Transcript E. coli

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
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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
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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
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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 ▲
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–
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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