Insights into pH-induced Metabolic Switch by Flux Balance Analysis
Supplementary Material
Marija Ivarsson, Heeju Noh, Massimo Morbidelli and Miroslav Soos*
Institute for Chemical and Bioengineering, Department of Chemistry and Applied Biosciences,
ETH Zurich, Zurich, Switzerland
*
Corresponding author. E-mail: miroslav.soos@chem.ethz.ch; HCI F137, Vladimir-Prelog-Weg
1, CH-8093 Zurich; Tel.: +41 44 6334659, Fax.: +41 44 6321082
1
Supplementary Table 1. List of target genes investigated by gene expression analysis
Gene Symbol
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
Hk1
Hk2
Pfkl
Pgam2
Pkm2
Pepck1
Pepck2
Ldha
Pcx
Pdha1
Pdha2
Dlat
Pdk1
Pdk2
Pdk3
Cs
Idh3a
Ogdh
Sdha
Fh1
Mdh1
Mdh2
Acly
Me1
Me2
Me3
Gpt2
Got1
Got2
Glud1
Gls
G6pdh
Name
hexokinase 1
hexokinase 2
phosphofructokinase, liver
phosphoglycerate mutase 2
pyruvate kinase muscle
phosphoenolpyruvate carboxykinase 1, cytosolic
phosphoenolpyruvate carboxykinase 2, mitochondrial
lactate dehydrogenase A
pyruvate carboxylase
pyruvate dehydrogenase E1 alpha 1
pyruvate dehydrogenase E1 alpha 2
dihydrolipoamide S-acetyltransferase
pyruvate dehydrogenase kinase, isoenzyme 1
pyruvate dehydrogenase kinase, isoenzyme 2
pyruvate dehydrogenase kinase, isoenzyme 3
citrate synthase
isocitrate dehydrogenase 3 alpha
oxoglutarate dehydrogenase
succinate dehydrogenase complex, subunit A
fumarate hydratase 1
malate dehydrogenase 1, NAD
malate dehydrogenase 2, NAD, mitochondrial
ATP citrate lyase
malic enzyme 1, cytosolic
malic enzyme 2, mitochondrial NADP dependent
malic enzyme 3, mitochondrial NAD dependent
glutamic pyruvic transaminase 2
glutamate oxaloacetate transaminase 1
glutamate oxaloacetate transaminase 2
glutamate dehydrogenase 1 (=GDH)
glutaminase
glucose-6-phosphate dehydrogenase
2
Supplementary Table 2: Reactions in the metabolic network used for flux balance analysis.
Glycolysis
1
2
3
4
5
GLC6P <-> F6P
F6P + ATP -> ADP + 2 GAP
GAP + NAD + ADP <-> [3PG] + NADH + ATP
3PG -> PEP
PEP + ADP + H -> PYR + ATP
6
7
8
9
Pentose Phosphate Pathway
GLC6P + 2 NADP + H2O -> RBL5P + 2 NADPH + CO2
RBL5P <-> R5P
RBL5P <-> X5P
2 X5P + R5P <-> 2 F6P + G3P
Pyruvate Metabolism
10
11
12
13
14
PYR + H -> PYR_m + H_m
PYR_m + CoA_m + NAD_m -> CO2 + AcCoA_m + NADH_m + H_m
PYR_m + HCO3_m + ATP_m -> OAA_m + ADP_m + Pi
MAL_m + NAD_m <-> PYR_m + NADH_m + H_m + CO2
MAL + NADP <-> PYR + NADPH + H + CO2
15
16
17
18
19
20
TCA Cycle
OAA_m + AcCoA_m + H2O <-> CIT_m + CoA_m
CIT_m + NAD_m + H2O <-> AKG_m + NADH_m + CO2 + H_m
AKG_m + NAD_m + CoA_m -> SUCCoA_m + NADH_m + H_m + CO2
SUCCoA_m + ADP_m + Pi_m + FAD_m <-> FUM_m + ATP_m + CoA_m + FADH2_m
FUM_m + H2O <-> MAL_m
MAL_m + NAD_m <-> OAA_m + NADH_m + H_m
21
22
23
24
25
Malate Aspartate Shuttle
OAA_m + GLU_m <-> AKG_m + ASP_m
AKG + ASP <-> OAA + GLU
ASP_m + GLU -> ASP + GLU_m
OAA + NADH + H <-> MAL + NAD
MAL + AKG_m <-> MAL_m + AKG
Glutaminolysis
26 GLN + H2O -> GLU + NH4
27 GLU_m + H2O + NAD_m <-> AKG_m + NADH_m + NH4
28 GLU <-> GLU_m
Acetyl CoA Formation
29 CIT + CoA + ATP + H2O -> AcCoA + OAA + ADP + Pi
3
Amino Acid Synthesis
30
31
32
33
34
GLU + PYR <-> AKG + ALA
3PG + NAD + GLU + H2O -> NADH + H + AKG + Pi + SER
GLU + ATP + H + NADPH + H + NADH -> ADP + NADP + Pi + H2O + NAD + PRO
SER + THF <-> GLY + MTHF + H2O
GLN + ASP + ATP + H2O <-> GLU + ASN + AMP + 2 Pi + H
Amino Acid Degradation
35 SER <-> PYR + NH4
36 THR + NAD + CoA -> GLY + AcCoA + NADH + H
37 ARG_m + H2O + AKG_m + NAD_m <-> 2 GLU_m + UREA_m + NADH_m + H_m
38 PRO_m + 2 H2O + NAD_m -> GLU_m + NADH_m + H_m
39 HIS_m + 3 H2O -> GLU_m + Formamide_m + NH4
40
LYS_m + NADPH + 2 AKG_m + 4 NAD_m + 2 H2O + 2 CoA_m -> NADP + 2 H_m + 4
NADH_m + 2 GLU_m + 2 CO2 + 2 AcCoA_m
41 PHE + NADPH + H + O2 -> TYR + NADP
42 TYR + AKG + 2 O2 + CoA + H2O <-> GLU + CO2 + 2 H + FUM_m + AcCoA + Acetate
43
VAL_m + AKG_m + CoA_m + 3 NAD_m + FAD_m + 3 H2O + ATP_m -> 2 H_m + GLU_m
+ SUCCoA_m + CO2 + 3 NADH_m + FADH2_m + ADP_m + Pi_m
44
ILE_m + AKG_m + 2 NAD_m + 2 CoA_m + FAD_m + H2O + HCO3_m + ATP_m -> GLU_m
+ SUCCoA_m + AcCoA_m + 2 NADH_m + CO2 + FADH2_m + ADP_m + Pi_m
45
LEU_m + AKG_m + NAD_m + 2 CoA_m + ATP_m + HCO3_m + H2O -> GLU_m +
NADH_m + CO2 + H_m + Pi_m + ADP_m + Acetate + 2 AcCoA_m
46 CYS + AKG + SO3 + O2 + H2O -> GLU + PYR + S2O3 + 2 H
47
MET + 2 ATP + H2O + SER + CoA + NAD + HCO3 -> NH4 + CYS + CO2 + NADH +
SUCCoA_m + ADP + Pi
Transport
48 CIT_m + MAL <-> CIT + MAL_m
49 PRO <-> PRO_m
50 ARG <-> ARG_m
PEP Regeneration
51 OAA_m + ATP_m -> ADP_m + PEP_m + CO2
52 PEP <-> PEP_m
Oxidative Phosphorylation
53 NADH_m + H_m + 0.5 O2 + 2.5 ADP_m + 2.5 Pi_m -> NAD_m + H2O + 2.5 ATP_m
54 FADH2_m + 0.5 O2 + 1.5 ADP_m + 1.5 Pi_m -> FAD_m + H2O + 1.5 ATP_m
4
Nucleotide Synthesis
55 R5P + ATP -> PRPP + AMP
56 PRPP + 2 GLN + GLY + ASP + 4 ATP + CO2 -> IMP + 2 GLU + FUM + 4 ADP + 2 H2O
57 IMP + ASP + 2 ATP + GTP -> dATP + FUM + 2 ADP + GDP
58 IMP + GLN + 3 ATP + NAD + 2 H2O -> dGTP + GLU + 2 ADP + AMP + NADH
59 HCO3 + NH4 + ASP + PRPP + 3 ATP + NAD -> dTTP + 3 ADP + CO2 + NADH
60 dTTP + GLN + ATP -> dCTP + GLU + ADP
5
Uptake or Production of Substrates (Measured Fluxes)
61 GLC6P + ADP + H -> GLC_ex + ATP
62 PYR + NADH + 2 H -> LAC_ex + NAD + H
63 GLN <-> GLN_ex
64 GLU <-> GLU_ex
65 ALA -> ALA_ex
66 SER -> SER_ex
67 GLY -> GLY_ex
68 ASP -> ASP_ex
69 ARG_m -> ARG_ex
70 HIS_m -> HIS_ex
71 CYS -> CYS_ex
72 THR -> THR_ex
73 ILE_m -> ILE_ex
74 VAL_m -> VAL_ex
75 PHE -> PHE_ex
76 TYR -> TYR_ex
77 LYS_m -> LYS_ex
78 LEU_m -> LEU_ex
79 PRO -> PRO_ex
80 MET -> MET_ex
81 ASN -> ASN_ex
0.0148 dATP + 0.0099 dCTP + 0.0099 dGTP + 0.0148 dTTP + 0.07 ATP + 0.033 dATP +
0.0551 dCTP + 0.0624 dGTP + 0.033 dTTP + 0.07 ATP + 0.6 ALA + 0.377 ARG_m + 0.359
ASP + 0.288 ASN + 0.145 CYS + 0.322 GLN + 0.386 GLU + 0.538 GLY + 0.143 HIS_m +
0.324 ILE_m + 0.564 LEU_m + 0.57 LYS_m + 0.138 MET + 0.219 PHE + 0.313 PRO + 0.43
SER + 0.386 THR + 0.044 TRP + 0.182 TYR + 0.416 VAL_m + 29.04 ATP + 0.279 GLC6P +
1.3148 ATP + 0.109 GAP + 1.9184 AcCoA + 1.70476 ATP + 3.40952 NADH + 3.51852 H +
82
0.109 NADH + 0.008 GAP + 0.0704 AcCoA + 0.06256 ATP + 0.12512 NADH + 0.13312 H +
0.008 NADH + 0.324 AcCoA + 0.324 ATP + 0.288 NADH + 0.288 H -> Biomass + 0.07 ADP +
0.07 Pi + 0.07 ADP + 0.07 Pi + 29.04 ADP + 29.04 Pi + 1.3148 ADP + 1.3148 Pi + 1.9184 CoA
+ 1.70476 ADP + 1.70476 Pi + 3.40952 NAD + 1.49112 H2O + 0.109 NAD + 0.0704 CoA +
0.06256 ADP + 0.06256 Pi + 0.12512 NAD + 0.05472 H2O + 0.008 NAD + 0.162 CoA + 0.324
ADP + 0.324 Pi + 0.288 NAD + 0.162 CO2
0.5768 ALA + 0.411919 ASN + 0.330176 ASP + 0.35901 GLN + 0.464684 GLU + 0.683611
GLY + 0.669275 PRO + 1.010565 SER + 0.198476 ARG_m + 0.232693 CYS + 0.204171
83 HIS_m + 0.30046 ILE_m + 0.574408 LEU_m + 0.54615 LYS_m + 0.114338 MET + 0.366898
PHE + 0.979228 THR + 0.19333 TRP + 0.251256 TYR + 0.796933 VAL_m + 39.89 ATP ->
MAB + 39.89 ADP + 39.89 Pi
6
Supplementary Figure 1. Flux balance model scheme. PEP regeneration pathway was only
active at pH 7.8 and therefore the fluxes OAAmPEPm and PEPmPEP were constrained under
all other conditions.
7
Supplementary Table 3. Measured specific consumption and production rates of amino acids
given in nmol/106 cells/h, and biomass and mAb given in µg/106 cells/h.
pH 7.2
Compound
Glc
Lac
Gln
Glu
Ala
Ser
Gly
Asp
Arg
His
Cys
Thr
Ile
Val
Phe
Tyr
Lys
Leu
Pro
Met
Asn
Biomass
mAb
WD 1.0
WD 1.5
-271.2 (±79.0) -162.1 (±27.0)
266.7 (±144.4) 35.3 (±23.8)
-39.2 (±7.6)
-75 (±17.1)
4.1 (±1.4)
8.1 (±4.7)
6.9 (±1.8)
13.2 (±0. 8)
-12.3 (±3.8)
-18.7 (±5.3)
-0.9 (±1.1)
0.4 (±2.3)
-0.5 (±0.1)
-0.7 (±0.1)
-4.1 (±1.9)
-5.7 (±2.6)
-1.9 (±0.8)
-2.8 (±1.1)
-1.3 (±0.5)
-2.4 (±1.8)
-4.9 (±1.4)
-7.6 (±0.1)
-9.3 (±4.1)
-10.3 (±2.9)
-7.8 (±2.6)
-9.3 (±1.5)
-2.8 (±1.1)
-3.7 (±1.5)
-2.4 (±0.9)
-4.2 (±1.2)
-5.2 (±0.7)
-8.7 (±1.2)
-9.2 (±3.8)
-11.8 (±4.0)
-4.5 (±2.0)
-4.3 (±2.3)
-2.6 (±1.1)
-3.1 (±1.5)
-3.5 (±0.9)
-4.3 (±1.4)
10.7 (±2.3)
11.4 (±1.9)
1.2 (±0.1)
1.3 (±0.3)
pH 6.8
pH 7.8
WD 1.9
WD 1.9
WD 1.9
-87.7 (±19.1)
6.7 (±10.7)
-7.7 (±2.6)
3.3 (±0.8)
4.4 (±1.8)
-3.8 (±0.9)
-0.4 (±0.5)
-0.5 (±0.1)
-1.5 (±0.3)
-0.5 (±0.3)
-0.6 (±0.2)
-1.6 (±0.5)
-5.8 (±1.7)
-3.9 (±0.9)
-0.9 (±0.3)
-0.7 (±0.1)
-1.6 (±0.4)
-5.4 (±0.7)
-2.5 (±1.3)
-0.9 (±0.2)
-1.2 (±0.1)
6.3 (±0.9)
0.6 (±0.1)
-31.5 (±16.2)
-57.6 (±17.4)
-12.8 (±5.1)
1.4 (±0.7)
5.7 (±2.2)
-0.4 (±0.1)
-1.2 (±0.7)
-0.3 (±0.0)
-0.8 (±0.5)
-0.5 (±0.5)
-1.4 (±0.3)
-1.5 (±1.0)
-3.1 (±1.1)
-2.7 (±1.8)
-0.8 (±0.6)
-1.5 (±0.7)
-1.0 (±0.5)
-3.6 (±1.7)
-1.1 (±0.2)
-0.5 (±0.3)
-1.1 (±0.5)
3.3 (±1.2)
0.9 (±0.2)
-196.2 (±1.4)
408.7 (±51.1)
-12.2 (±6.8)
6.6 (±0.5)
6.6 (±3.3)
-5.2 (±0.3)
5.8 (±0.2)
-0.7 (±0.1)
-1.0 (±0.3)
-0.8 (±0.2)
-2.0 (±0.1)
-0.5 (±0.1)
-5.3 (±0.4)
-4.4 (±0.5)
-0.7 (±0.1)
-0.9 (±0.1)
-1.7 (±0.6)
-4.6 (±0.3)
-2.6 (±0.9)
-0.8 (±0.1)
-1.2 (±0.0)
0.9 (±0.9)
0.5 (±0.1)
8
Biomass composition
The biomass composition of murine hybridoma cells averaged in previous work by Altamirano et
al. (2001), Selvarasu et al. (2010) and Xie and Wang (1994) was obtained using the relative
compositions from Sheikh et al. (2005) (Supplementary Table 4). The mole of each cellular
component can be calculated from the biomass weight. Dry cell weight of CRL 1606 cells was
considered as 250 pg/cell.5
Furthermore, diphosphatidyglycerol was not taken into consideration because of its negligible
proportion in cellular composition. Therefore, protein, DNA/RNA, carbohydrate, cholesterol, and
phospholipids (phosphoglycerides and sphingomyelin) were included in the equations of biomass
formation.
In addition to biomass synthesis, monoclonal antibody (mAb) synthesis was also considered in
our network by utilizing the amino acids composition of IgG1 from Quek et al. (2010)
(Supplementary Table 5). The theoretical amount of 4.3 ATP per mol amino acid is taken into
account for protein synthesis.
Monte Carlo error treatment method
The reported 95% confidence interval was compared to the confidence obtained by a Monte
Carlo error treatment method, where the error of the measured fluxes was assumed normally
distributed and 10% of the measured flux, except for biomass and antibody (12%).7 From the
resulting distributions 10 000 samples were randomly picked and used for FBA. The fluxes
calculated from FBA also followed normal distribution except some fluxes having the constraint
of an irreversible reaction. In those cases either the upper or lower bound was set to zero based on
their distribution results and constraints.
9
Supplementary Table 4: Averaged dry cell composition (protein, carbohydrates, nucleotides,
lipids) of murine hybridoma cells derived from literature 4.
Metabolite
nmol /μg DW
0.6
ALA
ARG
ASP
ASN
CYS
GLN
GLU
GLY
HIS
ILE
Protein
LEU
LYS
MET
PHE
PRO
SER
THR
TRP
TYR
VAL
0.377
0.359
0.288
0.145
0.322
0.386
0.538
0.143
0.324
0.564
0.57
0.138
0.219
0.313
0.43
0.386
0.044
0.182
0.416
Metabolite
Carbohydrates Glycogen
Nucleotides
Lipids
dAMP
dCMP
dGMP
dTMP
AMP
CMP
GMP
UMP
Cholesterol
Phosphatidylcholine
Phosphatidylethanolamine
Phosphatidylinostitol
Phosphatidylserine
Phosphatidylglycerol
Diphosphatidylglycerol
Sphingomyelin
nmol/μg DW
0.279
0.0148
0.0099
0.0099
0.0148
0.033
0.0551
0.0624
0.033
0.018
0.069
0.026
0.01
0.003
0.001
0.003
0.008
10
Supplementary Table 5: Amino acid composition of IgG1 derived from an average IgG1
protein sequence 6.
molar mass-H2O
g/mol AA
ALA
71.08
ASN
114.1
ASP
115.09
GLN
128.13
GLU
129.12
GLY
57.05
PRO
97.12
SER
87.08
ARG
156.19
CYS
103.14
HIS
137.14
ILE
113.16
LEU
113.16
LYS
128.17
MET
131.19
PHE
147.18
THR
101.1
TRP
186.21
TYR
163.18
VAL
99.13
sum (nmol AA/μg mAB)
mass fraction
g AA/g mAB
0.041
0.047
0.038
0.046
0.06
0.039
0.065
0.088
0.031
0.024
0.028
0.034
0.065
0.07
0.015
0.054
0.099
0.036
0.041
0.079
nmol AA/μg mAB
0.577
0.412
0.33
0.359
0.465
0.684
0.669
1.0106
0.198
0.233
0.204
0.3
0.574
0.546
0.114
0.367
0.979
0.193
0.251
0.797
9.264
11
Supplementary Table 6: Comparison of experimentally determined flux values from a 13-C
tracer study8 and calculated fluxes using maximization of ATP as the objective function. The
metabolic network built for FBA is based on the literature8 together with oxidative
phosphorylation pathway (FADH2 -> 1.5 ATP and NADHm/NADH -> 2.5 ATP). Pentose
phosphate pathway was excluded from the model resulting in 40 intracellular metabolites and 63
reactions. 19 extracellular fluxes were used for input in the model and 39 intracellular fluxes
described commonly in both the literature and model were compared by the goodness-of-fit. Chisquare test was performed to obtain the minimized variance weighted sum of squared residuals8,9.
Reactions
G6P <-> F6P
F6P -> DHAP + GAP
DHAP <-> GAP
GAP <-> [3PG] + NADH + ATP
[3PG] <-> PEP
PEP + ATP -> PYR
PYR + NADH <-> LAC
PYR <-> PYRm
PYRm -> AcCoAm + CO2 + NADHm
AcCoAm + OAAm -> CITm
CITm <-> AKGm + CO2 + NADHm
AKGm -> SUCm + CO2 + NADHm
SUCm <-> FUMm
FUMm <-> MALm + FADH2
MALm <-> OAAm + NADHm
MALm <-> PYRm + CO2 + NADHm
PYRm + CO2 + ATP -> OAAm
MALm <-> MAL
MAL <-> OAA + NADH
CITm <-> CIT
CIT + ATP -> AcCoA + OAA
AcCoA + 0.899 ATP -> FA
FA <-> FAm
FAm -> AcCoAm + 0.899 ATP
ALA <-> PYR
SER -> GLY + Cl
SER -> PYR
AKGm <-> GLU
GLU -> PRO
GLN -> GLU
ASP <-> OAAm
13
Calculated
C
95%
fluxes
experimental
confidence
(max ATP)
values
interval
204.9
203.2
168.4 236.4
204.9
203.2
168.6 237.2
204.9
202.8
168.2 237.2
409.8
406
338.1
475
409.8
406
338.1
475
409.8
406
338.1
475
289.8
289.8
240.4 347.5
113.5
109.7
36.9 182.1
142.04
138.3
67.9 207.2
158.2
154.4
81.7 248.2
158.2
154.4
80.6 238.4
182.3
178.6
106.3 259.3
189.1
185.3
111.8 270.1
191.2
187.4
113.8 266.5
162.6
133.9
74.3 213.1
28.6
53.5
32.5
84.8
0
24.9
7.1
56.5
-0.0338
0
-77
0
-0.0338
0
-77
0
0.0338
0
0
77
0.0338
0
0
77
1.6353
1.6
0.9
82.1
0
0
0
41.1
0
0
0
41.1
-6.6
-6.6
-7.3
-6.1
5.1
5.2
4.5
6.1
0.04
0
0
0.8
-24.1
-24.2
-30.5 -16.9
2.1
2.1
1.9
2.4
33.1
33.8
26.9
40.5
-4.5
-4.4
-5.1
-4.1
12
ASP -> ASN
2.2
THR -> AcCoA + GLY
1.6
MET + CO2 -> SUCm + CO2 + Cl + NADHm
0.7
VAL + CO2 -> SUCm + 2 CO2 + 3 NADHm +
2.2
FADH2
ILE + CO2 -> SUCm + AcCoAm + CO2 + 2
4.0
NADHm + FADH2
PHE -> FUMm + 2 AcCoAm + CO2
1.3
TYR -> FUMm + 2 AcCoAm + CO2
0.8
LEU + CO2 -> 3 AcCoAm + CO2 + NADHm +
2.7
FADH2
χ20.025 = 0.48 < h= 8.6 < χ20.975 = 9.5 (DF=4)
2.2
1.6
1.6
2
0.9
0.9
2.5
2.1
2.1
2.1
1
2.8
4
2.7
4.8
1.2
0.8
0.6
0.4
1.6
1.1
2.7
1.1
3.6
13
Supplementary Figure 2. Glutamine profile during standard batch cultivation. Glutamine
depletion was measured after 2.1 days of batch culture.
14
Supplementary Figure 3. Intracellular fluxes calculated by flux balance analysis at three
different time points (WD 1.0, WD 1.5, WD 1.9) for standard pH 7.2 culture. Numbers
correspond to reactions specified below.
15
Supplementary Figure 4. Comparison between intracellular fluxes at culture pH 7.2 and pH 6.8
calculated by flux balance analysis at WD 1.9. Numbers correspond to reactions specified below.
16
Supplementary Figure 5. Comparison between intracellular fluxes at culture pH 7.2 and pH 7.8
calculated by flux balance analysis at WD 1.9. Numbers correspond to reactions specified below.
Reactions 48-49 included to metabolic network as additional constraints according to gene
expression data.
17
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