Examples of the GC-MS spectrum of a synthetic amino acid mixture (standard), and intra cellular amino acids (in the current study) are shown in Figure 5.2. A and B. The details of ions used to detect each amino acid compound are shown in Table 5.1. The calibration curves for determination of each amino acid concentration are also shown in Appendix D (Section A). The relative abundance of each peak (amino acid) (see Figure 5.2.A) is used to calculate corresponding amino acid concentration. The detail GC-MS data of the current study is shown in Appendix D (Section B). Furthermore, a method to calculate amino acid concentrations, which are performed in Figure 5.3, is also presented in Appendix D (Section C). Finally, a summary of the results of this investigation are presented in Figure 5.3.
Table 5.1. The list of amino acids detected by GC-MS.
No Amino acid 1-Letter code Derivative compound Specific ions (m/z) 1 Alanine A Ala-(TBDMS)2 158; 232; 260
2 Glycine G Gly-(TBDMS)2 218; 246
3 Valine V Val-(TBDMS)2 186; 260; 288
4 Leucine L Leu-(TBDMS)2 200, 274, 302 5 Isoleucine I Ile-(TBDMS)2 200, 274, 302 6 Proline P Pro-(TBDMS)2 184; 258; 286
7 Methionine M Met-(TBDMS)2 218; 292; 320 8 Serine S Ser-(TBDMS)3 288; 262; 390 9 Threonine T Thr-(TBDMS)3 302; 376; 404 10 Phenylalanine F Phe-(TBDMS)2 234; 308; 336 11 Aspartic acid D Asp-(TBDMS)3 316; 390; 418 12 Glutamic acid E Glu-(TBDMS)3 272; 330; 432 13 Asparagine N Asn-(TBDMS)3 302; 315; 417 14 Glutamine Q Gln-(TBDMS)3 329; 357; 431 15 Arginine R Arg-(TBDMS)3 414; 442; 484 16 Histidine H His-(TBDMS)2 196; 338; 413 17 Tyrosine Y Tyr-(TBDMS)3 364; 438; 466 18 Tryptophan W Try-(TBDMS)3 302; 461; 489 19 Lysine K Lys-(TBDMS)3 329; 431; 488 20 Cysteine C Cys-(TBDMS)3 304; 378; 406
Figure 5.2. The ion chromatograph of a synthetic amino acid mixture (A) and intra cellular amino acids (B) analysed by GC/MS. The details of compound-specific ions are displayed in Table 5.1, as well as the peptide codes are also shown in Table 5.1. The inserted Figure illustrates the peak area was used to calculate the peptide concentration.
(A)
(B)
Ala
0 50 100 150 200 250 300 350
-2 0 2 4 6 8 10 12 14 Time (h)
Concentration (μg/g dry cell)
Gly
0 50 100 150 200 250 300 350
-2 0 2 4 6 8 10 12 14 Time (h)
Concentration (μg/g dry cell)
Val
0 50 100 150 200 250 300 350 400 450 500
-2 0 2 4 6 8 10 12 14 Time (h)
Concentration (μg/g dry cell)
Leu
0 100 200 300 400 500 600 700
-2 0 2 4 6 8 10 12 14 Time (h)
Concentration (μg/g dry cell)
Met
0 20 40 60 80 100 120 140 160 180
-2 0 2 4 6 8 10 12 14 Time (h)
Concentration (μg/g dry cell Ser
0 50 100 150 200 250 300 350 400
-2 0 2 4 6 8 10 12 14 Time (h)
Concentration (μg/g dry cell)
Thr
0 50 100 150 200 250 300 350 400 450 500
-2 0 2 4 6 8 10 12 14 Time (h)
Concentration (μg/g dry cell) Phe
0 100 200 300 400 500 600 700
-2 0 2 4 6 8 10 12 14 Time (h)
Concentration (μg/g dry cell)
Asp
0 200 400 600 800 1000 1200
-2 0 2 4 6 8 10 12 14 Time (h)
Concentration (μg/g dry cell)
Lys
0 100 200 300 400 500 600 700 800 900 1000
-2 0 2 4 6 8 10 12 14 Time (h)
Concentration (μg/g dry cell)
Arg
0 200 400 600 800 1000 1200
-2 0 2 4 6 8 10 12 14 Time (h)
Concentration (μg/g dry cell) His
0 100 200 300 400 500 600 700 800 900 1000
-2 0 2 4 6 8 10 12 14 Time (h)
Concentration (μg/g dry cell)
Tyr
0 200 400 600 800 1000 1200
-2 0 2 4 6 8 10 12 14 Time (h)
Concentration (μg/g dry cell) Trp
0 200 400 600 800 1000 1200
-2 0 2 4 6 8 10 12 14 Time (h)
Concentration (μg/g dry cell)
Figure 5.3. The concentrations of intracellular amino acids in cells grown with standard ( ) and VHG conditions with (z) or without ({) amino acid supplementation. All experiments were performed in triplicate.
A significant decrease of most amino acids under VHG conditions was found, especially for histidine, leucine, lysine, arginine, alanine, asparagine, tryptophan (see Figure 5.3 for details). However, the recovery of intracellular amino acids concentrations under VHG conditions with amino acid supplementations was faster at around 6 h in comparison with
the cultures without amino acid supplementation where the amino acid concentrations were recovered after 10 h (see Figure 5.3 for details). This was especially the case under VHG conditions with amino acid supplementation, where 10 h after being shocked by high osmotic stress (VHG), the intracellular amino acids were recovered and even reached a higher value than the control sample. The fluctuations of alanine, asparagine, phenylalanine and tyrosine concentration were significantly changed, whilst lysine, proline, threonine, arginine, and glycine were slightly changed. The concentrations of histidine, tryptophan, asparagine, valine were significantly decreased under VHG conditions, but these amino acids significantly recovered their concentrations under VHG conditions with amino acid supplementation, whilst other amino acids were showed slight changes in concentrations.
The faster recovery of intracellular amino acids led to an increase cell tolerance and then a decrease in the duration of the growth lag phase under VHG conditions (with amino acids supplementation) as shown in Figure 5.1. The data observed here are in agreement with the observations published recently when the addition of amino acids was used to study the effect of osmotic stress conditions (NaCl) on yeast [287]. When cells reached stationary phase, there was a decrease in the concentration intracellular amino acids (because of nutrition limitations). In the cells as illustrated in Figure 5.3 for the control sample at 10 and 12 h, whilst significant increase in the intracellular amino acid concentrations under VHG conditions (both with and without amino acid supplementation) were observed in these times. The concentrations of histidine, leucine, alanine, tyrosine, phenylalanine, and methionine were significantly increased after 6 h following the shock by VHG conditions for cells growing with amino acid supplementation, whilst these amino acids concentrations in cells growing without exogenous amino acid supplementation were increased after 10 h.
The increase of intracellular amino acid supplementation was in agreement with the increase of OD650 after 10 h for cells grown under VHG conditions.
It can be seen that after the supplementation of amino acids in VHG media, the OD was almost the same as the OD of the normal control samples at 12 h (after the stress condition
was applied) (see Figure 5.1). As detailed previously [27] (Section 4.4.7), there were a large number of proteins related to amino acids metabolism that were down-regulated under VHG conditions because of the inhibitions of high osmotic stress generated from high concentrations of both glucose and ethanol. To date, many transcriptional-level-based analyses for relative comparisons between normal and stress conditions have been performed [210, 289-291]. These results show that the osmotic stress tolerance in S.
cerevisiae cells was influenced by the up-regulation of genes involved the biosynthesis and accumulation of, for example, glycerol, trehalose, and glycogen. However, the roles of many other up-regulated genes cannot be determined under stress conditions [290, 291].
Moreover, the response of yeast to such stress conditions cannot be fully characterised because the up- or down-regulations of individual genes cannot directly decide and exert biological functions [287].
Previous studies have claimed that intracellular amino acid depletion led to a decrease of transport through cell membrane [292]. Furthermore, the delayed up-regulation of many proteins involved in the amino acids biosynthesis suggests that there is amino acid starvation under VHG conditions because of high osmotic stress, and the amino acid supplementation can aid cells to increase their tolerance under these conditions. The lack of amino acids might result from the demand of amino acids in cells to redistribute the intracellular concentration of many different enzymes to achieve the tolerance under this stress condition [287]. Therefore, a strain with high differential regulation of transport processes and amino acids biosynthesis might be a suitable strain for fermentation under VHG conditions. The decrease of amino acids uptake, together with the decrease of intracellular amino acids led to a sensitivity of cell growth under VHG conditions. The cellular amino acid uptake rates might be accelerated by the increase of exogenously supplied extracellular amino acids in the media (see Figure 5.3).
Most amino acids in yeast are found in the cytoplasm and various organelles including the mitochondria, vacuole, and the nucleus [293]. A large proportion of these amino acids are found in the vacuole, and compounds exchange between the cytoplasm and the vacuole pools [294]. Amino acid exchanges also occur in the mitochondria, since the biosynthesis of some amino acids are carried out in this organelle, as well as many proteins involved in the biosynthesis of these amino acids are also found here. For example, the conversion of glutamate to ornithine, an intermediate of arginine biosynthesis, is performed in the mitochondria. Therefore, the regulations of glutamate and arginine levels might involve this organelle, whilst orninine is exported from this organelle into cytosol [293]. Most amino acids such as serine, glycine, asparagine, methionine, threonine, tyrosine, phenylalanine, and histidine are exclusively derived from metabolite pools located in the cytosol. The biosynthesis of other amino acids such as alanine, valine, leucine, and isoleucine are required from pyruvate [18]. Glutamate, proline and arginine are synthesised from oxoglutamate generated from the mitochondria, while lysine is synthesised from both oxoglutamate and mitochondria AcCoA via the cytosolic homocitrate synthase [295]. The presence of amino acids originating from the media or produced in cells results in transport of the other amino acids from the cytoplasm and into other organelles such as the nucleus [293]. Histidine and lysine might indirectly influence arginine biosynthesis [293]. The increased concentrations of leucine, valine, asparagine, threonine, lysine, phenylalanine, and tyrosine might relate to proteolytic activity in the vacuole [296]. When high lysine levels were applied to the media, a high concentration of this amino acid was also found in the vacuole. It has been reported that this phenomenon was also similar for arginine, whose main function is to compensate for the level of this amino in the vacuole, with this compound being removed from the vacuole for the growth of cells when concentrations of arginine were limited in the media [297].
The changes in amino acid concentrations in specific subcellular compartments are triggers for the signal transmission in response to stress conditions. It has been shown that the
addition of amino acids in the media was rapidly sensed by the cells, and this information was relayed to the nucleus via a signalling pathway involving the activity of many amino acids biosynthesis proteins [298]. To date, an effect of enzyme on the size of amino acid pools were found for tyrosine, phenylalanine, lysine, arginine, and histidine, and these pools were increased 2 to 10 fold in response to starvation of one of several amino acids, whilst leucine and glutamate pools was slightly increased between 50 and 100% [296].
Moreover, tryptophan, methionine pools may increase under starvation conditions [299].
The accumulation of basic and aromatic amino acids might change the partitioning of other amino acids between the cytoplasm and vacuolar compartments. For example, the supplementation with lysine can increase the concentration of ornithine and arginine, moreover, the supplementation of arginine and histidine also has been shown to increase the concentrations of other amino acids in the cytosol [299]. Cysteine plays several important biological functions since it has redox properties of sulphur atoms in its side chain [300]. Furthermore, it serves as a key residue in the enzyme catalysis, protein oxidative folding [301] and trafficking [302], redox signalling and regulation [303]. As the supplementation of the basic amino acids in media was carried out here, more than half of the total amino acids was found and predominantly localized in the vacuole. As a result, it is likely that the accumulation of these amino acids led to increase widely the cytoplasmic fractions of other, limiting amino acids, as has been shown elsewhere [293]. However, the effects of all amino acids are not the same for the growth of S. cerevisiae. As an example, glycine is rapidly consumed by S. cerevisiae but it is also an inhibitor for both growth and fermentation, with this inhibition deriving from the inability of yeast to dispose of glyoxylate (two carbon skeleton) originating from glycine [304].