Transcript Dahlquist-Robic_MetabolicProbSpace_20070616

“Pathway-Pondering”
Metabolic 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
Classical Text Book Representation of Glycolysis
from Alberts et al. Molecular Biology of the Cell
Balancing the check book
• Carbons
• ATP
• NAD+/NADH
The “Two” Fates of Pyruvate
from Alberts et al. Molecular Biology of the Cell
Fermentation
TCA Cycle
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
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)
We Would Like to Use This Paradigm
When Teaching Metabolism (Thanks, Brian!)
Genes
Molecular Biology
Genetics
Phenotype
Individual,
Population,
Ecosystem
Biochemisty
Proteins
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
Metabolic Engineering Problem Space
https://engineering.purdue.edu/ChE/Research/Biochem/Biochem-01.jpg
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 >100 tons (potential for 1000 tons) a year
http://vitaminsbeautycare.com/images/Pyruva%20Powder.jpg
Chemical versus Biological Synthesis
of 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?
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)
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?
http://karamatsu.shinshu-u.ac.jp/lab/ferment/ikeda_e2.jpg
Central Carbon Metabolism in E. coli
Causey et al. (2004) PNAS 101: 2235-2240
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?
Cassey et al. Data Available for
Exploration in an MS Excel File
Growth Rate versus Pyruvate Production
Cell mass (g/L)
Growth rates of various strains
4.5
4
3.5
3
2.5
2
1.5
1
0.5
0
1
2
3
4
5
6
7
8
9
10
Strains
Production of pyruvate
800
Red = TC44
strain
700
Pyruvate (nM)
600
500
400
300
200
100
0
Strains
Data from Causey et al. (2004) PNAS 101: 2235-2240
analyzed by Srebrenka
Visualizing Pathways
Biwer et al. (2005) Ind Eng Chem Res 44: 3124-3133
Pyruvate Metabolism in E. coli (KEGG)
http://www.ecocyc.org
Mutations in E. coli TC44 strain shown in GenMAPP
Other Questions, Datasets, Tools
Genes
Molecular Biology
Genetics
Phenotype
Individual,
Population,
Ecosystem
Biochemisty
Proteins
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
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