Chasing the flux
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Chasing the flux:selecting target pathways through flux analysis of carbon metabolism
Vangelis Simeonidis
COBRA 2011, Reykjavik7 April 2023
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Luxembourg Centre for Systems Biomedicine
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LCSB new building
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Bioinformatics
Mechanisms of Parkinson´s DiseaseMechanisms of Parkinson´s Disease
Disease networks Disease networks
Gene-environment interactionsGene-environment interactions
Modelling and simulation
Computational biology
Personaliz
edMedicine
Transcriptomics Proteomics Metabolomics
Animal modelsHuman genetics
Chemical biologyImaging Chemical biologyImaging
LCSB strategy
http://wwwen.uni.lu/lcsb/
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Cell growth Protein purification Enzyme kinetics
Quantitativeproteomics
Quantitativemetabolomics
SBML model
Parameters(KM, Kcat)
Variables(metabolite and protein concentrations)
MCISB
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Kinetic modelling
Teusink et al. glycolysis model (Eur J Biochem 267:5313, 2000)
aims to characterize fully the mechanics of each enzymatic reaction
( ) GLK GLTind GLC tv v
dt
6 ( )2
..............................................................
GLK GLYCOGEN PGI TREHALOSEd G P tv v v v
dt
226 16
26 16
6 62 2
26 16 ( )0
6
6 ( )6 ( )6 ( ) 1
1 1 1PFK PFK PFK AK PFKF bP F bP AMP ATP
PFK PFK PFKF bP F bP AMP
PFK RR PFK PFK PFK PFK
F P ATP ATP F PPFK
C F bP C F P t C Keq Ci
K K KPFK PFKATP F P
g F P tF P tg Vm F P t
Km Km Km Kmv
LKm Km
2
26 16 6 6
2 2
2 22 26 ( )16 ( ) 6 ( )26
1
1 1 1 1
PFKATP
PFK PFKATP ATP
AKR
PFK PFK PFK PFK PFK PFK PFK PFKF bP F bP AMP ATP F P ATP ATP F P
C
Ki Km
g F P tF P t Keq F P tF bPK K K Ki K Km Km Km
2 2 2( ) 4 ( ) 2 ( ) 8 ( ) ( ) 4 ( )
2 8
AK AK AKAXP AXP AXP AXP
AK
P t Keq P t P t Keq P t P t Keq P t
Keq
2 2 22 ( ) 8 ( ) ( ) 4 ( )
1 4
AK AKAXP AXP AXP AXP
AK
P t Keq P t P t Keq P t
Keq
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Consensus yeast model
Nature Biotechnology 26, 1155 - 1160 (2008)
A consensus yeast metabolic network reconstruction obtained from a community approach to systems biology
Markus J Herrgård, Neil Swainston et al.
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Challenge
Which enzymes?
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General strategy
Brute-force approach:• Study all enzymes • Create complete map
But:• Gaps in the network
• Might end up with eg: >90% of enzymes, but <10% of flux
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General strategy
Flux-centric approach:• Identify where the
carbon flux goes• Prioritize pathways by
ranking higher ones that carry the most flux
• Will end up with eg: 20-30% of enzymes, but >90% of flux
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Flux Balance Analysis
Stoichiometric Matrix: signifies if and how a metabolite takes part in a certain reaction
AB…G
r1 r2 …. rn
a1
b1
….g1
a2
b2
….g2
….….….….
an
bn
….gn
Flux Vector: Each component represents the flux through the corresponding reaction
v1
v2
….vn
v
dA/dtdB/dt
….dG/dt
=
Steady State condition
00….0
=
Rn space Rm SpaceKer(S)
null vector 0
L1 ≤ v1 ≤ U1
L2 ≤ v2 ≤ U2
…..............Ln ≤ vn ≤ Un
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Experimental measurements (mmoles/hr/g DW) 1 2 3 AVE C% input flux for FBA
Carbon input flux as glucose 67.5 42.1 74.1 61.2 100.00 1.0000
qBiomass 10.3 9.4 8.7 9.5 15.46 0.0234
qCO2 (offgas) 16.0 12.3 20.9 16.4 26.78 1.6070
qEthanol (exometabolome) 29.5 16.9 34.8 27.1 44.20 1.3261
qAcetate (exometabolome) 0.5 0.4 0.5 0.5 0.76 0.0229
qAcetaldehyde (exometabolome) 0.2 0.1 0.2 0.2 0.27 0.0082
qGlycerol (exometabolome) 5.9 6.2 8.1 6.7 11.00 0.2199
qTrehalose (exometabolome) 0.2 0.2 0.2 0.2 0.33 0.0016
Exometabolome measurements
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Results Name flux glucose_transport__uniport 1.000 hexokinase_D_glucoseATP 1.000 glucose_6_phosphate_isomerase 0.957 fructose_bisphosphate_aldolase 0.931 phosphofructokinase 0.931 glyceraldehyde_3_phosphate_dehydrogenase 1.646 phosphoglycerate_kinase -1.646 enolase 1.437 phosphoglycerate_mutase -1.437 pyruvate_kinase 1.425 pyruvate_decarboxylase 1.349 glycerol_3_phosphate_dehydrogenase_NAD 1.059 glycerol_3_phosphate_dehydrogenase_FAD_mitochondrial 0.850 ethanol_reversible_transport -1.260 alcohol_dehydrogenase_ethanol -1.260 triose_phosphate_isomerase 0.721 CO2_transporter_via_diffusion -1.930 glycine_cleavage_complex_lipoamide_mitochondrial 0.183 phosphoserine_transaminase 0.209 glutamate_dehydrogenase_NADP -0.300
glycine_cleavage_system_lipoamide_irreversible_mitochondrial 0.183 glycine_cleavage_complex_lipoamide_mitochondrial 0.183 acetyl_CoA_hydrolase 0.051
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Results biomass production 15.55% D-Glucose exchange 100.00% Glucose-6-phosphate isomerase 100.00% Glucokinase 100.00% glucose-6-phosphate isomerase 95.67% fructose bisphosphate aldolase 93.09% phosphofructokinase 93.09% glyceraldehyde-3-phosphate dehydrogenase 82.32% phosphoglycerate kinase 82.32% enolase 71.93% phosphoglycerate mutase 71.93% pyruvate kinase 71.30% pyruvate decarboxylase 67.39% alcohol dehydrogenase 41.99% Ethanol exchange 41.99% triose phosphate isomerase 36.07% CO2 exchange 32.16% glycine cleavage complex lipoamide 30.70% ......................................................... ............ ......................................................... ............
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Elementary Flux Mode (EFM) analysisthe smallest sub-networks that allow a metabolic reconstruction network to function (in steady state)
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Which enzymes? GLC
DHAP
G6P
F6P
FDP
G3P
13PG
3PG
2PG
PYR
PEP
ACALDCO2
ETOH
AKG
3PHP
PSEP
GLU SER
GLY
CO2
GLYC3P
GLYC
OAA
ASP
G1P
UDPG
13BDGLCN
AC
MAN6P
MAN1P
GDPMANN
DOLMANP MANNAN
14GLUN
GLYCOGEN
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Flux percentage coverage
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Targeted selection allows rapid coverage
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Future plans
The method was used to select/prioritize the enzymes for kinetic models of yeast metabolism in MCISB
13C measurements of intracellular flux will constrain the problem further and match it to our growing conditions
Paper in preparation
Such crucial improvements are a necessary stepping stone for experimental design and to verify predictions
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Acknowledgments The MCISB team
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Acknowledgments The LCSB team