Chasing the flux:selecting target pathways through flux analysis of carbon metabolism

Vangelis Simeonidis

COBRA 2011, Reykjavik7 April 2023

Luxembourg Centre for Systems Biomedicine

LCSB new building

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/

Cell growth Protein purification Enzyme kinetics

Quantitativeproteomics

Quantitativemetabolomics

SBML model

Parameters(KM, Kcat)

Variables(metabolite and protein concentrations)

MCISB

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

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.

Challenge

Which enzymes?

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

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

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

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

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

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%  .........................................................  ............  .........................................................  ............

Elementary Flux Mode (EFM) analysisthe smallest sub-networks that allow a metabolic reconstruction network to function (in steady state)

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

Flux percentage coverage

Targeted selection allows rapid coverage

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

Acknowledgments The MCISB team

Acknowledgments The LCSB team

Thank you!

evangelos.simeonidis@uni.lu

@vangos