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Supplementary Materials for
Systems-level analysis of mechanisms regulating yeast metabolic flux Sean R. Hackett, Vito R. T. Zanotelli, Wenxin Xu, Jonathan Goya, Junyoung O. Park,
David H. Perlman, Patrick A. Gibney, David Botstein, John D. Storey, Joshua D. Rabinowitz*
*Corresponding author. Email: [email protected]
Published 28 October 2016, Science 354, aaf2786 (2016)
DOI: 10.1126/science.aaf2786
This PDF file includes:
Figs. S1 to S15 Tables S1 to S6 Captions for Tables S7 to S9 References
Other Supplementary Material for this manuscript includes the following: (available at www.sciencemag.org/content/354/6311/aaf2786/suppl/DC1)
Tables S7 to S9
Supplemental Tables
Pathway Rea
ctio
nsw
ithm
easu
red
enzy
mes
Tota
lpat
hway
reac
tions
Frac
tion
ofre
actio
nsw
ithm
easu
red
enzy
mes
Mea
sure
den
zym
es
Tota
lenz
ymes
Frac
tion
ofm
easu
red
enzy
mes
Glycolysis / Gluconeogenesis 16 16 100% 17 18 94%Citrate cycle (TCA cycle) 12 12 100% 12 12 100%Biosynthesis of amino acids 73 83 88% 63 75 84%Purine metabolism 37 52 71% 28 41 68%Pyrimidine metabolism 27 39 69% 22 31 71%All reactions 346 524 66% 304 510 60%
Table S1: Proteomic coverage of enzymes in major metabolic pathways. For each pathway, weshow both the fraction of reactions for which at least one isozyme was measured, as well as thetotal fraction of enzymes measured. Pathway annotations are based on KEGG.
58
#ye
ast
anno
tatio
nsin
BR
EN
DA
#no
n-ye
ast
anno
tatio
nsin
BR
EN
DA
Rea
ctio
nsR
egul
ator
Cla
ss
Ace
tola
ctat
eSy
ntha
se(+
)AT
P1
0G
old
stan
dard
Asp
arta
teK
inas
e(-
)Thr
eoni
ne(u
ltras
ensi
tive)
734
Gol
dst
anda
rdG
luta
mat
e5-
Kin
ase
(-)P
rolin
e1
23G
old
stan
dard
Hom
ocitr
ate
Synt
hase
(-)L
ysin
e8
18G
old
stan
dard
PRPP
Am
idot
rans
fera
se(-
)AM
P1
20G
old
stan
dard
Ade
nyla
teK
inas
e(-
)AM
P1
15Pr
evio
usly
valid
ated
inye
ast
Cys
tath
ioni
neB
eta-
Synt
hase
(+)S
AM
220
Prev
ious
lyva
lidat
edin
yeas
tG
uany
late
Kin
ase
(-)G
MP
52
Prev
ious
lyva
lidat
edin
yeas
tTr
ehal
ose-
phos
phat
eSy
ntha
se(-
)Ino
rgan
icPh
osph
ate
44
Prev
ious
lyva
lidat
edin
yeas
tO
rnith
ine
Car
bam
oyltr
ansf
eras
e(-
)Ala
nine
04
Bio
chem
ical
lyva
lidat
edPy
ruva
teD
ecar
boxy
lase
(-)P
heny
lpyr
uvat
e0
2B
ioch
emic
ally
valid
ated
Pyru
vate
Kin
ase
(-)I
soci
trat
e0
7B
ioch
emic
ally
valid
ated
(citr
ate)
Ade
nyla
teK
inas
e(+
)Thr
eoni
ne0
1N
otte
sted
Ald
olas
e(-
)AM
P0
7N
otte
sted
Asp
arta
teC
arba
moy
ltran
sfer
ase
(+)U
TP
02
Not
test
edC
itrat
eto
Cis
-aco
nita
te(-
)Iso
citr
ate
02
Not
test
edC
ysta
thio
nine
Gam
ma-
lyas
e(-
)Ala
nine
02
Not
test
edFu
mar
ate
(-)C
itrat
e0
3N
otte
sted
Glu
tam
ate
Deh
ydro
gena
se(N
AD
P)(-
)Qui
nolin
ate
04
Not
test
edH
omoc
itrat
eSy
ntha
se(-
)Qui
nolin
ate
10
Not
test
edM
alic
Enz
yme
(NA
D)
(-)A
MP
04
Not
test
edPh
osph
ofru
ctok
inas
e(+
)Fru
ctos
e1,
6-bi
spho
spha
te0
8N
otte
sted
Phos
phog
lyce
rate
Deh
ydro
gena
se(+
)Met
hion
ine
01
Not
test
edPh
osph
ogly
cera
teM
utas
e(-
)3-P
hosp
hogl
ycer
ate
12
Not
test
edS7
PPh
osph
ofru
ctok
inas
e(-
)Pho
spho
enol
pyru
vate
029
Not
test
edTr
ehal
ose-
phos
phat
ase
(+)F
6P0
1N
otte
sted
Treh
alos
e-ph
osph
atas
e(+
)Tre
halo
se0
1N
otte
sted
UT
PG
1PU
ridy
ltran
sfer
ase
(-)U
DP-
Glu
cose
011
Not
test
edA
TP-
Phos
phor
ibos
yltr
ansf
eras
e(-
)AM
P0
4B
ioch
emic
ally
inva
lidat
edD
AH
PSy
ntha
se(-
)PE
P0
2B
ioch
emic
ally
inva
lidat
edD
AH
PSy
ntha
se(-
)Phe
nylp
yruv
ate
03
Bio
chem
ical
lyin
valid
ated
Glu
cose
6-ph
osph
ate
Deh
ydro
gena
se(-
)AM
P0
3B
ioch
emic
ally
inva
lidat
edG
luco
se6-
phos
phat
eD
ehyd
roge
nase
(-)P
EP
03
Bio
chem
ical
lyin
valid
ated
Phos
phog
luco
nate
Deh
ydro
gena
se(+
)Asp
arta
te0
1B
ioch
emic
ally
inva
lidat
edPy
ruva
teK
inas
e(-
)AM
P0
7B
ioch
emic
ally
inva
lidat
ed
Tabl
eS2
:B
est-
supp
orte
dpr
edic
tions
ofph
ysio
logi
cal
regu
lato
rsof
yeas
tm
etab
olis
mba
sed
onSI
MM
ER
(N=
35).
The
12re
gula
tors
liste
dab
ove
the
line
have
been
bioc
hem
ical
lyva
lidat
edin
S.ce
revi
siae
.
59
1-by-1 testing of whether each metabolite as a regulator improves regulation-free model(based on fit only, no Bayesian analysis)
Does not improve Significantly improves(q > 0.1) (q < 0.1)
Gold standard 10 10BRENDA (non GS) 591 118Other metabolites 8509 2007
Top predicted regulator for each reaction based on 1-by-1 testing of all metabolites (basedon fit only, no Bayesian analysis)
Not top TopGold standard 20 0BRENDA (non GS) 707 2Other metabolites 10463 53No regulation 55 1
Top predicted regulator based on 1-by-1 testing of putative regulators in BRENDA (basedon fit only, no Bayesian analysis)
Not top TopGold standard 17 3BRENDA (non GS) 676 33No regulation 36 20
Top predicted and other supported regulators for each reaction based on Bayesian analysisand allowing for pairwise regulation and cooperativity
Not supported Top Other supported All supported(AICc better than no regulation)
Gold standard 10 5 5 10BRENDA (non GS) 662 30 17 47
Table S3: Effectiveness of SIMMER, with and without Bayesian analysis, in recapitulating goldstandard regulation. Bayesian analysis winnows down an unmanageable number of significantpredictions (2135) to a manageable number (57), while capturing an equal fraction of gold standardregulation.
60
Rea
ctio
nN
ame
Abb
revi
atio
nB
estS
uppo
rted
Reg
ulat
ion
R2
Alte
rnat
ive
Reg
ulat
ors
#R
egul
ator
sTe
sted
1,3-
beta
-glu
can
synt
hase
beta
-GS
none
0.52
83-
deox
y-D
-ara
bino
-he
ptul
oson
ate
7-ph
osph
ate
synt
heta
seD
AH
PSy
nth
(-)P
heny
lpyr
uvat
e&
(-)
Phos
phoe
nolp
yruv
ate
0.61
(-)D
-Ery
thro
se4-
phos
phat
e,(-
)L
-Phe
nyla
lani
ne,(
-)Pr
ephe
nate
,(-)
L-T
yros
ine,
(-)L
-Try
ptop
han
11
Ace
tola
ctat
esy
ntha
seA
LS
(+)A
TP
0.61
6
Ade
nyla
teki
nase
AD
K(-
)AM
P&
(+)L
-Thr
eoni
ne0.
85(+
)dA
TP,
(+)L
-His
tidin
e,(-
)Ph
osph
oeno
lpyr
uvat
e18
Arg
inin
osuc
cina
tely
ase
ASL
none
0.38
9A
rgin
inos
ucci
nate
synt
hase
ASS
none
0.64
3A
spar
agin
esy
ntha
seA
snSy
nth
none
0.42
5A
spar
tate
carb
amoy
ltran
sfer
ase
AT
Cas
e(+
)UT
P0.
5(+
)AT
P33
Asp
arta
teki
nase
Asp
K(-
)Thr
eoni
ne(u
ltras
ensi
tive)
0.77
(-)L
-Lys
ine,
(-)L
-Hom
oser
ine
18A
spar
tate
tran
sam
inas
eA
STno
ne0.
4212
AT
Pph
osph
orib
osyl
tran
sfer
ase
AT
P-PR
Tase
(-)A
MP
0.85
(-)L
-His
tidin
e4
Car
bam
oyl-
phos
phat
esy
ntha
seC
PSno
ne0.
6733
Aco
nita
seA
CO
(-)I
soci
trat
e0.
629
CT
Psy
ntha
se(N
H3)
CT
PSy
nth
none
0.45
7C
ysta
thio
nine
beta
-syn
thas
eC
BS
(+)S
-Ade
nosy
l-L
-met
hion
ine
0.62
5C
ysta
thio
nine
g-ly
ase
CB
L(-
)L-A
lani
ne0.
466
Fruc
tose
-bis
phos
phat
eal
dola
seA
LD
(-)A
MP
0.74
(-)A
DP
31Fu
mar
ase
FUM
(-)C
itrat
e0.
588
Glu
cose
6-ph
osph
ate
dehy
drog
enas
eG
6PD
(-)P
hosp
hoen
olpy
ruva
te&
(-)
AM
P0.
5414
Glu
tam
ate
5-ki
nase
G5K
(-)L
-Pro
line
0.62
4G
luta
mat
ede
hydr
ogen
ase
(NA
DP)
GL
DH
(-)Q
uino
linat
e0.
7649
Gly
cera
ldeh
yde-
3-ph
osph
ate
dehy
drog
enas
eG
APD
Hno
ne0.
7419
Gly
cero
l-3-
phos
phat
ede
hydr
ogen
ase
(NA
D)
G3P
DH
none
0.83
15
GM
Psy
ntha
seG
MP
Synt
hno
ne0.
923
Gua
nyla
teki
nase
GU
K(-
)GM
P0.
53
His
tidin
olde
hydr
ogen
ase
HD
Hno
ne0.
440
Hom
ocitr
ate
synt
hase
HC
S(-
)Qui
nolin
ate
&(-
)L-L
ysin
e0.
66(+
)dA
TP,
(-)L
-Arg
inin
e9
Mal
icen
zym
e(N
AD
)M
E(-
)AM
P0.
5932
Orn
ithin
eca
rbam
oyltr
ansf
eras
eO
TC
ase
(-)L
-Ala
nine
0.71
27O
rota
teph
osph
orib
osyl
tran
sfer
ase
OPR
Tase
none
0.72
16
Oro
tidin
e-5’
-pho
spha
tede
carb
oxyl
ase
OD
Cas
eno
ne0.
516
61
Rea
ctio
nN
ame
Abb
revi
atio
nB
estS
uppo
rted
Reg
ulat
ion
R2
Oth
erSu
ppor
ted
Reg
ulat
ion
#R
egul
ator
sTe
sted
Phos
phof
ruct
okin
ase
PFK
(+)D
-Fru
ctos
e1,
6-bi
spho
spha
te0.
59(-
)AM
P,(-
)Iso
citr
ate
27
Phos
phof
ruct
okin
ase
(S7P
)S7
PPF
K(-
)Pho
spho
enol
pyru
vate
0.5
27Ph
osph
oglu
cona
tede
hydr
ogen
ase
6PG
D(+
)L-A
spar
tate
0.62
18
Phos
phog
lyce
rate
dehy
drog
enas
ePH
GD
H(+
)L-M
ethi
onin
e0.
4910
Phos
phog
lyce
rate
kina
sePG
Kno
ne0.
719
Phos
phog
lyce
rate
mut
ase
PGM
(-)3
-Pho
spho
glyc
erat
e0.
534
Phos
phor
ibos
ylpy
roph
osph
ate
amid
otra
nsfe
rase
PPA
T(-
)AM
P0.
679
Phos
phor
ibos
ylpy
roph
osph
ate
synt
heta
sePR
PPSy
nth
none
0.9
20
Pyru
vate
deca
rbox
ylas
ePD
C(-
)Phe
nylp
yruv
ate
0.55
4
Pyru
vate
kina
sePy
K(-
)Iso
citr
ate
&(-
)AM
P0.
96(-
)Citr
ate,
(+)D
ihyd
roxy
acet
one
phos
phat
e,(+
)D-F
ruct
ose
1,6-
bisp
hosp
hate
,(+)
Rib
ose-
5-ph
osph
ate
44
Tran
sket
olas
e1
TK
T-1
none
0.59
3
Treh
alos
e-ph
osph
atas
eT
RE
P(+
)Fru
ctos
e6-
phos
phat
e&
(+)
Treh
alos
e0.
97
Treh
alos
e-ph
osph
ate
synt
hase
TPS
(-)P
hosp
hate
0.77
4Tr
iose
-pho
spha
teis
omer
ase
TPI
none
0.66
5U
TP-
gluc
ose-
1-ph
osph
ate
urid
ylyl
tran
sfer
ase
UT
PG
UT
(-)U
DP-
D-g
luco
se0.
66(-
)D-G
luco
se1-
phos
phat
e8
Tabl
eS4
:B
ests
uppo
rted
regu
latio
n(i
fan
y)fo
r46
reac
tions
whe
reth
efit
isac
cept
able
(R2>
0.35
);(+
)A
ctiv
ator
,(-)
Inhi
bito
r.B
est-
supp
orte
dre
gula
tion
isba
sed
onB
ayes
ian
anal
ysis
.Alte
rnat
ive
regu
lato
rsth
atar
eal
sost
atis
tical
lysu
ppor
ted
over
regu
latio
n-fr
eeki
netic
s(l
ower
AIC
cth
anno
regu
latio
n)ar
esh
own.
62
Reaction Name Regulation Support3-phosphoglycerate dehydrogenase (-) Serine Alternative regulation
Acetolactate synthase(+) ATP Strongest support(-) Valine Alternative regulation
Acetylglutamate kinase (-) Arginine No tested model is adequateAsparagine synthetase (-) Asparagine No regulation identifiedAspartate carbamoyltransferase (-) UTP Alternative regulation
Aspartokinase(-) Homoserine Supported(-) Threonine Strongest support
ATP phosphoribosyltransferase (-) Histidine SupportedCarbamoyl phosphate synthase (-) UTP No regulation identifiedCTP synthase (-) CTP No regulation identified
DAHP synthase(-) Phenylalanine Supported(-) Tyrosine Supported
Glutamate 5-kinase (-) Proline Strongest supportHomocitrate synthase (-) Lysine Strongest supportHomoserine kinase (-) Threonine No tested model is adequate
Phosphofructokinase(+) AMP Alternative regulation(-) ATP Alternative regulation(+) Fructose 2,6-P2 Not measured
PRPP amidotransferase (-) AMP Strongest supportPyruvate kinase (+) Fructose 1,6-P2 Supported
Table S5: Gold standard yeast metabolic regulation. 21 instances of gold standard metabolicregulation, involving 16 reactions, were manually obtained from Jones & Fink 1982, Sekine etal. 2007 and Fraenkel 2011 (33–35). Metabolite concentration data were available to evaluate20 of the 21 instances. “Strongest support” indicates unsupervised fitting of the cellular dataidentified the gold standard regulation as the best regulator. “Supported” indicates that the goldstandard regulator is better supported than an unregulated model, but less supported than anotherregulator. “No regulation identified” indicates that no tested regulator was better supported thanthe unregulated model. “Alternative regulation supported” indicates that the tested mechanism wasnot supported but other regulation is supported. “No tested model is adequate” indicates that noneof the tested regulators predicted measured flux well (R2 < 0.35 for all regulators).
63
Tested regulator is gold standard orbiochemically confirmed SIMMERprediction
Regulator is best-supportedphysiological regulator perSIMMER
True FalseTrue 12 23False 15 679
TPR, TP/(TP+FN): 0.44FPR, FP/(FP+TN): 0.03
Table S6: Comparison of the best-supported allosteric regulators per SIMMER with truephysiological regulators (as defined by gold standard + biochemically confirmed SIMMERpredictions). Conservatively, we assume that any untested predictions are false, thus true positivesmay be under-reported. Similarly, false negatives may be under-reported as some unpredictedregulation that is not included in the gold standard may nevertheless be physiologically important.
64
Table S7: Summary of 729 literature-reported activators and inhibitors (across all organisms) ofthe 56 studied reactions. The R2 between measured flux and the regulators kinetic model is listed.The AICc of each regulator is listed if this regulator significantly improves upon the fit based on1-by-1 testing without Bayesian analysis (q < 0.1). Note that higher R2 and lower AICc indicate abetter fit.
Table S8: Metabolic leverage for each of the 28 studied metabolic reactions that was adequatelyfit (R2 > 0.35) with the best-supported reaction equation either involving no regulation orbiochemically validated regulation. Best estimate reflects the metabolic leverage determined usingthe best estimate of each reactions kinetic parameters. To determine the robustness of the metabolicleverage measurements, these were repeated using 2000 different well-fitting kinetic parametersets. The interquartile range (IQR) and 95% credibility interval of these samples is reported.
Table S9: Experimentally measured metabolite and protein abundances and boundary fluxes.
65
Supplemental Figures
Uptake
ExcretionBiomass production
Slow Fasth
DR-1
Flux
(mm
oles
/ ho
ur /
ml c
ells)
Glucose
0.0
2.5
5.0
7.5
10.0
12.5
Ethanol
Glycerol
Acetate
Acetaldehyde
Lactate
Succinate
Orotate
05
101520
0.00
0.25
0.50
0.75
1.00
0.00
0.05
0.10
0.15
0.20
0.00
0.02
0.04
0.06
0.00
0.02
0.04
0.06
0.00
0.02
0.04
0.06
0.08
0.000.010.020.030.040.05
Amino Acids
Carbohydrates
Soluble metabolites
Polyphosphates
Nucleic Acids
Fatty Acids
0.00
0.05
0.10
0.15
0.20
0.00
0.05
0.10
0.15
0.0000.0250.0500.0750.1000.125
0.00
0.02
0.04
0.000
0.005
0.010
0.015
0.002
0.004
0.006
0.008
SMetabolism
P C N L UP C N L U
Figure S1: Experimentally determined rates of metabolite uptake, excretion and incorporation intobiomass for each chemostat condition. Fluxes are expressed per ml of cells based on packed cellvolume.
66
Pentose phosphatepathway
TCA cycle
Glycolysis
Carbohydratesynthesis
Amino acidsynthesis
RNA synthesis
P C N L USlow Fasth
DR-1
Rela
tive
Flux
Auxotrophyrelated
Fatty acidsynthesis
-3.0 -2.0 -1.0 0.0 1.0 2.0 3.0 4.0
-4.0
Figure S2: Fluxes through 233 reactions across 25 chemostats. Absolute flux in linear space wasnormalized on a per reaction basis through division by the median flux across conditions. Reactionswere hierarchically clustered using Pearson correlation.
67
Current Study (0.11 h−1)
CS
ENO
GAPDH
GLUT
GPI
HXK
IDH
OGDC
oxPPP
PCO
PDC
PDHPFK/ALD
PyK
SDH/FUM
TKK2●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
●
0.0
0.5
1.0
Jouhten et al. 2008 (0.10 h−1)
0.0 0.5 1.0 1.5Current Study (0.30 h−1)
ACS
CS
ENO
GAPDH
GLUT
ME
PCO
PDC
PDH
PFK/ALD
PyK
TKK1TKK2
TPI
●
●
●●
●
●
●●
●
●
●
●
●●
●
0.0
0.5
1.0
1.5 Frick and Wittm
an 2005 (0.30 h−1)
0.0 0.5 1.0 1.5
R2 = 0.93
R2 = 0.89
OGDC● oxPPP
TKK1
ACS
ME●
PGK ●●
●
Relative �ux in current study
Lite
ratu
re re
port
ed re
lativ
e �u
x
Figure S3: Measured fluxes based on flux balance analysis constrained by uptake, biomassproduction, and excretion data agree with previous 13C-based metabolic flux analysis. Fluxesthrough carbon-limited yeast chemostats reported in Frick and Wittman 2005 (DR = 0.3 h−1) using100% 1-13C-labeled glucose and in Jouhten et al. 2008 (DR = 0.1 h-1) using 10% U-13C-labeledglucose were compared to the carbon-limited chemostat with the most similar dilution rate fromthis study. In each case, the indicated flux is the average of the lower and upper confidence intervalof each reaction, normalized to glucose uptake rate.
68
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ACS
CS
ENO
GAPDH
GLUT
ME
OGDCoxPPP
PCO
PDC
PDH
PFK/ALD
PyK
TKK1
TKK2
TPI
ACS
CS
ENO GAPDH
GLUT
ME
OGDC
oxPPP
PCO
PDC
PDH
PFK/ALD
PyK
TKK1
TKK2
0.0
0.5
1.0
0.0 0.5 1.0 1.5Relative flux in current study and Jouhten et al. 2008
Rel
ative
flux
in F
rick
and
Witt
man
200
5 (0
.15
h-1) Current Study (0.16 h−1): R2 = 0.64
Jouhten et al. 2008 (0.10 h−1): R2 = 0.65
Figure S4: Comparison of fluxes in carbon limitation measured here by experimentally-constrainedflux balance analysis compared to two different 13C-based metabolic flux analysis approaches.Frick and Wittman 2005 report rapid acetyl-CoA production from acetate (and accordingly lowerPDH and higher ACS and PDC fluxes) than our measurements or those of Jouhten et al. 2008.Other fluxes generally agree.
69
Nucleotides
TCA cycle
Glycolysis
Ura3- related
Amino acids
P C N L USlow Fasth
DR-1
Rela
tive
met
abol
ite c
once
ntra
tions
(log
2) dGDP carbamoyl phosphate proline glutamate dCTP dTTP UDP-N-acetyl-glucosamine CTP UTP UDP-D-glucose PRPP 3-phospho-D-glycerate D-sedoheptulose-7-phosphate ATP NAD+ 6-phospho-D-gluconate ribose-phosphate GTP hexose-phosphate dATP 1,3-bisphospho-D-glycerate uridine deoxyadenosine GMP guanine hypoxanthine guanosine xanthine N-acetyl-glutamate D-gluconate riboflavin glycerate N-acetyl-glutamine deoxyguanosine cytosine cytidine inosine adenosine isocitrate citrate/isocitrate citrate a-ketoglutarate malate fumarate hydroxyphenylpyruvate trans,trans-farnesyl-diphosphate phenylpyruvate trehalose-6-phosphate trehalose/sucrose glutathione disulfide glutathione AMP D-glyceraldehyde-3-phosphate dihydroxyacetone-phosphate fructose-1,6-bisphosphate pyridoxine N-acetyl-glucosamine-1-phosphate pyruvate D-glucono-1,5-lactone-6-phosphate lactate glycine CDP dimethylglycine choline S-adenosyl-L-methionine homoserine cystathionine prephenate tyrosine tryptophan phenylalanine ornithine lysine threonine histidine arginine alanine valine leucine/isoleucine methionine NADH histidinol aspartate serine glutamine citrulline asparagine NADP+ argininosuccinate ADP D-erythrose-4-phosphate acetyl-CoA orotidine-phosphate N-carbamoyl-L-aspartate orotate dihydroorotate FMN succinate thiamine quinolinate aconitate cyclic AMP FAD 3-hydroxy-3-methylglutaryl-CoA nicotinate pantothenate
-3.0
-2.0
-1.0
0.0
1.0
2.0
3.0
Figure S5: Concentrations of 106 metabolites across 25 chemostats. Metabolite relativeabundances were centered by row mean and hierarchically clustered using Pearson correlation.
70
−4
−2
0
2
4
−4 −2 0 2 4log2 (n0.05/ 15 N−p0.05) R1
log 2
(n0.
05/ 15
N−p
0.05
) R2
25 50 75 100Counts
R2 = 0.74
Figure S6: Reproducibility of nutrient-induced changes in peptide abundance. As a representativeexample, a slow growth nitrogen-limited chemostat (n0.05) is compared to an internal referenceslow-growth phosphorous-limited 15N-labelled chemostat (15N-p0.05). For each of two technicalreplicates derived from independent digestion, fractionation and mass spectrometry analysis of asingle biological sample, the relative abundance of unlabelled experimental peptides relative to15N-labelled reference peptides is shown.
71
P C N L USlow Fasth
DR-1
Peptidases, amino acidpermeases
Mitochondrial& Electron
Transport Chain
Amino acidsynthesis
Ribosomal
Phosphatases
p0.
05
p0.
11
p0.
16
p0.
22
p0.
30
gap
c0.
05
c0.
11
c0.
16
c0.
22
c0.
30
gap
.1
n0.
05
n0.
11
n0.
16
n0.
22
n0.
30
gap
.2
L0.
05
L0.
11
L0.
16
L0.
22
L0.
30
gap
.3
u0.
05
u0.
11
u0.
16
u0.
22
u0.
30
GENE43X GENE42X GENE318X GENE242X GENE418X GENE1033X GENE238X GENE285X GENE981X GENE33X GENE365X GENE199X GENE195X GENE573X GENE249X GENE674X GENE751X GENE821X GENE478X GENE886X GENE246X GENE151X GENE321X GENE466X GENE239X GENE640X GENE516X GENE447X GENE364X GENE641X GENE14X GENE1072X GENE178X GENE615X GENE588X GENE767X GENE686X GENE671X GENE216X GENE499X GENE1149X GENE1075X GENE23X GENE644X GENE351X GENE790X GENE49X GENE903X GENE186X GENE32X GENE513X GENE346X GENE122X GENE366X GENE296X GENE720X GENE488X GENE1059X GENE1087X GENE668X GENE1056X GENE360X GENE692X GENE883X GENE979X GENE63X GENE748X GENE103X GENE960X GENE395X GENE600X GENE87X GENE387X GENE811X GENE487X GENE552X GENE620X GENE762X GENE902X GENE1017X GENE363X GENE770X GENE159X GENE971X GENE1023X GENE451X GENE830X GENE682X GENE924X GENE104X GENE241X GENE514X GENE548X GENE555X GENE1045X GENE61X GENE527X GENE110X GENE476X GENE530X GENE205X GENE444X GENE831X GENE45X GENE606X GENE683X GENE1109X GENE175X GENE399X GENE152X GENE687X GENE768X GENE702X GENE796X GENE907X GENE155X GENE304X GENE782X GENE179X GENE426X GENE526X GENE97X GENE244X GENE921X GENE12X GENE278X GENE913X GENE462X GENE13X GENE832X GENE994X GENE105X GENE18X GENE1122X GENE89X GENE691X GENE1042X GENE1018X GENE398X GENE333X GENE666X GENE114X GENE113X GENE408X GENE352X GENE368X GENE274X GENE220X GENE27X GENE372X GENE219X GENE717X GENE240X GENE0X GENE142X GENE524X GENE905X GENE171X GENE725X GENE338X GENE275X GENE281X GENE129X GENE210X GENE48X GENE630X GENE235X GENE651X GENE31X GENE40X GENE638X GENE1153X GENE564X GENE663X GENE509X GENE405X GENE824X GENE1006X GENE1085X GENE660X GENE675X GENE769X GENE621X GENE1001X GENE1032X GENE24X GENE349X GENE959X GENE817X GENE1117X GENE144X GENE468X GENE859X GENE472X GENE116X GENE861X GENE570X GENE1053X GENE1071X GENE1019X GENE221X GENE1119X GENE117X GENE694X GENE906X GENE778X GENE802X GENE891X GENE457X GENE603X GENE628X GENE367X GENE904X GENE894X GENE491X GENE211X GENE108X GENE464X GENE215X GENE41X GENE764X GENE52X GENE383X GENE473X GENE745X GENE517X GENE715X GENE2X GENE36X GENE714X GENE330X GENE53X GENE882X GENE1140X GENE124X GENE812X GENE847X GENE920X GENE292X GENE995X GENE677X GENE276X GENE806X GENE391X GENE91X GENE592X GENE705X GENE44X GENE708X GENE348X GENE109X GENE567X GENE676X GENE672X GENE494X GENE71X GENE201X GENE544X GENE822X GENE154X GENE761X GENE851X GENE34X GENE1145X GENE153X GENE191X GENE273X GENE86X GENE1028X GENE1097X GENE938X GENE1116X GENE69X GENE862X GENE177X GENE593X GENE435X GENE863X GENE829X GENE613X GENE655X GENE932X GENE1082X GENE125X GENE927X GENE865X GENE1151X GENE181X GENE1014X GENE402X GENE525X GENE1106X GENE427X GENE685X GENE631X GENE710X GENE25X GENE634X GENE59X GENE1147X GENE605X GENE680X GENE11X GENE99X GENE520X GENE693X GENE532X GENE646X GENE577X GENE1049X GENE4X GENE354X GENE869X GENE966X GENE208X GENE952X GENE855X GENE284X GENE121X GENE808X GENE563X GENE218X GENE75X GENE673X GENE578X GENE899X GENE961X GENE576X GENE820X GENE547X GENE887X GENE443X GENE433X GENE836X GENE1073X GENE1144X GENE916X GENE697X GENE934X GENE407X GENE625X GENE776X GENE1054X GENE765X GENE956X GENE234X GENE459X GENE931X GENE167X GENE703X GENE386X GENE792X GENE1099X GENE481X GENE1150X GENE192X GENE226X GENE252X GENE622X GENE51X GENE250X GENE133X GENE756X GENE632X GENE884X GENE943X GENE848X GENE834X GENE940X GENE300X GENE781X GENE617X GENE835X GENE787X GENE174X GENE213X GENE506X GENE695X GENE753X GENE1102X GENE203X GENE439X GENE135X GENE939X GENE1081X GENE150X GENE165X GENE534X GENE362X GENE1039X GENE232X GENE559X GENE964X GENE77X GENE130X GENE482X GENE500X GENE571X GENE1047X GENE280X GENE604X GENE50X GENE656X GENE518X GENE1079X GENE90X GENE410X GENE436X GENE359X GENE394X GENE127X GENE295X GENE303X GENE412X GENE669X GENE1040X GENE1025X GENE259X GENE343X GENE9X GENE149X GENE879X GENE302X GENE1041X GENE431X GENE308X GENE323X GENE726X GENE217X GENE253X GENE317X GENE126X GENE67X GENE228X GENE385X GENE22X GENE332X GENE21X GENE225X GENE1163X GENE1155X GENE799X GENE970X GENE1092X GENE107X GENE948X GENE156X GENE554X GENE741X GENE1154X GENE545X GENE846X GENE15X GENE801X GENE60X GENE819X GENE890X GENE138X GENE803X GENE1105X GENE257X GENE629X GENE1134X GENE310X GENE452X GENE777X GENE441X GENE490X GENE200X GENE533X GENE1024X GENE128X GENE54X GENE454X GENE623X GENE993X GENE206X GENE949X GENE1000X GENE160X GENE455X GENE585X GENE826X GENE119X GENE279X GENE797X GENE357X GENE493X GENE168X GENE897X GENE115X GENE353X GENE1111X GENE946X GENE65X GENE933X GENE299X GENE502X GENE1048X GENE1004X GENE987X GENE260X GENE289X GENE679X GENE723X GENE1013X GENE416X GENE204X GENE335X GENE843X GENE659X GENE1043X GENE1101X GENE421X GENE1010X GENE355X GENE701X GENE1148X GENE1136X GENE1107X GENE336X GENE955X GENE958X GENE984X GENE541X GENE1022X GENE277X GENE414X GENE98X GENE146X GENE643X GENE64X GENE1132X GENE914X GENE784X GENE339X GENE214X GENE1074X GENE161X GENE929X GENE844X GENE164X GENE925X GENE262X GENE985X GENE413X GENE393X GENE419X GENE1020X GENE963X GENE118X GENE92X GENE270X GENE56X GENE607X GENE81X GENE5X GENE536X GENE804X GENE519X GENE306X GENE839X GENE147X GENE1034X GENE256X GENE591X GENE649X GENE85X GENE875X GENE667X GENE72X GENE504X GENE1062X GENE1129X GENE1128X GENE422X GENE290X GENE561X GENE1050X GENE794X GENE814X GENE854X GENE448X GENE901X GENE501X GENE699X GENE608X GENE1171X GENE16X GENE1141X GENE689X GENE1139X GENE132X GENE264X GENE314X GENE637X GENE180X GENE258X GENE440X GENE445X GENE434X GENE908X GENE754X GENE752X GENE449X GENE759X GENE134X GENE551X GENE361X GENE798X GENE162X GENE805X GENE969X GENE369X GENE716X GENE755X GENE850X GENE148X GENE893X GENE231X GENE896X GENE224X GENE999X GENE376X GENE579X GENE1069X GENE57X GENE371X GENE465X GENE866X GENE263X GENE590X GENE347X GENE612X GENE170X GENE1113X GENE272X GENE315X GENE522X GENE66X GENE1015X GENE1110X GENE568X GENE973X GENE1012X GENE996X GENE1091X GENE19X GENE531X GENE1037X GENE737X GENE818X GENE892X GENE140X GENE187X GENE197X GENE1100X GENE1009X GENE6X GENE586X GENE665X GENE460X GENE587X GENE301X GENE785X GENE329X GENE95X GENE558X GENE828X GENE379X GENE424X GENE297X GENE546X GENE245X GENE251X GENE370X GENE584X GENE601X GENE633X GENE80X GENE864X GENE1089X GENE690X GENE992X GENE867X GENE1114X GENE881X GENE101X GENE233X GENE873X GENE965X GENE311X GENE381X GENE474X GENE453X GENE1X GENE20X GENE538X GENE62X GENE267X GENE582X GENE598X GENE1077X GENE786X GENE838X GENE480X GENE967X GENE788X GENE505X GENE1090X GENE84X GENE652X GENE358X GENE316X GENE550X GENE120X GENE458X GENE196X GENE345X GENE78X GENE614X GENE475X GENE658X GENE678X GENE589X GENE528X GENE529X GENE184X GENE420X GENE508X GENE374X GENE1080X GENE657X GENE597X GENE565X GENE982X GENE1121X GENE28X GENE293X GENE173X GENE661X GENE1052X GENE471X GENE871X GENE403X GENE727X GENE738X GENE8X GENE877X GENE930X GENE1066X GENE736X GENE700X GENE766X GENE813X GENE912X GENE1096X GENE942X GENE1029X GENE1167X GENE182X GENE599X GENE1007X GENE535X GENE878X GENE719X GENE734X GENE467X GENE816X GENE469X GENE1008X GENE926X GENE1061X GENE209X GENE3X GENE642X GENE880X GENE265X GENE331X GENE483X GENE511X GENE1108X GENE635X GENE237X GENE417X GENE429X GENE337X GENE619X GENE953X GENE944X GENE540X GENE521X GENE758X GENE898X GENE923X GENE247X GENE757X GENE324X GENE553X GENE1133X GENE1064X GENE664X GENE713X GENE503X GENE735X GENE749X GENE287X GENE334X GENE1031X GENE542X GENE707X GENE709X GENE840X GENE35X GENE137X GENE1094X GENE74X GENE889X GENE704X GENE1078X GENE286X GENE823X GENE972X GENE1174X GENE227X GENE742X GENE390X GENE512X GENE139X GENE596X GENE662X GENE874X GENE539X GENE809X GENE791X GENE450X GENE572X GENE1065X GENE377X GENE1088X GENE111X GENE470X GENE1076X GENE485X GENE954X GENE998X GENE1057X GENE825X GENE950X GENE507X GENE523X GENE885X GENE136X GENE356X GENE415X GENE47X GENE654X GENE312X GENE96X GENE193X GENE342X GENE88X GENE344X GENE549X GENE94X GENE269X GENE322X GENE102X GENE212X GENE616X GENE853X GENE918X GENE325X GENE93X GENE688X GENE375X GENE537X GENE670X GENE1103X GENE243X GENE618X GENE849X GENE978X GENE176X GENE248X GENE320X GENE991X GENE733X GENE496X GENE636X GENE712X GENE650X GENE327X GENE37X GENE207X GENE442X GENE437X GENE497X GENE401X GENE574X GENE68X GENE722X GENE172X GENE106X GENE194X GENE141X GENE380X GENE123X GENE841X GENE158X GENE1060X GENE1030X GENE58X GENE461X GENE1126X GENE350X GENE288X GENE456X GENE313X GENE837X GENE294X GENE845X GENE653X GENE888X GENE411X GENE498X GENE477X GENE510X GENE740X GENE169X GENE1131X GENE698X GENE479X GENE562X GENE38X GENE595X GENE645X GENE560X GENE1142X GENE980X GENE977X GENE1003X GENE746X GENE945X GENE268X GENE1002X GENE772X GENE743X GENE611X GENE789X GENE1035X GENE706X GENE166X GENE326X GENE298X GENE1118X GENE1127X GENE222X GENE112X GENE1104X GENE1124X GENE29X GENE397X GENE229X GENE291X GENE917X GENE230X GENE858X GENE26X GENE340X GENE261X GENE223X GENE583X GENE721X GENE307X GENE378X GENE373X GENE1152X GENE76X GENE202X GENE990X GENE10X GENE189X GENE868X GENE404X GENE569X GENE1068X GENE1137X GENE957X GENE1051X GENE79X GENE937X GENE581X GENE627X GENE1083X GENE100X GENE495X GENE515X GENE430X GENE271X GENE747X GENE900X GENE7X GENE624X GENE55X GENE73X GENE860X GENE131X GENE1058X GENE438X GENE750X GENE951X GENE305X GENE744X GENE190X GENE1175X GENE1138X GENE556X GENE1146X GENE17X GENE729X GENE857X GENE780X GENE1016X GENE988X GENE283X GENE827X GENE594X GENE409X GENE1063X GENE774X GENE70X GENE1112X GENE1130X GENE626X GENE328X GENE711X GENE1180X GENE1036X GENE183X GENE941X GENE1183X GENE1086X GENE876X GENE1046X GENE763X GENE610X GENE730X GENE1098X GENE983X GENE282X GENE718X GENE309X GENE341X GENE1125X GENE648X GENE647X GENE895X GENE236X GENE760X GENE739X GENE157X GENE783X GENE400X GENE425X GENE810X GENE1157X GENE188X GENE1177X GENE396X GENE406X GENE856X GENE724X GENE1185X GENE731X GENE566X GENE795X GENE815X GENE915X GENE254X GENE1178X GENE446X GENE989X GENE39X GENE1162X GENE909X GENE145X GENE732X GENE484X GENE389X GENE392X GENE266X GENE432X GENE773X GENE1093X GENE681X GENE489X GENE1186X GENE728X GENE1161X GENE1172X GENE1005X GENE1169X GENE46X GENE1176X GENE1011X GENE423X GENE82X GENE609X GENE1182X GENE198X GENE143X GENE486X GENE639X GENE602X GENE986X GENE935X GENE1179X GENE1164X GENE793X GENE1160X GENE1070X GENE83X GENE1168X GENE775X GENE382X GENE1095X GENE974X GENE1166X GENE1184X GENE771X GENE1115X GENE1156X GENE1173X GENE1026X GENE910X GENE1165X GENE1158X GENE1044X GENE1055X GENE1181X GENE928X GENE975X GENE1170X GENE1159X GENE936X GENE872X GENE968X GENE1135X GENE833X GENE800X GENE1027X GENE852X GENE163X GENE575X GENE919X GENE1021X GENE1038X GENE580X GENE976X GENE255X GENE384X GENE185X GENE30X GENE696X GENE842X GENE492X GENE684X GENE388X GENE557X GENE319X GENE962X GENE1084X GENE870X GENE922X GENE911X GENE947X GENE1123X GENE543X GENE997X GENE779X GENE807X GENE463X GENE1120X GENE1143X GENE428X GENE1067X
-3.0
-2.0
-1.0
0.0
1.0
2.0
3.0
Rel
ativ
e pr
otei
n co
ncen
tratio
ns (l
og2)
Figure S7: Concentrations of 1,187 proteins across 25 chemostats. Protein relative abundanceswere centered by row mean and hierarchically clustered using Pearson correlation.
72
0%
25%
50%
75%
100%
Trans
cript
s T
rans
cript
s of
mea
sure
d pr
otein
s
Prot
eins
Enzy
mes
Met
aboli
tes
% V
aria
nce
Expl
aine
dLimitation Limitation x growth−rate Growth−rate
Figure S8: Systematic variation of transcripts, proteins, enzymes and metabolites due to specificgrowth rate versus identity of the limiting nutrient. Variance explained was determined for log-transformed abundances using ANOVA. F-values were summed across each gene, metabolite, etc.,and normalized to 100% for each data type.
73
TCA cycle
Glycolysis
Carbohydratesynthesis
Amino acidsynthesis
Auxotrophyrelated
P C N L USlow Fasth
DR-1
-3.0
-2.0
-1.0
0.0
1.0
2.0
3.0
Rela
tive
enzy
me
conc
entr
atio
ns (l
og2)
Figure S9: Concentrations of 370 enzymes across 25 chemostats. Enzyme relative abundanceswere centered by row mean and hierarchically clustered using Pearson correlation.
74
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−8
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0
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0.00 0.05 0.10 0.15 0.20Noise added
Δ A
ICc
Figure S10: Choice of top regulator is generally robust to measurement error. Using pyruvatekinase activated by fructose 1,6-bisphosphate and inhibited by citrate as an example, differentamounts of noise were added to both regulators and impact on overall support was assessed(∆AICc). Noise added refers to the standard deviation of random Gaussian noise added to thelog2 concentrations of each regulator, in each condition. 20 simulations for each noise level wereperformed and the change in AICc (relative to no noise added) across these simulations is shown(lower numbers indicate better fit). Increasing noise occasionally increases the fit, but generallydecreases the average fit’s quality. The red line indicates the mean difference between the topregulator prediction for a gene and when one exists, the second best supported prediction.
75
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ata
Geometric mean value (log10) from BRENDAparameter interval contains BRENDA averageparameter interval consistent given BRENDA uncertaintyparameter estimate is inconsistent with BRENDA
p < 0.0001
79 61 35***
0.0
0.2
0.4
0.6
0.8
1.0
Substrate Product Regulator
[S][S] +Km
A B
Figure S11: Quantitative affinities from fitting cellular data generally align with biochemicalliterature. (A) Fitted metabolite affinities based on cellular data are generally consistent withliterature biochemical data. Using the best-fitting form for each reaction, affinities inferred usingSIMMER were compared to those reported in BRENDA. Uncertainty exists both in estimatedaffinities from SIMMER (accounted for by the 95% credibility interval, the Bayesian analogof a confidence interval) and in reported affinities from BRENDA. BRENDA affinities weresummarized by the geometric mean of affinities across multiple studies (log10) and the associatedSD. For 74% of substrates or products, the SIMMER CI contained the BRENDA mean. For89% of metabolites, the SIMMER CI overlapped with the BRENDA CI (mean ± 2 SD). (B)Extent of substrate, product, and regulator saturation based on fitted affinities. The violin plotwas generated by determining an occupancy value for each metabolite (j) and enzyme (i) in everycondition (k). To determine if substrates, products and regulators show trends in occupancy, themedian occupancy of each metabolite-enzyme pair was first found. The median occupancies of79 substrates, 61 products and 35 regulators were then separately compared to their Log-Uniformexpectation using a Kolmogorov-Smirnov test (*** p < 0.001).
76
CP
Citrulline
Pi
OrnithineArg3
Flux
Car
ried
(a.u
.)
Flux
Car
ried
(a.u
.)
P C N U
jrMM � jMeasured?
Alan
ine
(mM
)
h-1
0.05 0.30
kcat [Arg3] Arg3 [ORN][CP] − [CIT][Pi]Keq
1[ORN]KORN
+[CIT]KCIT
+ 1[CP]KCP
+ [Pi]KPi
+KORN KCP
jrMM = Inhi
bito
r
AlajrMM � jMeasured?
0.02.55.07.5
10.012.5
01020304050
0.0
0.5
1.0
1.5
0
50
100
Citr
ullin
e (1
00μM
)
Phos
phat
e (m
M)
Car
bam
oyl
phos
phat
e (a
.u.)
Orn
ithin
e (1
00μM
)Ar
g3 (a
.u.)
Con
cent
ratio
n
0
0.5
1
1.5
2
0
0.5
1
1.5
2 Michaelis−Menten Fit (95% CI)Measured Flux
R2 = 0.58 R2 = 0.70
Michaelis−Menten Fit (95% CI)Measured Flux
P C N U P C N U
P C N U
[Ala] KAla
jrMM =Ala jrMM 1+
●●● ●
●●
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●
●
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0%
25%
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75%
100%
3 10 30 100
[Ala](mM)
Frac
tion
of m
axim
um re
actio
n ve
loci
ty
K =14.8 mMi
CP
Low alanineLow pyrimidines
Orn
Asp
Arg
UMP
Ala
High alanineLow pyrimidines
Orn
Asp
Arg
UMP
Ala
CP
Low alanineHigh pyrimidines
Orn
Asp
Arg
UMP
Ala
CPOTCase
ATCase
A B C
D E
Figure S12: Ornithine carbamoyltransferase is regulated by alanine. (A) Substrate, productand enzyme concentrations for the ornithine carbamoyltransferase reaction which synthesizesthe arginine precursor citrulline. (B) Michaelis-Menten equation relating concentrations to fluxand extent of agreement between measured fluxes and the best Michaelis-Menten fit. (C)Concentrations of the reaction inhibitor alanine and extent of agreement between measured fluxesand the best Michaelis-Menten fit after including alanine as a regulator. Chemostats that areleucine-limited are not shown because of aphysiological ornithine accumulation (∼10-fold aboveother limiting nutrients at slow specific growth rates). Alanine is still predicted as the top regulatorof ornithine carbamoyltransferase when all 25 chemostats are considered. (D) Biochemicalconfirmation that physiological concentrations of alanine inhibit Arg3. (E) Inhibition of Arg3can in principle direct carbamoyl phosphate towards the synthesis of pyrimidines under conditionsof relative amino acid excess.
77
Acetaldehyde
CO2
Pyruvate Pdc1,5,6
Flux
Car
ried
(a.u
.)
Flux
Car
ried
(a.u
.)
jrMM � jMeasured?
h-1
0.05 0.30
kcat [Pdci] Pdci [Pyruvate] − [Acetald][CO2]
Keq
1[Pyruvate]
KPyruvate
+[Acetald]KAcetald
+ 1[CO2]KCP
+KPyruvate
jrMM =�i = 1
[1,5,6]
Phe-PyrjrMM � jMeasured?
Con
cent
ratio
n
[Phe-Pyr] KPhe-Pyr
jrMM =Phe-Pyr jrMM 1+
0
5
10
15
0.0
0.5
1.0
1.5
0.30.60.9
Pdc1
(a.u
.)Pd
c5 (a
.u.)
Pdc6
(a.u
.)Py
ruva
te (m
M)
0
1
2
Michaelis−Menten Fit (95% CI)Measured Flux
R2 = 0.38
0
1
2
R2 = 0.55
Phen
ylpy
ruva
te (m
M)
Inhi
bito
r
P C N L U P C N L U P C N L U
P C N L U
Michaelis−Menten Fit (95% CI)Measured Flux
0.1 mM Pyruvate 0.3 mM Pyruvate 1 mM Pyruvate
3 mM Pyruvate 10 mM Pyruvate 30 mM Pyruvate−0.3
0.0
0.3
0.6
0.0
0.5
1.0
1.5
2.0
0
2
4
0
3
6
9
12
0
5
10
15
0
5
10
15
0.1
0.3 1 3 10 0.1
0.3 1 3 10 0.1
0.3 1 3 10
Phenylpyruvate Concentration (mM)
Rea
ctio
n Ve
loci
ty
A
Nitrogen &carbon replete
Glucose
Nitrogen limited,carbon replete
EtOHPDC
Normal fermentation
PyrPhe
Phe-pyr
Glucose
EtOHPDC
PyrPhe
Phe-pyr Phe-EtOH
quorum sensing,morphological
changes leading to “foraging behavior”
B C
D E
0
2
4
6
Pdc1
Pdc1 +
10 m
M Pyruva
te
Pdc1 +
10 m
M Pheny
lpyruv
ate
Ion
Cou
nt (m
illion
s)
Acetaldehyde Phenylacetaldehyde
Pdc1
Pdc1 +
10 m
M Pyruva
te
Pdc1 +
10 m
M Pheny
lpyruv
ate
F
78
Figure S13 (previous page): Pyruvate decarboxylase is regulated by phenylpyruvate. (A) Substrateand enzyme concentrations for the pyruvate decarboxylase reaction which directs pyruvate towardsethanol production. The reaction products acetaldehyde and carbon dioxide were not measured.(B) Michaelis-Menten equation relating concentrations to flux and extent of agreement betweenmeasured fluxes and the best Michaelis-Menten fit. (C) Concentrations of the reaction inhibitorphenylpyruvate and extent of agreement between measured fluxes and the best Michaelis-Mentenfit after including phenylpyruvate as a regulator. (D) Biochemical confirmation that physiologicalconcentrations of phenylpyruvate inhibit Pdc1. (E) Incubation of Pdc1 with pyruvate leads toacetaldehyde accumulation, while incubation with phenylpyruvate leads to phenylacetaldehydeaccumulation. (F) During low nitrogen conditions, phenylpyruvate accumulation slows pyruvatedecarboxylase and thus ethanol excretion, while itself being converted to the quorum sensingmolecule phenylethanol.
79
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Figure S14: The primary determinant of cellular metabolic reaction rates is metaboliteconcentrations, irrespective of whether one analyzes only validated regulation or the top SIMMER-predicted regulation of each reaction. Analysis limited to validated yeast regulation is shown inmain text Figure 6. The present figure is identical to main text Figure 6, but also includes allSIMMER-predicted regulation that is reported in Table S2.
80
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Figure S15: Distribution of metabolic leverage between substrate/product, enzyme, and allostericregulators is robust to uncertainty in kinetic parameters (e.g., Km, Ki). In main text Figure 6,we show a single value without error estimates for the metabolic leverage of each player in eachreaction. This value is calculated based on the most likely parameter set for that reaction. Toassess the impact of uncertainty in these kinetics parameters, we randomly sampled, from thedistribution of well-fitting parameters (the posterior distribution), 20 sets of kinetic parameters foreach reaction. For each reaction from Figure 6, the metabolic leverage calculated based on each ofthese 20 parameter sets is shown here.
81
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