“Pathway-Pondering” Metabolic Engineering Problem Space June 16, 2007 BioQUEST Summer Workshop Srebrenka Robic Department of Biology Agnes Scott College Kam Dahlquist Department of Biology Loyola Marymount University

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 TCA Cycle Fermentation

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)

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

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

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

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?

Central Carbon Metabolism in E. coli Causey et al. (2004) PNAS 101:

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 Red = TC44 strain Data from Causey et al. (2004) PNAS 101: analyzed by Srebrenka

Biwer et al. (2005) Ind Eng Chem Res 44: Visualizing Pathways

Pyruvate Metabolism in E. coli (KEGG)

Mutations in E. coli TC44 strain shown in GenMAPP

Genes Proteins Phenotype Molecular Biology Biochemisty Genetics Other Questions, Datasets, Tools Individual, Population, Ecosystem

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 EcoCyc at MPD at (limited but has thermodyanmic information) GenMAPP software at