To estimate how long the expressions distributions of the proteome took to recover, samples were taken at 0, 2, 10, and 12 h. These sampling times were compared to a control sample harvested before VHG conditions (at 0 h) and amino acid supplementation were
initiated. After the reliability and statistical cut-off criteria detailed in the materials and methods were applied, a number of important quantitative proteomic observations are summarised in Table 5.2.
Table 5.2. Relative expression levels of proteins discussed here.
115:114 116:114 117:114 Protein Protein name
Ratio EF Ratio EF Ratio EF
Glycolysis pathway
Hxk1p Hexokinase +1.9 1.1 +1.6 1.1 +1.3 1.1 Hxk2p Hexokinase +1.2 1.2 +1.5 1.2 +1.3 1.2 Glk1p Glucokinase +2.1 1.1 +1.4 1.1 +1.7 1.1 Pgi1p Glucose-6-phosphate isomerase +1.3 1.0 +1.3 1.0 +1.1 1.0 Pfk1p Phosphofructokinase alpha
subunit
+1.1 1.1 +1.4 1.1 +1.2 1.1 Pfk2p phosphofructokinase beta
subunit
-1.1 1.1 +1.3 1.1 +1.3 1.1 Tdh1p Glyceraldehyde-3-phosphate
dehydrogenase
+1.3 1.0 -1.1 1.0 1.0 1.0 Tdh2p Glyceraldehyde-3-phosphate
dehydrogenase
-1.3 1.1 -1.2 1.1 -1.1 1.1 Tdh3p Glyceraldehyde-3-phosphate
dehydrogenase
-1.3 1.1 -1.2 1.1 -1.1 1.1 Pgk1p 3-phosphoglycerate kinase +1.2 1.0 -1.2 1.1 -1.2 1.0 Eno1p Enolase I +1.1 1.1 -1.1 1.1 -1.1 1.1 Eno2p Enolase II -1.3 1.1 1.0 1.1 -1.1 1.1 Pdc1p Pyruvate decarboxylase -1.1 1.0 +2.6 1.0 +2.8 1.0 Adh1p Alcohol dehydrogenase +2.0 1.1 +4.6 1.1 +4.6 1.1 Adh4p Alcohol dehydrogenase
isoenzyme IV
+1.6 1.4 +2.3 1.5 +1.8 1.2 Fba1p Aldolase -1.1 1.0 -1.1 1.1 -1.4 1.1 Gpd1p Glycerol-3-phosphate
dehydrogenase
+1.3 1.1 +1.9 1.1 +2.0 1.1 Hor2p DL-glycerol-3-phosphatase +1.3 1.2 +2.1 1.2 2.0 1.2
Starch and sucrose metabolism Ugp1p Uridinephosphoglucose
pyrophosphorylase
+1.4 1.2 +1.6 1.1 +1.6 1.2
115:114 116:114 117:114 Protein Protein name
Ratio EF Ratio EF Ratio EF Tps1p 56 kD synthase subunit of
trehalose-6-phosphate
synthase/phosphatase complex
+1.4 1.2 +1.3 1.1 +1.4 1.1
Tps2p Trehalose-6-phosphate phosphatase
+1.3 1.1 +1.4 1.3 +1.7 1.7 Pgm2p Phosphoglucomutase +1.9 1.1 +1.7 1.1 +1.7 1.2 Gph1p Glycogen phosphorylase +1.9 1.2 +1.4 1.4 +1.4 1.5 Tsl1p 123 kD regulatory subunit of
trehalose-6-phosphate
synthase/phosphatase complex
+1.3 1.1 +1.3 1.1 +1.4 1.1
Pentose phosphate pathway Tal1p Transaldolase +1.1 1.4 +1.0 1.2 +1.1 1.2 Tkl1p Transketolase -1.7 1.2 -1.1 1.2 +1.9 1.1 Gnd1p 6-phosphogluconate
dehydrogenase
-1.1 1.1 +1.1 1.1 +2.0 1.1
Aminoacyl-tRNA biosynthesis (translation)
Krs1p Lysyl-tRNA synthetase +1.2 1.7 +3.2 1.4 +2.6 1.1 Frs1p Phenylalanyl-tRNA synthetase -1.1 1.1 +2.2 1.2 +2.2 1.1 Cdc60p Cytosolic leucyl tRNA
synthetase
-1.3 1.5 +2.2 1.2 +1.9 1.3 Ses1p Seryl-tRNA synthetase +1.1 1.2 +1.8 1.4 +1.6 1.2 Dps1p Aspartyl-tRNA synthetase,
cytosolic
+1.1 1.1 +2.1 1.2 +1.9 1.2 Ded81p Asparaginyl-tRNA synthetase -1.2 1.4 +1.8 1.0 +1.5 1.3 Msk1p Mitochondrial lysine-tRNA
synthetase
+1.0 1.1 +1.7 1.1 +1.6 1.4 Grs1p Glycyl-tRNA synthase +1.2 1.1 +2.7 1.1 +2.5 1.1
Citrate cycle (TCA cycle)
Idp1p Mitochondrial form of NADP- specific isocitrate
dehydrogenase
-1.3 2.4 +1.3 2.2 +1.4 2.1
Mdh1p Mitochondrial malate dehydrogenase
-1.4 1.2 -1.4 1.2 -1.3 1.1 Aco1p Aconitase, mitochondrial -1.2 1.2 1.0 1.2 +1.1 1.2 Cit1p Citrate synthase 1.0 1.1 1.0 1.1 +1.2 1.1 Cys3p Cystathionine gamma-lyase +1.1 1.3 +1.7 1.3 +1.6 1.3
115:114 116:114 117:114 Protein Protein name
Ratio EF Ratio EF Ratio EF Cys4p Cystathionine beta-synthase -1.5 1.2 -2.4 1.4 -1.9 1.3 Aat2p Aspartate aminotransferase,
cytosolic
+1.4 1.2 +2.3 1.6 +1.6 1.2 Aro4p 3-deoxy-D-arabino-
heptulosonate 7-phosphate (DAHP) synthase isoenzyme
-1.2 1.4 -1.2 1.2 -1.2 1.2
Ura1p Dihydroorotate dehydrogenase 1.0 1.6 +2.7 1.1 +1.8 1.2 Ura2p Carbamoyl-phophate synthetase,
aspartate transcarbamylase, and glutamine amidotransferase
+3.5 1.7 +4.6 2.3 +5.3 1.8
Ade12p Adenylosuccinate synthetase +1.4 1.0 +1.1 1.6 +1.4 1.1 Ilv3p Dihydroxyacid dehydratase -2.5 1.1 +2.5 1.2 +2.2 1.5 Ilv5p Acetohydroxyacid
reductoisomerase
-1.8 1.1 -1.7 1.1 +1.8 1.2 Bat1p Mitochondrial branched-chain
amino acid aminotransferase
-2.7 1.4 -2.1 1.2 +2.3 1.3 Leu4p Alpha-isopropylmalate synthase -1.9 2.0 -2.0 1.3 +1.8 1.4 Ald3p Aldehyde dehydrogenase +1.8 1.3 +1.8 1.4 +2.6 1.2 Ald4p Mitochondrial aldehyde
dehydrogenase
-1.1 1.1 -1.2 1.1 -1.1 1.1 Ald6p Cytosolic Aldehyde
Dehydrogenase
+1.7 1.6 +1.6 1.4 +2.4 1.5 Erg10p Acetoacetyl CoA thiolase -1.6 1.1 -1.1 1.3 -1.1 1.5 His4p Histidinol dehydrogenase +1.4 1.2 1.0 1.7 +2.2 1.8 Ctt1p Cytoplasmic catalase T +1.4 1.2 +1.6 1.2 +1.9 1.2 Sah1p Putative S-adenosyl-L-
homocysteine hydrolase
-1.6 1.2 +1.1 1.2 1.0 1.3 Met6p Cobalamin-independent
methionine synthase
-3.0 1.1 -2.6 1.1 +2.7 1.1 Gln1p Glutamine synthetase -1.6 6.2 -1.4 12.4 +1.9 5.9 Gdh1p NADP-specific glutamate
dehydrogenase
-1.6 1.8 +1.3 1.5 +1.7 1.5 Hom6p Homoserine dehydrogenase -1.1 2.8 -1.1 2.1 1.0 1.5 Aro8p Aromatic amino acid
aminotransferase
+1.1 1.2 +1.5 1.2 +1.4 1.2 Gre3p A keto-aldose reductase +1.2 1.1 +1.3 1.2 +1.3 1.1
Nucleotide metabolism
Adk1p Adenylate kinase -1.1 1.2 -1.2 1.2 +1.9 1.2
115:114 116:114 117:114 Protein Protein name
Ratio EF Ratio EF Ratio EF Ysa1p Homolog to serendipity protein -1.1 1.2 -1.2 1.2 -1.2 1.2 Rnr4p Ribonucleotide reductase +1.2 1.3 1.0 1.3 1.0 1.2 Ylr432wp Ylr432wp -1.5 1.4 -1.2 1.1 -1.2 1.4 Ynk1p Probable nucleoside-
diphosphate kinase
-1.4 1.1 -1.1 1.1 -1.1 1.1 Guk1p Guanylate kinase -1.5 6.1 -1.2 1.8 -1.3 1.7 Rnr2p Small subunit of ribonucleotide
reductase
+1.3 1.4 -1.2 2.2 -1.2 1.6
Energy metabolism
Tfp1p Vacuolar ATPase V1 domain subunit A (69 kDa)
+2.1 1.2 +2.3 1.2 +1.8 1.2 Atp2p F(1)F(0)-ATPase complex beta
subunit, mitochondrial
+2.6 1.6 +3.0 1.9 +3.2 1.8 Vma2p vacuolar ATPase V1 domain
subunit B (60 kDa)
+2.9 1.4 +3.3 1.4 +2.6 1.5 Atp1p Mitochondrial F1F0-ATPase
alpha subunit
+2.3 1.3 +2.8 1.3 +2.8 1.3 Ipp1p Inorganic pyrophosphatase -1.2 1.1 1.1 1.1 1.0 1.1 Cdc19p Pyruvate kinase -1.2 1.0 -1.1 1.1 -1.3 1.1
Cell division, cycle, homeostasis
Ubc9p Ubiquitin-conjugating enzyme -1.8 1.7 -2.4 1.5 +2.6 2.4 Bmh1p Homolog of mammalian 14-3-3
proteins
-1.2 1.2 -1.3 1.1 -1.1 1.2 Bmh2p Homolog of mammalian 14-3-3
proteins
-1.2 1.2 -1.3 1.1 -1.1 1.2 Ydr516cp Ydr516cp +1.5 1.8 +1.4 15.4 +1.2 138 Sps100p Cell wall-related secretory
glycoprotein
+1.1 1.1 +1.1 1.2 +1.3 1.2 Tpm1p Tropomyosin -1.2 1.2 +1.2 1.3 +1.0 14.1 Yor177cp Yor177cp 1.0 1.1 -1.1 1.1 1.0 1.5 Act1p Actin +1.4 1.1 +1.6 1.1 +1.6 1.1 Bud6p Actin interacting protein -1.3 1.1 +1.5 1.2 +1.9 1.1 Cof1p Cofilin, actin binding and
severing protein
+1.2 1.1 +1.5 1.2 +1.5 1.1 Cdc48p Microsomal ATPase 1.0 1.3 -1.1 1.2 +1.2 1.2 Wtm1p Transcriptional modulator +1.3 1.2 1.0 1.3 1.0 1.3 Mbp1p Transcription factor -1.3 2.8 -1.4 2.9 -1.3 2.1
115:114 116:114 117:114 Protein Protein name
Ratio EF Ratio EF Ratio EF Trx2p Thioredoxin -1.5 1.2 -1.8 1.2 -1.6 1.1 Tsa1p Thioredoxin-peroxidase +1.2 1.1 +1.3 1.0 +1.2 1.1 Ahp1p Alkyl hydroperoxide reductase +1.2 1.1 +1.1 1.1 +1.2 1.1 Sod1p Cu, Zn superoxide dismutase -1.7 1.4 -1.1 1.3 +1.2 1.2 Yor129cp Yor129cp 1.0 1.2 1.1 1.2 -1.1 1.3
Response to stress
Ssc1p Mitochondrial matrix protein -1.2 1.1 1.0 1.1 +1.8 1.1 Htb1p Histone H2B +1.2 1.2 -1.3 1.1 -1.1 1.2 Hsc82p constitutively expressed heat-
shock protein
1.0 1.4 +1.4 1.5 +1.1 1.4 Din7p involved in DNA repair -2.0 4.4 +1.4 1.7 +2.0 1.9 Sti1p Heat-shock protein -1.3 1.1 1.0 1.2 -1.1 1.1 Hsp12p 12 kDa heat-shock protein -1.7 1.1 +1.2 1.1 +2.0 1.1 Sod2p Manganese-containing
superoxide dismutase
-1.4 2.3 +1.1 1.4 -1.1 2.4 Hsp26p Heat-shock protein 26 -1.8 1.1 +1.6 1.2 +2.0 1.1 Ssb2p Heat-shock protein of HSP70
family
-1.5 1.1 -1.3 1.1 -1.2 1.1 Mcr1p NADH-cytochrome b5 reductase -1.1 1.3 -1.2 1.4 -1.1 1.4 Hsp104p 104 kDa heat-shock protein -1.8 1.1 -1.2 1.1 +2.1 1.1 Sse1p HSP70 family member -1.4 1.1 -1.3 1.1 +1.6 1.2 Ssb1p Cytoplasmic member of the
HSP70 family
-1.5 1.1 -1.3 1.1 +1.7 1.2 Rps31p Ribosomal protein S31 +1.2 1.1 +1.5 1.1 +1.5 1.2 Ssa2p Member of 70 kDa heat-shock
protein family
-1.2 1.3 +1.1 1.2 +1.5 1.2 Hsp82p 82 kDa heat-shock protein +1.2 1.1 +1.5 1.1 +1.5 1.1 Ssa1p Heat-shock protein of HSP70
family
+1.3 1.2 +1.1 1.2 +1.3 1.2 Hsp60p Mitochondrial chaperonin -1.2 1.1 +1.1 1.1 +1.2 1.1 Ssa4p Member of 70 kDa heat-shock
protein family
-1.2 1.2 1.0 1.2 +1.5 1.2 Dak1p Putative dihydroxyacetone
kinase
+1.1 1.3 +1.3 1.3 +1.8 1.2 Rhr2p DL-glycerol-3-phosphatase +1.2 1.3 +1.2 1.7 +1.5 1.3
Figure 5.4. The expression of proteins at 2, 10, and 12 h under VHG conditions with amino acid supplementation compared to standard conditions (at 0 h). Red, green and yellow colours are representatives for numbers of down-, up-, un- regulated proteins, respectively.
In previous work [27] (Chapter 4), most proteins relating to amino acid metabolism, and heat-shock showed decreases in their relative abundances in repose to VHG conditions compared to lower glucose fermentation control conditions. Here, to improve and understand the cell growth under VHG conditions by improving the media quality, proteins with unchanged expression levels were also considered, since these proteins were previously down-regulated under VHG conditions [27]. The expressions of important proteins summarised in Table 5.2 (according to their functions) versus times are depicted in Figure 5.4. From Figure 5.4, we can see that most involved in glycolysis proteins were up- regulated (compared to the control sample), where 22%, 39%, and 33% of detected glycolysis proteins were up-regulated at 2 h, 10 h and 12 h, respectively. Whilst the remaining glycolysis proteins were unchanged in expression. No detected glycolysis proteins were down-regulated in out sample time period. Together with the up-regulation of glycolysis proteins, the relative expressions of proteins involved in the pentose phosphate pathway were also increased. These data suggest that the glycolysis pathway (and by implication glycolysis protein activities) was not repressed by this stress condition, and the enhancement of protein abundance here led to an increased yield of ethanol as seen at 10 h
and 12 h (see Section 5.4.9 for details). This phenomenon was similar for proteins involved in starch and sucrose metabolism, and energy metabolism. Another surprise here was the differential expression of proteins relating to amino acids metabolism, since there was a recovery in the expression of these proteins observed 10 h and then 12 h after the cells were shocked by VHG conditions. 38% of the detected amino acids metabolism related proteins were down-regulated at 2 h following the glucose shock; however, this ratio was reduced to 27% by 10 h, and then significantly to only 8% at 12 h post shock. Up-regulation of several proteins relating to amino acids metabolism was also observed. In this case, only 12% of up-regulated proteins were found at 2 h, this ratio increased to 31% at 10 h, and then significantly to 54% at 12 h post glucose shock (see Figure 5.4 for details). 29% of detected proteins with putative functions involved in responding to stress conditions were down- regulated at 2 h. However, these proteins recovered their levels of expression, and even increased in expression (up to 14%) by 10 h, and then increased to 43% at 12 h post shock.
Unsurprisingly, most detected proteins involved with aminoacyl-tRNA biosynthesis demonstrated unchanged expressions at 2 h, and subsequently increased expression dramatically by up to 100% at 10 h and then 12 h.
The expressions of many of the proteins are discussed in the following sections in the context with metabolite measurements. These discussions will supply corroborative evidence for the benefit of amino acid supplementation to cultures fermenting under VHG conditions with the aim of enhancing ethanol production.