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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 (200
4) 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 ge
ne 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 Secondary metabolite Xenobiotics
2. Genetic Information Processing 3. Environmental Information Processing4. 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
Pathways Genes Reactions Compounds Metabolic Chart Expression Viewer
Database Search
Advanced Database Search
BLAST
Project Overview
Guided Tour
Publications
Update History
Advisory Board
Credits
Software/Data Download
User Support
Subscribe to Mailing List
Gene Cross-Reference
Contact Us
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...]
New Users
Take the guided tour of the EcoCyc web site, or read "The Ecocyc Database" [PDF].
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 Leaders
Peter D. Karp | Julio Collado-Vides | John Ingraham | Ian Paulsen | Milton Saier
Editor Emerita
Monica Riley
EcoCyc KB Statistics by Year
20042003
2002
2001
2000
1999
Description
Pathways 182 176 164 165 165 159 Number of metabolic plus signaling pathways. Excludes super-pathways.
Reactions 35473177
2862
2604
2115
946Number of reactions -- includes metabolic reactions, transport reactions, reactions involving binding of transcription factors to their binding sites.
Enzymes 1132 992 918 905 884 629 Number of enzymes that catalyze biochemical reactions.
Transporters 197 169 168 162 158 13 Number of transporters.
Protein comments
33951929
1030
921 846 486 Number of proteins that contain comments.
Genes 44914477
4393
4393
4393
4390
Number of genes, including some that have not been pinned to the DNA sequence.
Transcription Units
931 828 724 629 NA NA Number of transcription units -- includes operons and single-gene transcription-units.
Citations 86966223
3701
3508
3208
1944
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 reactionGlucose (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, f
umB, 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
370 genes downregulated
Metabolic genes
Cell replication genesTranscription genesTranslation 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 Manipulationin Metabolic Engineering
• Coenzyme A (CoA)/ Acetyl-Coenzyme A (Acetyl-CoA)
• Nicotinamide adenine dinucleotide (NAD+)
Increase Carbon Flux from Pyruvate to Acetyl-CoA
Overexpression of panthothenate kinase
Supplementation of pantothenic acid
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
Expression
of pnaK
Coexpression of
pnaK and PDH
Conclusion Chapter for Metobolic Networks