           !"#$%&'() *        +   , (+   * + E. coli metabolic capabilities       +   , (+  How can we identify multiple metabolic manipulations for producing a desired product and also computationally evaluate of the consequences of potential network modifications? * + How can we systematically select the appropriate set of pathways/genes to recombineinto existing production systems?DesiredPhenotypeE. coli metabolic capabilitiesHow can we identify gene knockouts that will force biochemical overproductionby coupling it with cell growth?DesiredPhenotypeE. coli metabolic capabilitiesE. coli metabolic capabilities    µµµµ PEPG6PF16PF6P13P2DG3PDGL2PDGLPEPPYROAMALSUCCOACITAKGGAPICITDHAPGLCPYRD6PGL RL5PX5PS7PE4P F6PGAPR5PD6PGCFUMACCOASUCCACTPACLACETHPEPG6PF16PF6P13P2DG3PDGL2PDGLPEPPYROAMALSUCCOACITAKGGAPICITDHAPGLCPYRD6PGL RL5PX5PS7PE4P F6PGAPR5PD6PGCFUMACCOASUCCACTPACLACETHEscherichia coliChemical Process Plant *     *    + - " ! !     DNASequenceGenesProteins (Enzymes)Metabolic PathwaysCellular PhysiologyIncreasing Complexity          DNA SequenceGenesEnzymes(Gene Products)Metabolism andCellular PhysiologyAutomated SequencingGenomicsDNA MicroarraysTranscriptomics2-D Protein ArraysProteomics  !       Genome Annotation:DNA sequenceORFs identificationGenesORFs assignmentGenes ProductsFunctionGenomeDatabaseList of reactionsPathwayDatabaseOrganism’s metabolismMetabolic Reconstruction:ORF = open reading frame, a short fragment of DNA that is translated into RNA message Manual curationWet LabLiterature ReviewOrganism-Specific Model Construction:  " #    $  % Biodegradation of XenobioticsMetabolism of Complex CarbohydratesNucleotide MetabolismMetabolism of Complex LipidsCarbohydrate MetabolismMetabolism of Other Amino AcidsAmino Acid MetabolismLipid MetabolismMetabolism of Cofactors and VitaminsEnergy MetabolismBiosynthesis of Secondary MetabolitesGlucoseGlc-6-PFru-6-P&  '  (  )* + , ,  -Central Metabolism in E. coli.+ + /0" 1Glucose G6P    "                         !"!#"!"!$   $  % Reaction network:2B CA Bv1v2Stoichiometric matrix: ABCv1v2-1 01 -20 1Mass balance:dt= v1– 2 v2d[B]Steady-state assumption:=jjijvS0i = set of metabolites (chemical species)j = set of reactionsmmol BgDW⋅⋅⋅⋅hrv ≡≡≡≡Metabolic Flux:Biomass Composition.  # 4D3C2B(mmol / gDW)4D3C2B(mmol / AAxtBCDDxtbiomassbiomassbiomassbAbDv1v2v3vbiovbiovbio“Cell boundary”AAxtBCDDxtbiomassbiomassbiomassbAbDv1v2v3vbiovbiovbio“Cell boundary”Given:(1) Stoichiometry of the network(2) Cellular composition information(3) Substrate uptake ratebA=mmolgDW*hr-101000-4100-3010-2-1-11000-11000-100-010--1-11000-1=Optimization Model:Maximizevbiosubject to00-1000-105 unknowns > 4 equationsvbiov1v2v3bDv1,v2,v3,vbio,bD> 0D3.33310DAAxtB CDbiomassbiomassbiomass“Cell boundary”AAxtB CDxtbiomassbiomassbiomass1004.444“Cell