Zero flux through newly added pathway

[Nandini Kannoju]

Hello everyone,

I hope you're all doing well.

This is regarding the pathway which I'm working on. Firstly, I added all the required metabolites, followed by incorporating the first reaction of the pathway, which produces the first intermediate. However, after optimizing the model for this added reaction, I observed that there is zero flux through it.

Both the lists included the newly added metabolites and the reaction, along with the other metabolites and reactions that were already present in the model. I'm currently facing an issue with achieving the desired flux through the added pathway.

Could anyone provide some insights or suggestions on how we can proceed further to successfully incorporate the pathway and ensure the model produces the desired metabolites?

Your input would be greatly appreciated.

Here is the code

clc;

clear all;

initCobraToolbox;

changeCobraSolver('glpk');

% Load SBML model using readCbModel without the 'SBML' argument

model = readCbModel('iCGB21FR.xml');

% Add p-coumarate to the model using the struct's ID

model = addMetabolite(model, 'p_coumarate[c]',...

'metName', 'Trans 4 Hydroxycinnamate C9H7O3',...

'metFormula', 'C9H7O3',...

'Charge', -1, ...

'csense', 'E');

% Add protocatechuate to the model using the struct's ID

model = addMetabolite(model, 'protocatechuate[c]',...

'metName', '3,4-Dihydroxybenzoate',...

'metFormula', 'C7H5O4',...

'Charge', -1, ...

'csense', 'E');

% Add beta-carboxy cis cis muconate to the model using the struct's ID

model = addMetabolite(model, 'beta_carboxy_muconate[c]',...

'metName', 'Cis,cis-Butadiene-1,2,4-tricarboxylate',...

'metFormula', 'C7H3O6',...

'Charge', -3, ...

'csense', 'E');

% Add beta_keto_adipate_enol_lactone to the model using the struct's ID

model = addMetabolite(model, 'beta_keto_adipate_enol_lactone[c]',...

'metName', '5-Oxo-4,5-dihydrofuran-2-acetate',...

'metFormula', 'C6H5O4',...

'Charge', -1, ...

'csense', 'E');

% Add beta_ketoadipate to the model

model = addMetabolite(model, 'beta_ketoadipate[c]',...

'metName', '3-Oxoadipate',...

'metFormula', 'C6H6O5',...

'Charge', -2, ...

'csense', 'E');

% Add beta_ketoadipyl_CoA to the model

model = addMetabolite(model, '3-Oxoadipyl-CoA[c]',...

'metName', '3-Oxoadipate',...

'metFormula', 'C27H37N7O20P3S',...

'Charge', -5, ...

'csense', 'E');

% Add the exchange reaction for p-coumarate

model = addReaction(model, 'EX_p_coumarate[e]', 'metaboliteList', {'p_coumarate[c]'},...

'stoichCoeffList', -1);

% model = addReaction(model, 'EX_p_coumarate_e', 'reactionFormula', 'p_coumarate <=>');

%Add reaction between p-coumarate and 4-hydroxybenzoate

model = addReaction(model,'PCOUM2HBA','reactionFormula','p_coumarate[c] => 4-Hydroxybenzoate');

%Add reaction between 4-hydroxybenzoate and protocatechuate

model = addReaction(model,'HBA2PRPC','reactionFormula','4-Hydroxybenzoate => protocatechuate[c] ');

%Add reaction between protocatechuate and beta carboxy cis cis muconate

model = addReaction(model,'PRPC23CM','reactionFormula','protocatechuate[c] + O2 => beta_carboxy_muconate[c]');

%Add reaction between beta carboxy cis cis muconate and 4-Carboxymuconolactone

model = addReaction(model,'3CM24CM ','reactionFormula','beta_carboxy_muconate[c] => 4-Carboxymuconolactone[c]');

% Add reaction between 4-Carboxymuconolactone and beta keto adipate enol lactone

model = addReaction(model, '4CM2BKAL', 'reactionFormula', '4-Carboxymuconolactone[c] => beta_keto_adipate_enol_lactone[c]');

% Add reaction between beta_keto_adipate_enol_lactone and beta_ketoadipate

model = addReaction(model, 'BKAL2BKAD', 'reactionFormula', 'beta_keto_adipate_enol_lactone[c] => beta_ketoadipate[c]');

% Add reaction between beta_ketoadipate and beta_ketoadipyl_CoA

model = addReaction(model, 'BKAD2BKACoA', 'reactionFormula', 'beta_ketoadipate[c] => beta_ketoadipyl_CoA[c]');

% Add reaction between beta_ketoadipyl_CoA and Succinyl-CoA

model = addReaction(model, 'BKACoA2SUC', 'reactionFormula', 'beta_ketoadipyl_CoA => Succinyl-CoA + Acetyl-CoA');

% Diffusion reactions

%model = changeRxnBounds(model, {'HCO3E'}, 0, 'b');

model = changeRxnBounds(model, {'GLCtex'}, 0, 'b');

model = changeRxnBounds(model, {'FRUtex'}, 0, 'b');

%model = changeRxnBounds(model, {'GLUtex'}, 0, 'b');

%model = changeRxnBounds(model, {'HCO3tex'}, 0, 'b');

model = changeRxnBounds(model, {'ALAtex'}, 0, 'b');

%model = changeRxnBounds(model, {'ARGtex'}, 0, 'b');

model = changeRxnBounds(model, {'GLYtex'}, 0, 'b');

%model = changeRxnBounds(model, {'GLNtex'}, 0, 'b');

%model = changeRxnBounds(model, {'HIStex'}, 0, 'b');

%model = changeRxnBounds(model, {'LEUtex'}, 0, 'b');

%model = changeRxnBounds(model, {'LYStex'}, 0, 'b');

%model = changeRxnBounds(model, {'PROtex'}, 0, 'b');

%model = changeRxnBounds(model, {'SERtex'}, 0, 'b');

model = changeRxnBounds(model, {'UREAtex'}, 0, 'b');

%model = changeRxnBounds(model, {'GLCGLYCtex'}, 0, 'b');

model = changeRxnBounds(model, {'SUCRtex'}, 0, 'b');

model = changeRxnBounds(model, {'PTRCtex'}, 0, 'b');

model = changeRxnBounds(model, {'SPMDtex'}, 0, 'b');

%model = changeRxnBounds(model, {'CYNTtex'}, 0, 'b');

model = changeRxnBounds(model, {'CITtex'}, 0, 'b');

%model = changeRxnBounds(model, {'MALtpp'}, 0, 'b');

%model = changeRxnBounds(model, {'MALtex'}, 0, 'b');

%model = changeRxnBounds(model, {'AKGtex'}, 0, 'b');

%model = changeRxnBounds(model, {'SUCCtpp'}, 0, 'b');

%model = changeRxnBounds(model, {'SUCCtex'}, 0, 'b');

%model = changeRxnBounds(model, {'PYRtex'}, 0, 'b');

%model = changeRxnBounds(model, {'FUMtpp'}, 0, 'b');

%model = changeRxnBounds(model, {'FUMtex'}, 0, 'b');

model = changeRxnBounds(model, {'LDH_D'}, 0, 'b');

% Set the uptake rate for glucose:

model = changeRxnBounds(model, {'EX_glc__D_e'}, -20, 'b');

for j = 1:1:3

% Set the uptake rate for p-coumarate:

model = changeRxnBounds(model,'EX_p_coumarate_e',-j,'b');

% Set the objective function to biomass reaction:

model = changeObjective(model, 'Growth');

% Perform FBA to calculate biomass production:

fbasolution = optimizeCbModel(model,'max');

biomass_production_values(j) = fbasolution.x(1485);

bketoadipate_values(j) = fbasolution.x(1546);

end

disp(bketoadipate_values);

disp(biomass_production_values);

[Diana El Assal]

Hi Nandini,

This is a common issue encountered when adding new pathways. You could try to identify blocked reactions and dead end metabolites or use relaxedFBA by setting the objective function as one of your blocked reactions to identify the causes. 

https://opencobra.github.io/cobratoolbox/stable/modules/analysis/exploration/index.html

https://opencobra.github.io/cobratoolbox/stable/modules/analysis/relaxedFBA/index.htm

https://opencobra.github.io/cobratoolbox/stable/modules/reconstruction/modelGeneration/index.html

Best wishes,

Diana

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[Ines Thiele]

Note that you have errors in at least these reactions:

model = addReaction(model,'PCOUM2HBA','reactionFormula','p_coumarate[c] => 4-Hydroxybenzoate');

model = addReaction(model, 'BKACoA2SUC', 'reactionFormula', 'beta_ketoadipyl_CoA => Succinyl-CoA + Acetyl-CoA');

while the first one might be fine as the product appears as substrate in another reaction, it is unlikely that  Succinyl-CoA + Acetyl-CoA are connected to the rest of your network bc 1. they would be present as met abbr rather than names and 2. no compartment is specified

------
Ines Thiele, PhD
Professor in Systems Biomedicine
ERC grant recipient
University of Galway Foundation Research Leadership Programme Awardee
Principal Investigator, APC Microbiome Ireland 

Principal Investigator, Ryan Institute

Principal Investigator, Galway Neuroscience Centre

University of Galway
School of Medicine
School of Biological and Chemical Sciences | Microbiology, 

Orbsen Building | Galway, Ireland

thielelab.eu,
GoogleScholarProfile,
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