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.