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Escherichia coli SYSTEMS BIOLOGY Suh-Chin Wu Institute of Biotechnolog y National Tsing Hua Univer sity
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Page 1: Escherichia coli SYSTEMS BIOLOGY Suh-Chin Wu Institute of Biotechnology National Tsing Hua University.

Escherichia coli SYSTEMS BIOLOGY

Suh-Chin Wu

Institute of Biotechnology

National Tsing Hua University

Page 2: 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

Page 3: Escherichia coli SYSTEMS BIOLOGY Suh-Chin Wu Institute of Biotechnology National Tsing Hua University.

Part II: Metabolic Networks

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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.

Page 5: Escherichia coli SYSTEMS BIOLOGY Suh-Chin Wu Institute of Biotechnology National Tsing Hua University.
Page 6: Escherichia coli SYSTEMS BIOLOGY Suh-Chin Wu Institute of Biotechnology National Tsing Hua University.

“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)

Page 7: Escherichia coli SYSTEMS BIOLOGY Suh-Chin Wu Institute of Biotechnology National Tsing Hua University.

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,

Page 8: Escherichia coli SYSTEMS BIOLOGY Suh-Chin Wu Institute of Biotechnology National Tsing Hua University.

Measuring Metabolomics

Page 9: Escherichia coli SYSTEMS BIOLOGY Suh-Chin Wu Institute of Biotechnology National Tsing Hua University.
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Principal Criteria for Predicting the System of Metabolic Pathways

• Coverage of known metabolic reactions and pathways

• Predictive capability

• Amenability to large-scale computation

Page 13: Escherichia coli SYSTEMS BIOLOGY Suh-Chin Wu Institute of Biotechnology National Tsing Hua University.

Paradigm shift from metabolic pathways to networks and neighborhoods!

Page 14: Escherichia coli SYSTEMS BIOLOGY Suh-Chin Wu Institute of Biotechnology National Tsing Hua University.
Page 15: Escherichia coli SYSTEMS BIOLOGY Suh-Chin Wu Institute of Biotechnology National Tsing Hua University.
Page 16: Escherichia coli SYSTEMS BIOLOGY Suh-Chin Wu Institute of Biotechnology National Tsing Hua University.

Data Bases of Metabolic Pathways

• EMP (WIT)• EcoCyc• KEGG• BRENDA• UMBBD• ENZYME• PathDB• SoyBase• BioPathways• Biocarta• GenomeKnowledgeBase• ERGO

Page 17: Escherichia coli SYSTEMS BIOLOGY Suh-Chin Wu Institute of Biotechnology National Tsing Hua University.

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. 

Page 18: Escherichia coli SYSTEMS BIOLOGY Suh-Chin Wu Institute of Biotechnology National Tsing Hua University.

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

                                      

  

Page 19: Escherichia coli SYSTEMS BIOLOGY Suh-Chin Wu Institute of Biotechnology National Tsing Hua University.

KEGG Database

• Kyoto Encyclopedia for Genes and Genomes (1995)

• www.genome.ad.jp/kegg/• Employs graph theoretic

concepts and define binary relations

Page 21: Escherichia coli SYSTEMS BIOLOGY Suh-Chin Wu Institute of Biotechnology National Tsing Hua University.

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)

Page 22: Escherichia coli SYSTEMS BIOLOGY Suh-Chin Wu Institute of Biotechnology National Tsing Hua University.

                                                                                                                           

Page 23: Escherichia coli SYSTEMS BIOLOGY Suh-Chin Wu Institute of Biotechnology National Tsing Hua University.

BRENDA Database

• BRaunschweig Enzyme Databases

• http://www.brenda.uni-koeln.de

Page 24: Escherichia coli SYSTEMS BIOLOGY Suh-Chin Wu Institute of Biotechnology National Tsing Hua University.

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

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   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

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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.

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  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)

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  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

Page 31: Escherichia coli SYSTEMS BIOLOGY Suh-Chin Wu Institute of Biotechnology National Tsing Hua University.

            

  E. coli K-12 Pathway: peptidoglycan biosynthesis     

Page 32: Escherichia coli SYSTEMS BIOLOGY Suh-Chin Wu Institute of Biotechnology National Tsing Hua University.

            

  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)

Page 34: Escherichia coli SYSTEMS BIOLOGY Suh-Chin Wu Institute of Biotechnology National Tsing Hua University.

            

  E. coli K-12 Pathway: gluconeogenesis

Page 36: Escherichia coli SYSTEMS BIOLOGY Suh-Chin Wu Institute of Biotechnology National Tsing Hua University.

            

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

Page 38: Escherichia coli SYSTEMS BIOLOGY Suh-Chin Wu Institute of Biotechnology National Tsing Hua University.

            

  E. coli K-12 Pathway: mixed acid fermentation

   

Page 39: Escherichia coli SYSTEMS BIOLOGY Suh-Chin Wu Institute of Biotechnology National Tsing Hua University.

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.

Page 40: Escherichia coli SYSTEMS BIOLOGY Suh-Chin Wu Institute of Biotechnology National Tsing Hua University.

           

  

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

Page 41: Escherichia coli SYSTEMS BIOLOGY Suh-Chin Wu Institute of Biotechnology National Tsing Hua University.

            

  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

Page 42: Escherichia coli SYSTEMS BIOLOGY Suh-Chin Wu Institute of Biotechnology National Tsing Hua University.

              E. coli K-12 Pathway: glycolysis I

Page 44: Escherichia coli SYSTEMS BIOLOGY Suh-Chin Wu Institute of Biotechnology National Tsing Hua University.

            

 

E. coli K-12 Pathway: superpathway of glycolysis, pyruvate dehydrogenase, TCA, and glyoxylate bypass

Page 45: Escherichia coli SYSTEMS BIOLOGY Suh-Chin Wu Institute of Biotechnology National Tsing Hua University.

            

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

Page 46: Escherichia coli SYSTEMS BIOLOGY Suh-Chin Wu Institute of Biotechnology National Tsing Hua University.

            

  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

Page 47: Escherichia coli SYSTEMS BIOLOGY Suh-Chin Wu Institute of Biotechnology National Tsing Hua University.

            

  E. coli K-12 Pathway: glyoxylate cycle

Page 48: Escherichia coli SYSTEMS BIOLOGY Suh-Chin Wu Institute of Biotechnology National Tsing Hua University.

            

  E. coli K-12 Pathway: pyruvate dehydrogenase

Page 49: Escherichia coli SYSTEMS BIOLOGY Suh-Chin Wu Institute of Biotechnology National Tsing Hua University.

            

  E. coli K-12 Pathway: pyruvate oxidation pathway

Page 50: Escherichia coli SYSTEMS BIOLOGY Suh-Chin Wu Institute of Biotechnology National Tsing Hua University.

E. coli K-12 Pathway: superpathway of glycolysis, pyruvate dehydrogenase, TCA, and glyoxylate bypass

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E. coli K-12 Pathway: superpathway of glyoxylate bypass and TCA

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  E. coli K-12 Pathway: TCA cycle -- aerobic respiration

Page 53: Escherichia coli SYSTEMS BIOLOGY Suh-Chin Wu Institute of Biotechnology National Tsing Hua University.

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

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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

Page 59: Escherichia coli SYSTEMS BIOLOGY Suh-Chin Wu Institute of Biotechnology National Tsing Hua University.

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

Page 60: Escherichia coli SYSTEMS BIOLOGY Suh-Chin Wu Institute of Biotechnology National Tsing Hua University.

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

Page 61: Escherichia coli SYSTEMS BIOLOGY Suh-Chin Wu Institute of Biotechnology National Tsing Hua University.

Acetate as the Carbon Source

• Acetate converted to acetyl-CoA, further metablized through the glyoxylate shunt and the TCA cycle

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Page 64: Escherichia coli SYSTEMS BIOLOGY Suh-Chin Wu Institute of Biotechnology National Tsing Hua University.

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

Page 65: Escherichia coli SYSTEMS BIOLOGY Suh-Chin Wu Institute of Biotechnology National Tsing Hua University.

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

Page 66: Escherichia coli SYSTEMS BIOLOGY Suh-Chin Wu Institute of Biotechnology National Tsing Hua University.

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%) ▼

Page 67: Escherichia coli SYSTEMS BIOLOGY Suh-Chin Wu Institute of Biotechnology National Tsing Hua University.

354 genes upregulated

370 genes downregulated

Metabolic genes

Cell replication genesTranscription genesTranslation genes

Page 68: Escherichia coli SYSTEMS BIOLOGY Suh-Chin Wu Institute of Biotechnology National Tsing Hua University.

Metabolic pathways connecting pyruvate with fermentation acids in E. coli.

Page 69: Escherichia coli SYSTEMS BIOLOGY Suh-Chin Wu Institute of Biotechnology National Tsing Hua University.

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.

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Co-factor Manipulationin Metabolic Engineering

• Coenzyme A (CoA)/ Acetyl-Coenzyme A (Acetyl-CoA)

• Nicotinamide adenine dinucleotide (NAD+)

Page 72: Escherichia coli SYSTEMS BIOLOGY Suh-Chin Wu Institute of Biotechnology National Tsing Hua University.

Increase Carbon Flux from Pyruvate to Acetyl-CoA

Overexpression of panthothenate kinase

Supplementation of pantothenic acid

Page 73: Escherichia coli SYSTEMS BIOLOGY Suh-Chin Wu Institute of Biotechnology National Tsing Hua University.

Intracellular Acetyl-CoA Level

Page 74: Escherichia coli SYSTEMS BIOLOGY Suh-Chin Wu Institute of Biotechnology National Tsing Hua University.
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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

Page 76: Escherichia coli SYSTEMS BIOLOGY Suh-Chin Wu Institute of Biotechnology National Tsing Hua University.

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)

Page 77: Escherichia coli SYSTEMS BIOLOGY Suh-Chin Wu Institute of Biotechnology National Tsing Hua University.

YBS121:ackA-pta deletion mutant

Expression

of pnaK

Coexpression of

pnaK and PDH

Page 78: Escherichia coli SYSTEMS BIOLOGY Suh-Chin Wu Institute of Biotechnology National Tsing Hua University.

Conclusion Chapter for Metobolic Networks


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