2.2. Modification of genome-scale metabolic models
One of the important effects of the variation in the intracellular pH is
the change of the ionization state of metabolites and metabolites
contain certain particular functional groups that can lose or obtain
protons at various pH levels. To modify the metabolic networks at
different pH values, Marvin view software 19.2, 2019, developed by
ChemAxon (http://www.chemaxon.com) was used to estimate the charge of
metabolites based on their pKa for intended pH value. The mol files of
each metabolite, which are generally classified as data files in plain
text format containing molecular details, atom, bonds, co-ordinates, and
communication detail, were obtained from Chebi
(https://www.ebi.ac.uk/chebi) and PubChem
(https://pubchem.ncbi.nlm.nih.gov/) databases.
The change of metabolites charge can result in the disruption of the
charge balance of reactions and so, the charge balance of the reactions
was automatically checked at different pH levels according to the method
presented in Figure 1. Initially, the proton was removed from each
reaction in the case of presence. Then, charges of the metabolites at
the right and left side of each reaction were separately summed, and the
total charge of the right side was subtracted from the left side. If it
was positive (negative), the proton with the coefficient of the
subtracted value was added to the left (right) side of the reaction. The
reaction was left unchanged if the subtracted value was zero. The charge
balance of transport reactions was not changed because the proton is
mechanistically added to the reactions (e.g., simple transport). The
charge of the exchange reactions was not changed because they are pseudo
reactions that exchange metabolites between the cell and the
environment. The accuracy of the method was evaluated by comparing the
modified model with the original model at pH=7, and the charge balance
of some reactions was not changed due to the fact that these reactions
exchange protons between subsystems to transfer materials (e.g.,
mitochondria to cytosol). S. cerevisiae, Z. mobilis, and E.
coli contained 8, 2, and 6 kinds of these reactions, respectively.
These reactions were balanced separately, which are shown in Table S1
and S2.
By changing metabolite charges at
different pH levels, the charge balance of each reaction would be
changed, which affects in proton exchange rates. To determine the
sensitivity of growth to the proton exchange rate at different pH
levels, developed models could be analyzed with robustness analysis
which can assess the optimal value of the objective function by varying
the flux of a reaction and indicating the sensitivity of the objective
function to changes in a particular reaction. In this regard, the
sensitivity of cell growth to proton exchange has been assessed via
robustness analysis. Mat files of modified metabolic models are also
provided in Supplementary File 3.