Overall, it can be said that the differential regulation of physiological parameters resulted in the differential regulation of the biological parameters (for example biomass), and one of the triggers for these oscillations is the generation and consumption of energy (known as a
redox balance) in yeast under VHG conditions [407, 423]. The stimulation of glucose under VHG conditions led to an acceleration of the glycolysis pathway, generating high energy in cells such as ATP, NADH, glycogen and trehalose as discussed elsewhere [27] (see Chapters 4, 5 and 6). The accumulation and consumption of these compounds may not occur simultaneously, since they require time to regulate intracellular metabolisms to respond to the inhibition of both ethanol production and high osmotic stress generated by the VHG conditions. This led to the formation of a lag phase in the oscillations in fermentation parameters, as discussed above. When the yeast cells adapted to the stress conditions, a relative balance of fermentation parameters would be established. These comments are discussed more detail in the following sections.
7.4.4.1. The regulation of ATP during continuous fermentation under VHG conditions
At the beginning of the fluctuations (when an increase of ethanol production and a corresponding decrease in residual glucose concentration occurred), a significant increase of most of the glycolysis proteins was found, especially for most proteins playing a role in generating ATP, NADH, glycogen and trehalose (see Figure 7.5.A and B). The differential expression of these proteins was evidence for the increase in ethanol concentration (together with this, the increase in glycerol produced was also observed), and whist a decrease of biomass (viability) was also found. In contrast to this phenomenon, the relative expression of the glycolysis pathway proteins was lower at the end of the fluctuations.
Therefore, factors playing main functions in generating this phenomenon are addressed here.
-2 -1 0 1 2 3 4 5 6
Hxk1p Hxk2p Pfk1p Pfk2p Tdh1p Tdh2p Pyk1p Pyk2p Adh1p Adh3p Adh4p
Protein name
Protein expression (fold)
(A)
0 1 2 3 4 5
Atp1p Atp2p Atp3p Atp16p Vma13p Vma2p Vma5p Vma6p Pma2p
Protein name
Protein expression (fold)
(B)
-3 -2 -1 0 1 2 3 4 5 6
Rpt1p Rpt4p Rpt6p His1p Lys1p Leu2p Ssa1p Ssa2p Ssb1p Ydj1p Sti1p
Protein name
Protein expression (fold)
(C)
Figure 7.5. The regulation of proteins relating to the fluxes of glycolysis (A), F-ATPases and V-ATPases (B), and other proteins (C) at the sampling time of ( ) 132 h, ( ) 144 h, and ( ) 165 h compared to 120 h.
Under micro-anaerobic fermentation conditions, ATP is known as one of the most essential free energy sources in S. cerevisiae. During their life, S. cerevisiae cells use more than 50%
of their generated ATP for processes other than the anabolism of biomass [428]. Moreover, ATP also plays a major role as a regulator between anabolism and catabolism [428], and this compound is synthesised from ADP and Pi (inorganic phosphate) mainly via the glycolysis pathway (Figure 7.8). During continuous fermentation processes, the ATP level decreased 25% from the 120th h to the 144th h (see Table 7.4). The decrease in ATP might relate to an increase in phosphorylation of glucose as discussed in [429], leading to up- regulation of hexokinases Hxk1p (+1.9), Hxk2p (+1.8) at the 144 h sampling time compared to 120 h. When a higher glucose concentration was available in the media, a decrease in the ATP/ADP ratio was found (ATP/ADP ratio decreased from 2.95 (at 132 h) to 1.01 (at 144 h)) (see Table 7.4 for details). The ATP concentration remained low at the
. . . . . .
144 and 156 h, but the ATP/ADP ratio recovered from 1.01 at 144 h to 2.25 at 156 h (almost the same ratio at 120 h (2.0)). The decrease in ATP/ADP resulted in a decreasing of the free energy of ATP hydrolysis (see the regulation of F-ATPases and V-ATPases for details), because of glucose diffusion into cells. The decrease of ATP can be reproduced if the biosynthesis pathway is more strongly stimulated by an increase of pyruvate concentration (derived from the glycolysis pathway) than is the TCA. This was confirmed by an increase of biomass at the 156th h, together with an increase of ATP concentration from 1.8 mg/g dry weight (at 144 h) to 2.4 mg/g dry weight at 156 h (Table 7.4). This can be explained since, as mentioned above, hexokinases increased their relative expression, leading to an increase intracellular glucose, but the relative expression of proteins Adh1p, Adh3p, Adh4p were not increased significantly. As a result, the ethanol concentration at 156 h was lower that at 120 h (see Figure 7.6). This is due to the biosynthesis of amino acids, which might be accelerated at 156 h compared to 120 h. The consuming of ATP (anabolic) pathways is stimulated in other ways than via the ATP level, and of course more strongly than the production of ATP (catabolic) pathways. Moreover, the anabolism is sensitive to its substrates since the regulation of these substrates is demanded by their products [430]. The ATP level is regenerated if the sensitivities of the consumption of ATP are included. Therefore, the increase of ATP of glycolytic flux generated net ATP production. This was seen at the 132th h (see Table 7.4). The fall in ATP concentration is compensated for a stronger activation of ATP consuming processes, and this ATP
Table 7.4. Concentrations of ATP and ADP, and ratio of ATP/ADP versus times.
Time (h) ATP (mg/g dry weight) ADP (mg/g dry weight) ATP/ADP*
120 3.8 1.6 2
132 4.2 1.2 2.95
144 1.8 1.5 1.01
156 2.4 0.9 2.25
*The ratio ATP/ADP was calculated by the equation:
ATP/ADP = (ATP g/507.2 g)/(ADP g/427.3 g).
consumption results in an anabolism, for example the biosynthesis of amino acids, and biomass observed at the 156th h compared to the 144th h. The decrease of ATP results from an inhibition of the TCA cycle and oxidative phosphorylation [431], this is confirmed by a significant decrease of pO2 (see Figure 7.7) in the media between 144 h and 156 h, suggesting that cells consumed more O2 for the biosynthesis of amino acids and then anabolism of biomass. The increase of intracellular glucose led to net ATP consumption.
Once a decrease in ATP occurred, the balance of ATP production and consumption would be restored to keep the bioenergetic balance in the cells that can be seen at the 156th h (see Table 7.4).
50 60 70 80 90 100 110 120 130 140 150
96 108 120 132 144 156 168 180
Time (h)
4.0 4.5 5.0 5.5 6.0 6.5
Glucose, and ethanol concentrations (g/L) Glycogen, and biomass concentrations (g/L)114 115 116 117
Figure 7.6. The figure is reproduced from 7.3.A to illustrate the sampling times as well as the labeling samples for proteomic analysis. The concentrations of ethanol (), glycerol ({), and residual glucose (¡) as well as biomass (z) for continuous fermentation under VHG condition at dilution rate D2 = 0.025 h-1.
0 20 40 60 80 100
108 120 132 144 156 168
pO2 (%)
Time (h)
Figure 7.7. The regulation of pO2 in the bioreactor.
The differential regulation of glycolysis proteins
It is fair to say that the oscillations of fermentative parameters resulted from the fluxes of some main metabolic pathways in S. cerevisiae, especially the glycolysis pathway. The triggers of glycolytic oscillations are the relative balance between ATP and ADP, NAD+ and NADH, performed via the ratio NAD+/NADH, ATP/ADP, and the PFK (phosphofructokinase)/adenosine nucleotide system, as well as the anabolism and catabolism of biomass. The generation and consumption of ATP/ADP, and NAD+/NADH in S. cerevisiae are briefly outlined in Figure 7.8. Furthermore, the differential regulation of glycolysis is focused on 3 main steps, which are catalysed by hexokinases (Hxk1p, Hxk2p), phosphofructokinases (Pfk1p, Pfk2p), and pyruvate kninases (Pyk1p, Pyk2p). Among these proteins, the phosphofructokinase plays the most important role in the regulation of glycolytic flux; other proteins, for example pyruvate kinases and hexokinases also play major roles in the regulation of the glycolysis pathway.
ATP plays a negative effect on glycolysis, in which Pfk1p, Pfk2p and Pyk1p, Pyk2p are the main target for ATP to inhibit [432] (see Figure 7.8). Pyk1p, Pyk2p are activated by fructose-1,6-bis-phosphate and inhibited by ATP [433], whilst hexokinase (Hxk1p, Hxk2p) is competitively inhibited by trehalose-6- phosphate [434]. So far, Pyk1p and Pyk2p were considered as the single rate-limiting step of glycolysis, however, this concept failed when it was used to in an attempt to enhance the flux through this pathway by overexpression of these proteins [435, 436]. In this situation under investigation here, the relative expressions of Pyk1p and Pyk2p were increased at 132 h compared to 120 h, followed by a decrease at the 144th h compared to the 120th h. However, their relative expression recovered at 156 h, nearly reaching the same concentration observed at 120 h (see Figure 7.5.A for details). The up-regulation of these proteins might result from an increased relative abundance of fructose-1,6-bis-phosphate, since the expressions of Pfk1p and Pfk2p also increased at 132 h compared to 24 h. However, unlike the fluctuations of Pyk1p and Pyk2p, the expressions of Pfk2p and Pfk2p decreased at both 144 h and 156 h. If the expressions of Hxk1p and
Hxk2p were examined in this context, we can see that at the 144th h Hxk1p and Hxk2p increased their expression, but both Pfk1p, Pfk2p, and Pyk1p, Pyk2p decreased in expression, leading to a decrease of ethanol conversion from glucose (69.2 g/L of ethanol at the 144th h compared to 76.4 g/L of ethanol at the 120th h). One reasonable explanation for the differential regulation of these proteins is that the high expression of Hxk1p and Hxk2p led to an increase in glucose phosphorylation, and most products of this process (glucose-6- phosphate) were driven to the synthesis of glycogen and trehalose. Moreover, the syntheses of these compounds were important in responding to stress conditions (high ethanol formed from the first state of oscillation (at 120 h and 136 h) and high glucose in the media). When resistant to this stress condition, cells accelerated their proliferation and of course viability.
Although no proteins involved in the cell cycle were detected in this study to support this, the cell proliferation and viability were determined via biomass as well as cell viability.
This was confirmed by an increase of biomass after 144 h, since there were increases in biomass as well as cell viability at 156 h compared to 144 h (6.0 g dry weight/L of biomass with 90% live cells at 156 h compared to 5.3 g dry weight/L of biomass with 70 % cell live at 144 h).
Moreover, the down-regulation of Pfk1p and Pfk2p at the end of oscillation state (at 114 h and 156 h) might be also affected by the inhibition of citrate compounds (diverted from the TCA cycle) since there was a significant decrease of pO2 (see Figure 7.7) in the media between 144 and 156 h (when compared to 120 h), suggesting that the TCA cycle was accelerated in this period. Furthermore, Pfk1p and Pfk2p are sensitive to a number of allosteric regulators, such as ATP, AMP, fructose-6-phosphate, and fructose-2,6-phosphate, fructose-1,6-bis-phosphate [135, 437] (see also Figure 7.8). However, the activation of Pfk1p and Pfk2p by fructose-6-phosphate is mainly important during a transition process, such as, for example, a change of carbon source [438, 439].
Fructose-6P Glucose-6P
Fructose-1,6BP Pfk1p Pfk2p
Glyceral-3P
1,3-DP-glycerate
3-P-glycerate
P-enolpyruvate
Pyruvate
Acetaldehyde Glucose
Ethanol DHacetone-P
Glycerol-3-P
NAD+ + Pi
NADH
NADH
NAD+ Pgk1p NADH
NAD+
Gpm1p Eno1p Eno2p
Pdc5p Pdc6p Pgi1p
Fba1p ATP
ADP
Pyk1p Pyk2p
ADP
ATP ADP
ATP HXk1p Hxk2p
ATP
ADP
P-93
Acetyl CoA
Adh1p Adh2p Tdh1p Tdh2p
Glycerol Gpd1p
Hor2p
Citrate
Isocitrate
2-Oxoglutarate
S-Succinyldi- hydrolipoamide Succinyl-CoA
Succinate Fumarate
Malate
Oxaloacetate Mdh1p
Kgd2p Ldp1p
Cdc25p
Ras2p
Cyr1p
AMP cAMP
ADP ATP
Adk1p
RNA DNA Rpb5p Pol12p
GTP GDP
Glucose-1P
Glycogen UDP-glucose
Aminolse Gsy1p
???
Gph1p
Trehalose-6P Trehalose
Tps1p Tps2p
Oxidative Phosphorylation
Atp2p Atp16p Atp3p
Pma2p
H+
NADH NAD+
NADH NAD+
Histidine NADH
NAD+ His2p
Leucine NAD+ NADH Leu2p
Lysine NADH NAD+
Lys1p
Nth1p
Figure 7.8. The diagram illustrates the formation and consumption of ATP, ADP, NAD+, NADH in glycolysis and other pathways. The effects of metabolites on activity of proteins functioned in the regulator of glycolytic flux are also included. The green dotted line is representative for the activation of a metabolite on the protein activity, and red dotted line is for the de-activation of a metabolite on the protein activity.
ATP and adenine nucleotides are also major regulators of glycolytic flux. These compounds act as substrates or products of most important reactions that are known key factors for the control of the rate of glycolysis pathway, such as hexokinase, phosphofructokinase, and pyruvate kinase steps, and AMP is an activator for phosphofructokinase [135]. Finally, the ATP consumption rate (or ATP demand) plays a role in governing the rate of glycolysis flux [440]. The correlation between intracellular ATP and glycolytic flux was found to be strongly negative, in which the lower the ATP concentration, the higher the rate of glycolysis [441]. However, it has been shown that the ATP/ADP ratio, and trehalose-6- phosphate levels do not influence glycolytic activity [432]. It is also very difficult to determine whether ATP plays a role in controlling the regulation of glycolysis pathway, or the ATP levels are simply a consequence of glycolysis pathway. If the ATP levels are as a consequence of the glycolysis pathway, it also means that the increase of glycolysis pathway flux leads to increase the concentrations of ATP [432]. Therefore, there is a correlation between the accumulation of glycogen, trehalose and glycolytic flux. In this work, the carbohydrate storage compounds were accumulated when there were decreases of most trigger factors for glycolytic flux such as Pfk1p, Pfk2p, Pyk1p, and Pyk2p.
In summary, most of the energy gained in S. cerevisiae cells from the glycolysis pathway where ADP, ATP, NAD+, NADH are formed and consumed was a main factor for the differential regulations of both the glycolysis pathway and glycolysis proteins. During the continuous fermentation process, ATP and NADH are generated via glycolysis pathway. In the upper portion of the glycolysis pathway, ATP is consumed by hexokinases and fructokinases (see Figure 7.8 for details). Therefore, when high amount of ATP is formed and accumulated in cells, the glycolysis will be decelerated, and this pathway will be accelerated when cells require more ATP (this might happen when high concentration of ADP or AMP are formed). ATP is required for some processes such as anabolism or catabolism. The fluctuations of ATP, and ADP concentrations resulted in the fluctuations of proteins expressions such as Pfk1p, Pfk2p (phosphofructokinases), and Pyk1p, Pyk2p
(pyruvate kinases), Hxk1p, Hxk2p (hexokinases). The expressions of these proteins versus time are discussed detail above. Finally, the correlative mechanism of between glycolytic flux and carbohydrate accumulation discussed above was the main trigger for the fluctuation of fermentative parameters during ethanol continuous fermentation under VHG conditions.
7.4.4.2. The expression fluctuations of proteins relating to oxidative phosphorylation Of 9 detected proteins involved in the oxidative phosphorylation process, 4 belong to F- type H+- transporting ATP chains including Atp1p (alpha chain), Atp2p (beta chain), Atp3p (gamma chain), Atp16p (delta chain), 4 belong to vacuolar H+-ATPases (V-ATPases) consisting of Vma13p (subunit H of the 8-subunit V1 peripheral membrane domain), Vma2p (subunit B of the 8-subunit V1 peripheral membrane domain), Vma5p (subunit C of the 8-subunit V1 peripheral membrane domain), Vam6p (subunit D of the 5-subunit V0 interal membrane domain), and 1 (Pma2p) belongs to the plasma membrane H+-ATPases.
The expression fluctuations of these proteins were noted since they relate to catalysis and transport of ATP.
The structure of F-ATPase proteins detected here is shown in Figure 7.9. The functions of these ATPase proteins are essential for the synthesis of ATP as well as mitochondrial maintenance, but these proteins might not be important for yeast life. However, the deletions of these genes (ATP1, 2, 3 and 16) results in a decrease in the growth rate and an inability to survive on non-fermentable carbon sources [442]. The catalytic role of these proteins is to form ATP from ADT and Pi by utilizing proton motive force, and their functions may have other important associations or localizations [443] in which an interaction with adenine nucleotide translocase has been found [444]. As a function of time the relative abundance of these proteins increased from 132 to 156 h compared to 120 h as seen in Figure 7.5.B. The highest ratio was found for Atp2p (+3.3) at the 140th h compared to 120 h. Together with the high expression of Atp2p, Atp1p was also up-regulated with
increasing time. The relative expression of ratios Atp3p and Atp16p were almost the same ratio at each sampling time. The high differential expression of these proteins at 132 h compared to 120 h agreed with the increased expression of most glycolysis proteins that led to an increase in amount of ATP (4.2 mg/g dry weight) at 132 h compared to (3.8 mg/g dry weight) at 120 h (as discussed above). But at 156 h, a slight increase of these proteins’
differential expressions was found. This information is also in agreement with the discussion above, since there was an increased glycolytic rate at 132 h and 156 h compared to 120 h.
Figure 7.9. The structures of F- and V-ATPases in the relationship with proteins detected in this study. The Figure was modified from [445, 446]. For F-ATPase, the synthesis of ATP occurs in F1 coupling with the transport of proton through F0 from lumen/extracellular.
For the For V-ATPase, the V1 domain is response for ATP hydrolysis and drives proton transport through the V0 domain from cytoplasm.
The V-ATPase is known as an ATP-dependent proton pumps [445], and the structure of V- ATPase proteins detected in this study is shown in Figure 7.9. V-ATPase includes two separate domains (V0 – an integral domain, and V1 – a peripheral domain), while V0 is responsible for proton translocation, and V1 is responsible for ATP hydrolysis [445]. Most
ATPase proteins (Vma13p, Vma2p, and Vma5p) detected here belong to the V1 subunit, while only one ATPase protein (Vma6p) was found that belongs to the V0 subunit. There was a similar pattern of protein expression behaviour as a function of time in comparison to the expressions of F-ATPase proteins discussed above (see Figure 7.5.B for detailed comparisons). Moreover, the expression of Pma2p was important to reflect the rhythm of protons transported out of the cells, since this the function of this protein is to pump protons out of the cells. Moreover, Pma2p also functions as a regulator of cytoplasmic pH and plasma potential, and the differential expression of this protein was +3.5, +1.8, and +2.5 at the sampling time of 132 h, 144 h, and 156 h compared to 120 h. The rhythm of the abundance of this protein was almost synchronised with the behavioural patterns of most F- ATPases and V-ATPases detected and discussed here.
The viability of S. cerevisiae cells depends strongly on many cellular processes via membrane-bounded organelles including the endoplasmic reticulum, nucleus, golgi apparatus, endosomes, vacuolar compartment, mitochondria and peroxisomes [447]. During the life of S. cerevisiae, there are many proteins synthesised that require targeting to membranes to transport across or insert into them, and the main purpose of these processes is growth, and an increase of the surface area and hence the cell volume [447]. Most of the proteins detected here belonging to these organelles are discussed in the following sub- sections.
The endoplasmic reticulum
The relative expressions of proteins Ssa1p and Ssa2p (cytosolic form of Hsp70 family) are important for the targeting of the endoplasmic reticulum, since these proteins are known to be one of the most important factors for membrane targeting in yeast [447]. It has been shown that the deletion of these proteins resulted in a decrease in the import of carboxypeptidase Y (CPY) and α-factor (α-F) precursors into the endoplasmic reticulum, as well as the F1-ATPase β-subunit (F1-β) into the mitochondria [448]. In the other words, the
functions of these proteins are regulated to SRP-dependent co-translational protein- membrane targeting and translocation. Moreover, the regulation of Ssb1p (also a member of the cytosolic form of Hsp70 family) was similar to the regulation of Ssa1p and Ssa2p (see Figure 7.5.C for detail), and this regulation might be also involved in the folding of newly-synthesised polypeptide chains [449]. In brief, the main function of the Hps70 family is to expedite the protein translation across membranes, and prevent the cell from protein denaturation under stress conditions, as well as to assist in folding of nascent polypeptides [450]. All heat-shock proteins belonging to the Hsp10 family consist of a N- terminal ATPase domain and a C-terminal peptide binding domain [451]. Although the ATP activity of these proteins is low, their ATP activity is stimulated by interaction with the Dnaj co-chaperones Ydj1p and Sti1p [451]. The expressions of both Ydj1p, and Sti1p were fluctuated with the expressions fluctuations of Ssa1p and Ssa2p. Ydj1p localized in membrane may stimulate the release of substrate to the translocation machinery [452].
Although so far, the ATPase activity of Ssa1p and Ssa2p is regulated by ATP turnover (alternatively between 2 states), in the presence of ATP (ATP-bound state), the peptide exchange is fast with low affinity, that is low with high affinity in the presence of ADP (ADP-bounded state) [451], the expression of these proteins (Ssa1p, and Ssa2p) was constantly up-regulated at the sampling times between 132 and 156 h compared to 120 h (see Figure 7.5.C for details). The differential protein expressions may have helped cells re- activate damaged proteins under VHG conditions and high ethanol concentration. As a result, an increase of biomass as well as cell viability was found at 156 h compared to 144 h.
Mitochondrial proteins
The main function of the mitochondria is to supply ATP formed by oxidative phosphorylation for cells [453], and to provide precursors for the biosynthesis of many cellular metabolites [454], as well as being central regulators of programmed cell death [455]. Only few mitochondrial proteins are essential for the viability of S. cerevisiae [456].