Dynamics of gene expression associated with stress response
Yeast cells suffer from multiple stresses during CVEF, such as ethanol
inhibition, osmotic stress by high residual glucose, and redox stress
from imbalanced energy metabolism (Auesukaree, 2017; Burphan et al.,
2018; Caspeta, Castillo, & Nielsen, 2015). The metabolism of storage
carbohydrates, such as trehalose, glycogen, and glycan, was investigated
to help determine tolerance to environmental stress (Babazadeh et al.,
2017; Li et al., 2009). During the oscillation, significant changes were
observed for the expression level of genes related with stress response,
such as oxidative stress response related genes SOD1, SOD2, CTT1,
OYE2 and GRX8 , and the HSP family
(HSP10,12,26,31,42,60,82 ). As shown in Figure 5, it was difficult
to draw a unified change rule for all, but most of the stress response
genes were highly expressed at P4, when ethanol levels kept increasing
in the broth and was about to achieve the maximum value. At the same
time, the glucose level approached its minimum and ORP value was at an
intermediate level, thus those two factors could not exert severe
stresses on cells, suggesting that a large proportion of cellular
response to stresses were triggered by ethanol inhibition in P4.
HSPs are a highly conserved family of molecular chaperone proteins that
play a role in the aggregation, assembly, transport, and folding of
proteins (Muhlhofer et al., 2019). Due to the common effect of multiple
stresses during CVEF on disturbing protein structures, the upregulation
of HSPs is observed upon exposures to all of these stresses, making the
expression of HSPs overall higher. HSP10 and HSP60 are
mitochondrial matrix co-chaperonin and chaperone ATPase, which
cooperates with each other, so their expression levels are similar.HSP26 and HSP42 are small heat shock proteins that form
oligomers to suppress unfolded proteins aggregation. The expression
levels of these HSPs are consistent with changes in ethanol
concentration, indicating that cellular protein denaturation was
triggered by ethanol stress.
In addition, it seems that the ethanol oscillation was mainly
responsible for the expression changes of SOD1 . During the VHG
fermentation, S. cerevisiae exhibited increased levels of
intracellular Reactive Oxygen Species (ROS) and oxidative damage to cell
structures. Superoxide dismutases (SODs), the major ROS-scavenging
enzymes, are classified into two groups according to their subcellular
localization and metal cofactors, namely cytosolic Cu/Zn-SOD (Sod1p) and
mitochondrial Mn-SOD (Sod2p). Although both SOD1 and SOD2are highly expressed in the whole oscillation for the tolerances to ROS
stress, the average and the variance of SOD1 expression is 21.6%
and 53.7% higher than that of SOD2 . This may be due to the
production of ethanol in the cytoplasm, which brings greater ROS stress
to this cellular compartment.
Although ethanol is a major stress trigger, we have also found additive
effects of multiple stress conditions on the expression of some stress
genes, making these genes highly expressed during the entire oscillation
cycle, especially at P3 and P4 to cope with the oxidative stress caused
by high osmotic pressure and strong ethanol inhibition. For example, the
oxidative stress response gene OYE2 (NADPH dehydrogenase) and the
glutathione-independent methylglyoxalase gene HSP31 were
initially up-regulated in response to the osmotic stress caused by high
glucose concentration (P2/P1), were subsequently further up-regulated
due to osmotic pressure and ethanol stress (P3/P2) and finally expressed
at its highest level at P4 due to strong ethanol stress.