3.2. Applying the systemic approach for improving ethanol
production of S. cerevisiae
To find candidate reactions for S. cerevisiae , the correlated
reactions with ethanol production at pH=5 were identified using the
Pearson correlation coefficient (between fluxes of each reaction and
ethanol production) in the range of proton exchange rate between -2 and
23 mmolgDCW-1h-1. This range was
determined based on robustness analysis (Figure 3b), considering that
the metabolic model of pH=5 was robust for growth compared to the two
other models and maximum ethanol is produced at optimal growth in this
range (Figure S8). So, flux distributions at optimal growth in the
selected range were determined and correlated reactions with ethanol
production were determined (Supplementary File 2). These 252 reactions
are key reactions that indicate the manner of using metabolism for
maximal ethanol production during optimal growth. 67 reactions with
positive coefficient are candidates for up-regulation and 185 with
negative coefficient are predicted for down-regulation. The Pearson
correlation coefficients between these reactions and growth indicate
that the correlated reactions with ethanol production are non-growth
associated and vice versa (Supplementary File 2). So, the correlation
coefficients predict that up and down-regulation of candidate reactions
for overproduction of ethanol results in growth reduction(Naghshbandi et
al., 2019; Pagliardini et al., 2013).
For more screening of the key reactions and determining the important
genes affected by the pH change, PCA was performed using fluxes of the
key reactions at pH levels of 5, 6 and 7. Figure 4a illustrated a clear
distinction between pH=5, and the other three pH models were achieved
based on the first PC. Only the first two components have been
considered as they demonstrate the high percentage of variation between
pH=5 and other pH levels: 90.18% for PC1 and PC2 (Figure 4b). The
decomposition of data by PCA indicates the contribution of each
correlated metabolic reaction to the differentiation of models at
various pH values. Among the 252 key reactions, 12 reactions
including ILETA, ME2m, PDHm,
ICDHyr, CSm, ACONTm, DESAT16, NDPK1, THRD_L, ICL, AGTi, and MDH were
essential for the discrimination of pH models based on the PCA results
presented in Figure 4c. These reactions were selected to evaluate the
effects of regulators on their enzymes on ethanol production. Table S6
provides information for these 12 reactions in detail.
It can be seen that various approaches have been suggested by the
systemic approach for ethanol overproduction. Figure 5 shows the
comprehensive connection of each proposed reaction, which is suitable
for up-regulation or down-regulation. For instance, through observing
the model reactions, mitochondrial pyruvate is not capable of converting
to acetaldehyde and must enter the cytosol for conversion to ethanol.
Thus, according to the PDHm reaction (Table S7), coenzyme A reacts with
the mitochondrial pyruvate to produce acetyl coenzyme A. The product of
this reaction is then converted to citrate by the CSm reaction and the
produced citrate is responsible for providing isocitrate via ACONTm
reaction. Cytosolic isocitrate is generated by mitochondrial isocitrate
from the CITtcm and then converted to 2-oxoglutarate by the ICDHyr
reaction. This metabolite is further produced to cytosolic pyruvate by
the ALATA_L reaction, which leads to acetaldehyde by PYRDC. Eventually,
acetaldehyde is reduced to ethanol by the ALCD2ir.
The predicted redirecting flux to the TCA cycle under acidic conditions
is expected. Isocitrate dehydrogenase (ICDHyr), citrate synthase (CSm),
Aconitate hydratase (ACONTm), which were ethanol-associated (or
non-growth associated) reactions and Malic enzyme NADP mitochondrial
(ME2m), with growth correlated coefficient (or non-ethanol associated),
were the identified candidate reactions in TCA. The predictions of the
systemic approach were in compliance with fluxes determined by
experimental data, where Blank et al.(Blank & Sauer, 2004) showed that
malic enzyme and mitochondria were more active under acidic conditions,
as was clear from metabolic reactions located at crucial branch points
in central metabolism. Furthermore, previous studies have supported the
predicted effects of some reactions. These were the bottleneck reactions
for both growth and ethanol production at pH=5. Isocitrate dehydrogenase
(ICDHyr) reaction, which is encoded by IDP2, converts isocitrate to
α-ketoglutarate, Scalcinati et al.(Scalcinati et al., 2012) showed that
overexpression of IDP2 has resulted in an increased NADPH production to
supply the required energy for biomass and yeast growth. The alanine
glyoxalate aminotransferase (AGTi) reaction, which encoded by AGX1,
converts glyoxalate and alanine to glycine and pyruvate. Chidi et
al.(Chidi, Rossouw, & Bauer, 2016) experimentally confirmed that the
deletion of AGX1 increased pyruvic acid, which led to improved ethanol
production.