by oxygen (Marion!)
• Relative amounts and recycling (consumed vs.
  recycled catalytic amounts)
• Invertebrates- diversity of metabolism
• Link metabolism with evolution

(Audience responses)
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We Would Like to Use This Paradigm When Teaching
Metabolism (Thanks, Brian!)
Genes
Molecular Biology
Genetics
Individual, Population, Ecosystem
Proteins
Phenotype
Biochemisty
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Learning Objectives

• Energetics
• storage of energy in bonds
• controlled release of chemical energy
• Oxidation/reduction
• links between carbon metabolism and recycling of
  redox agents
• Connections and coupling of various processes
• flux of chemical intermediates
• connections between different pathways (anabolism
  and catabolism)
• Regulation
• feedback loops
• subcellular location
• gene regulation
• Diversity of metabolism
• variation within populations
• variation between species
• biogeochemical cycles

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Metabolic Engineering Problem Space
https//engineering.purdue.edu/ChE/Research/Bioche
m/Biochem-01.jpg
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Who Needs a Bucket of Pyruvate?

• Food additive, nutriceutical, and a weight
  control supplement
• Starting material for synthesis of
  pharmaceutically active ingredients (amino acids,
  Trp, Ala, and L-DOPA)
• Starting point for other industrial fermentations

World market volume gt100 tons (potential for 1000
tons) a year
http//vitaminsbeautycare.com/images/Pyruva20Powd
er.jpg
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Chemical versus Biological Synthesisof Pyruvate

• CHEMICAL SYNTHESIS
• Synthesis from tartarate (pyrolysis) involves
  toxic organic solvents
• Cost 8650/ton
• BIOLOGICAL SYNTHESIS
• Green synthesis
• Typically made in E. coli or Torulopsis glabrata
  (yeast)
• Cost 1255/ton

Can we do better than that? Can we improve the
biological production of pyruvate?
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Pathway Pondering

• What do you need to know?
• Is there variation from organism to organism in
  rates of production of pyruvate?
• Is there is an easy chemical modification of
  pyruvate that sequesters it from the organism?
• At what temperature/pH do you need to extract,
  grow culture?
• Is there a way to extract without damaging
  organism (recylcable and ongoing fermentation)?
• If pyruvate is link in a pathway, you need to
  shut off the next step, take it out of oxygen
  environment.
• Can different pathways coming into pyruvate come
  in at different rates so start with something
  else besides glucose?
• What regulatory agency does this have to go under?

(Audience responses)
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Pathway Pondering

• How is pyruvate made in E. coli?
• What are some of the possible fates of pyruvate
  in E. coli?
• Is pyruvate production optimized in E. coli?
• What steps would you modify if you wanted to
  engineer an E. coli strain that makes more
  pyruvate?
• How would you engineer a different microorganism
  to produce more pyruvate?

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http//karamatsu.shinshu-u.ac.jp/lab/ferment/ikeda
_e2.jpg
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Central Carbon Metabolism in E. coli
Causey et al. (2004) PNAS 101 2235-2240
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Thinking Like a Bioengineer

• What makes a good pyruvate producing strain?
• What parameters might you want to measure and how
  would you compare your strain to already existing
  strains?
• How might you model the cost of production?
• How would you take into account the environmental
  impact?
• How do you engineer the strain without killing it?

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Cassey et al. Data Available for Exploration in
an MS Excel File
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Growth Rate versus Pyruvate Production
Red TC44 strain
Data from Causey et al. (2004) PNAS 101
2235-2240 analyzed by Srebrenka
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Visualizing Pathways
Biwer et al. (2005) Ind Eng Chem Res 44 3124-3133
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Pyruvate Metabolism in E. coli (KEGG)
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http//www.ecocyc.org
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Mutations in E. coli TC44 strain shown in GenMAPP
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Other Questions, Datasets, Tools
Genes
Molecular Biology
Genetics
Individual, Population, Ecosystem
Proteins
Phenotype
Biochemisty
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Other Questions, Datasets, Tools

• What are the differences between pyruvate
  pathways in other organisms (Saccharomyces,
  Lactobacilli, etc.) compared to E. coli?
• How would you engineer other organisms for
  pyruvate production?
• Analyzing cost and environmental impact of
  pyruvate synthesis
• Evolution of metabolic pathways
• Metagenomics, meta metabolic pathways in
  ecosystems, bioremediation

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Metabolism Pathway Databases

• KEGG at http//www.genome.ad.jp/kegg/
• EcoCyc at http//ecocyc.org/
• MPD at http//www.gwu.edu/mpb/ (limited but has
  thermodyanmic information)
• GenMAPP software at http//www.GenMAPP.org