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Escherichia coli SYSTEMS BIOLOGY Suh-Chin Wu Institute of Biotechnology National Tsing Hua University The Escherichia Coli Paradigm • Part I: Bacterial Cell Structure • Part II: Metabolic Networks Part II: Metabolic Networks References • EcoCyc (http://ecocyc.org) – Karp et al. Nucleic Acid Research (2002) 30(1), 56-58. • Goodacre et al., Trends in Biotechnology (2004) 22(5), 245-252. • Holms, FEMS Microb Rev (1996) 19, 85-116. • Oh et al., Journal of Biological Chemistry (2002) 277(15), 13175-13182. • Ravishankar et al., Biotechnology Progress (2004) 20, 692-697. “Metabolomics” • “the quantitative complement of all of the low molecular weight molecules present in cells in a particular physiological or developmental state” (Oliver, 1998) • “only of those native small molecules that are participants in general metabolic reactions and that are required for the maintenance, growth and normal function of a cells” (Beecher, 2003) Fields of Metabolomics • Complementary to transcriptomics and proteomics • Metabolic control theory – metabolic flux • Metabolic control experiments • “Downstream” results of gene expression – Example, metabolic fluxes not regulated by gene expression alone, Measuring Metabolomics Principal Criteria for Predicting the System of Metabolic Pathways • Coverage of known metabolic reactions and pathways • Predictive capability • Amenability to large-scale computation Paradigm shift from metabolic pathways to networks and neighborhoods! Data Bases of Metabolic Pathways • • • • • • • • • • • • EMP (WIT) EcoCyc KEGG BRENDA UMBBD ENZYME PathDB SoyBase BioPathways Biocarta GenomeKnowledgeBase ERGO EMP Database • A collaborative effort between Argonne National Laboratory and The Institute of Theoretical Biophysics (Russian Academy of Sciences, Puschino, Russia), led by Prof. E. Selkov. • It represents a key resource for developing detailed metabolic reconstructions for newly sequenced genomes far more rapidly than researchers even a few years ago would have thought possible. • EMP/MPW database currently contains 28, 100 records, which encode the full factual content of 17, 500 publications describing more than 8, 000 organisms. • The database contains information on 3, 900 enzymes, including data on enzyme specificity, enzymological constants, purification protocols, regulation, inhibitors and activators. EcoCyc Database • EcoCyc is a bioinformatics database that describes the genome and the biochemical machinery of E. coli K12 MG1655. • Metabolism. EcoCyc describes all known metabolic pathways and signal-transduction pathways of E. coli. It describes each metabolic enzyme of E. coli, including its cofactors, activators, inhibitors, and subunit structure – Pathways (220) – Reactions (3547) • Characterized relationships in a hierarchical manner KEGG Database • Kyoto Encyclopedia for Genes and Genomes (1995) • www.genome.ad.jp/kegg/ • Employs graph theoretic concepts and define binary relations KEGG PATHWAY Database Current knowledge on molecular interaction networks, including metabolic pathways, regulatory pathways, and molecular complexes Go to: 1. Metabolism Carbohydrate Energy Lipid Nucleotide Amino acid Other amino acid Glycan PK/NRP Cofactor/vitamin Sec ondary metabolite Xenobiotics 2. Genetic Information Processing 3. Environmental Information Processing 4. Cellular Processes 5. Human Diseases See also: KO (KEGG Orthology) BRENDA Database • BRaunschweig Enzyme Databases • http://www.brenda.uni-koeln.de UMBBD Database • University of Minnesota Database of Biocatalysis and Biodegradation • http://umbbd.ahc.umn .edu • Industrial application and bioremidiation with information on xenobiotic compound interconversions Encyclopedia of Escherichia coli K12 Genes and Metabolism EcoCyc Home Project Overview EcoCyc is a scientific database for the bacterium Escherichia coli K12 MG1655. The EcoCyc project performs literature-based curation of the entire genome, and of transcriptional regulation, transporters, and metabolic pathways. [more...] Database Search Advanced Database Search New Users Take the guided tour of the EcoCyc web site, or read "The Ecocyc Database" [PDF]. BLAST Pathways Genes Reactions Compounds Metabolic Chart Expression Viewer New Data Content •E. coli O157:H7 and Shigella flexneri are the newest knowledge bases in the BioCyc collection. •Our EcoCyc curation update project is progressing. Of the 4479 polypeptides within EcoCyc, 3395 now have comments or citations or are components of a complex that has a comment or citations. The database now contains 8696 citations. •The full EcoCyc release history is available here. Update Frequency The EcoCyc web site is updated quarterly. A new version that you can install locally on your computer is released semiannually (supported platforms: PC/Windows, PC/Linux, Sun workstation). [ Full EcoCyc release history ] Project Overvie w Guided Tour Publications Update History Advisory Board Credits Software/Data Downloa d Project Leaders Peter D. Karp | Julio Collado-Vides | John Ingraham | Ian Paulsen | Milton Saier Editor Emerita Monica Riley User Support Subscribe to Mailing Lis t Gene Cross-Reference Contact Us EcoCyc KB Statistics by Year 2004 200 3 20 02 200 1 200 0 19 99 Description Pathways 182 176 16 4 165 165 15 9 Number of metabolic plus signaling pathways. Excludes super-pathways. Reactions 3547 317 7 28 62 260 4 211 5 94 6 Number of reactions -- includes metabolic reactions, transport reactions, reactions involving binding of transcription factors to their binding sites. Enzymes 1132 992 91 8 905 884 62 9 Number of enzymes that catalyze biochemical reactions. Transporters 197 169 16 8 162 158 13 Number of transporters. Protein comments 3395 192 9 10 30 921 846 48 6 Number of proteins that contain comments. Genes 4491 447 7 43 93 439 3 439 3 43 90 Number of genes, including some that have not been pinned to the DNA sequence. Transcription Units 931 828 72 4 629 NA NA Number of transcription units -- includes operons and single-gene transcription-units. Citations 8696 622 3 37 01 350 8 320 8 19 44 Number of distinct references cited in EcoCyc. Release Notes for EcoCyc Version 8.1 Released on June 23, 2004. E. coli K-12 Class: Pathways Parent Classes: Generalized-Reactions Child Classes: Biosynthesis (97) , Degradation/Utilization/Assimilation (72) , Generation of precursor metabolites and energy (20) , Signal transduction pathways (22) , Super-Pathways (31) E. coli K-12 Class: Biosynthesis Parent Classes: Pathways Child Classes: Amines and Polyamines (3) , Amino acids (36) , Aminoacyl-tRNAs (1) , Aromatic Compounds (0) , Cell structures (10) , Cofactors, Prosthetic Groups, Electron Carriers (27) , Fatty Acids and Lipids (9) , Hormones (0) , Metabolic Regulators (1) , Nucleosides and Nucleotides (7) , Other (2) , Secondary Metabolism (0) , Siderophores (1) , Sugar Derivatives (0) , Sugars and Polysaccharides (4) E. coli K-12 Pathways Class: Cell structures Parent Classes: Biosynthesis Child Classes: Plant cell structures (0) Instances: colanic acid building blocks biosynthesis , dTDP-rhamnose biosynthesis , enterobacterial common antigen biosynthesis , GDP-mannose metabolism , KDO biosynthesis -- including transfer to lipid IVA , lipid-A-precursor biosynthesis , O-antigen biosynthesis , peptidoglycan biosynthesis , superpathway of KDO2-lipid A biosynthesis , UDP-N-acetylglucosamine biosynthesis E. coli K-12 Pathway: peptidoglycan biosynthesis E. coli K-12 Class: Biosynthesis Parent Classes: Pathways Child Classes: Amines and Polyamines (3) , Amino acids (36) , Aminoacyl-tRNAs (1) , Aromatic Compounds (0) , Cell structures (10) , Cofactors, Prosthetic Groups, Electron Carriers (27) , Fatty Acids and Lipids (9) , Hormones (0) , Metabolic Regulators (1) , Nucleosides and Nucleotides (7) , Other (2) , Secondary Metabolism (0) , Siderophores (1) , Sugar Derivatives (0) , Sugars and Polysaccharides (4) E. coli K-12 Pathways Class: Sugars and Polysaccharides Parent Classes: Biosynthesis Instances: gluconeogenesis , glycogen biosynthesis , trehalose biosynthesis and degradation -- low osmolarity , trehalose biosynthesis I E. coli K-12 Pathway: gluconeogenesis E. coli K-12 Class: Pathways Parent Classes: Generalized-Reactions Child Classes: Biosynthesis (97) , Degradation/Utilization/Assimilation (72) , Generation of precursor metabolites and energy (20) , Signal transduction pathways (22) , Super-Pathways (31) E. coli K-12 Class: Generation of precursor metabolites and energy Parent Classes: Pathways Child Classes: Chemoautotrophic energy metabolism (0) , Fermentation (1) , Glycolysis (3) , Methanogenesis (0) , Other (0) , Pentose phosphate pathways (3) , Photosynthesis (0) , Respiration (6) , TCA cycle (6) Instances: Entner-Doudoroff pathway , superpathway of glycolysis and Entner-Doudoroff E. coli K-12 Pathways Class: Fermentation Parent Classes: Generation of precursor metabolites and energy Instances: mixed acid fermentation E. coli K-12 Pathway: mixed acid fermentation Major Fermentation Acids in E. coli • • • • • • The major fermentation acids excreted by E. coli include acetate, formate, D-lactate, and succinate. A high concentration of fermentation acids limits growth, and acetate induces the RpoS regulon associated with entry into stationary phase. Above pH 7, the favored fermentation products are acetate (with ethanol) and formate. Production of acetate and formate is maximal in the absence of oxygen or other respiratory electron acceptors, but oxygenated cultures also excrete significant amounts of acetate and formate, a significant concern for bioreactors. As pH falls, E. coli limits internal acidification from metabolism by producing lactate instead of acetate plus formate, by reuptake and activation of acetate to acetyl-coenzyme A (CoA) to enter the tricarboxylic acid (TCA) cycle, and by conversion of formate to H2 and CO2. The mechanisms of regulation and the responses to high concentrations of different acids remain unclear. E. coli K-12 Class: Generation of precursor metabolites and energy Parent Classes: Pathways Child Classes: Chemoautotrophic energy metabolism (0) , Fermentation (1) , Glycolysis (3) , Methanogenesis (0) , Other (0) , Pentose phosphate pathways (3) , Photosynthesis (0) , Respiration (6) , TCA cycle (6) Instances: Entner-Doudoroff pathway , superpathway of glycolysis and Entner-Doudoroff E. coli K-12 Pathways Class: Glycolysis Parent Classes: Generation of precursor metabolites and energy Instances: glycolysis I , methylglyoxal pathway , superpathway of glycolysis, pyruvate dehydrogenase, TCA, and glyoxylate bypass E. coli K-12 Pathway: glycolysis I E. coli K-12 Pathway: methylglyoxal pathway E. coli K-12 Pathway: superpathway of glycolysis, pyruvate dehydrogenase, TCA, and glyoxylate bypass E. coli K-12 Class: Generation of precursor metabolites and energy Parent Classes: Pathways Child Classes: Chemoautotrophic energy metabolism (0) , Fermentation (1) , Glycolysis (3) , Methanogenesis (0) , Other (0) , Pentose phosphate pathways (3) , Photosynthesis (0) , Respiration (6) , TCA cycle (6) Instances: Entner-Doudoroff pathway , superpathway of glycolysis and Entner-Doudoroff E. coli K-12 Pathways Class: TCA cycle Parent Classes: Generation of precursor metabolites and energy Instances: glyoxylate cycle , pyruvate dehydrogenase , pyruvate oxidation pathway , superpathway of glycolysis, pyruvate dehydrogenase, TCA, and glyoxylate bypass , superpathway of glyoxylate bypass and TCA , TCA cycle -- aerobic respiration E. coli K-12 Pathway: glyoxylate cycle E. coli K-12 Pathway: pyruvate dehydrogenase E. coli K-12 Pathway: pyruvate oxidation pathway E. coli K-12 Pathway: superpathway of glycolysis, pyruvate dehydrogenase, TCA, and glyoxylate bypass E. coli K-12 Pathway: superpathway of glyoxylate bypass and TCA E. coli K-12 Pathway: TCA cycle -- aerobic respiration Metabolic Flux Analysis of Central Metabolic Pathways • • • • • Glycolysis (Emden-Meyerhof pathway, EMP) Pentose phosphate pathway (PPP) Pyruvate dehydrogenase (PD) Phosphoenolpyruvate carobxylase (PEPC) TCA cycle – Entner-Doudoroff pathway (EDP) – Phophoketolase pathway • Electron transfer system (ETS) – ATP • Cofactors – NADP/NADPH, CoA/Acetyl-CoA, FDH/FADH, ATP/ADP/AMP Metabolic pathway Networks of E.coli • Allowing the simulation and optimization of specific pathways to achieved a desirable phenotype – redirection of carbon flow for metabolite production Carbon Sources Used by E. coli • E. coli physiology has been experimentally studied for more than three decades on the sore carbon source of glucose or acetate. • Important to biotechnology industry – Acetate accumulation in medium during industrial fermentation – An obstacle to reach high cell density cultivation Glucose as the Carbon Source • E. coli uptakes glucose using the phosphotransferase system (PTS) • PTS is present in most facultative anaerobic bacteria but not in eucaryotic cells • PTS net reaction Glucose (outside) + PEP G6P + pyruvate NOT Glucose (outside) +ATP G6P + ADP • PTS is also responsible for the transport of fructose, mannose, manitol, sucrose, glucitol Acetate as the Carbon Source • Acetate converted to acetyl-CoA, further metablized through the glyoxylate shunt and the TCA cycle Global Expression Profiling for Acetate-grown E. coli – (1) • Acetate uptake ▲ – acs • Acetyl-coA malate ▲ – aceBAK, glcB • TCA cycle ▲ – gltA, icdA, acnA, acnB, sucABCD, sdhc-DAB, fumA, fumB, fumC, mdh • Gluconeogenesis ▲ – pckA, ppsA, sfcA, maeB • Pta-ackA pathway ▼ – used for acetate secretion during growth on glucose Global Expression Profiling for Acetate-grown E. coli – (2) • Glycolysis ▼ – pfkA, fba, gnpA, epd, pgk, eno, pykF, ppc • Pentose pathway ▼ – zwf, gnd • Pyr Acetyl-coA (pyruvate dehydrogenase) ▼ – aceEF • Glucose transport ▼ – ptsHI-crr, ptsG Global Expression Profiling for Acetate-grown E. coli – (3) • Transport genes for other carbon sources ▲ – – – – – – – • Galactose ABC transport operon (mglBAC) Ribose uptake gene operon (rhsD, rhsACB) N-acetyl-d-glucose-amide transport subunit (nagE) Arginine ABC transport gene (argT) C4 dicarboxylate transporter gene (cdtA) Atogatose metabolic gene (gatYZ) Maltose translocating gene (lamB) Glyoxylate-relate metabolic pathways ▲ – Glucolate metabolism – Allatoine metabolism • Genes involved in cell machinery – Cell structure, DNA replication, transcription, translation • 16 genes (3.3%) ▲ • 99 genes (20%) ▼ – ribosomal proteins • S1-S21, L1-L25, L27-L36, EF-Tu subunits, EF-TS, EF-G • 19/40 genes (70%) ▼ 354 genes upregulated Metabolic genes 370 genes downregulated Cell replication genes Transcription genes Translation genes Metabolic pathways connecting pyruvate with fermentation acids in E. coli. Expression Profiles of Glyoxylate Metabolic Genes in Acetate But also those involved in other glyoxylate-related metabolic pathways such as glycolate and allatoine metabolism are up-regulated. Co-factor Manipulation in Metabolic Engineering • Coenzyme A (CoA)/ Acetyl-Coenzyme A (Acetyl-CoA) • Nicotinamide adenine dinucleotide (NAD+) Increase Carbon Flux from Pyruvate to Acetyl-CoA Supplementation of pantothenic acid Overexpression of panthothenate kinase Intracellular Acetyl-CoA Level Expression of PanK Coexpression of PDH and PanK NO CHANGE in Acetyl-CoA However, higher carbon flux through the acetyl-CoA node by coexpressing both PDH and PanK Inactivation to increase intracellular CoA/acetyl-CoA levels • At the pyruvate node – the lactate production pathway (ldh) • At the acetyl-CoA node – the acetate production pathway (ackA-pta) YBS121: ackA-pta deletion mutant Conclusion Chapter for Metobolic Networks