32
www.sciencemag.org/content/354/6311/aaf2786/suppl/DC1 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

Supplementary Materials forscience.sciencemag.org/highwire/filestream/685969/... · Supplemental Tables Pathway with enzymes ay reactions reactions enzymes enzymes enzymes of enzymes

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Page 1: Supplementary Materials forscience.sciencemag.org/highwire/filestream/685969/... · Supplemental Tables Pathway with enzymes ay reactions reactions enzymes enzymes enzymes of enzymes

www.sciencemag.org/content/354/6311/aaf2786/suppl/DC1

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

Page 2: Supplementary Materials forscience.sciencemag.org/highwire/filestream/685969/... · Supplemental Tables Pathway with enzymes ay reactions reactions enzymes enzymes enzymes of enzymes

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

Page 3: Supplementary Materials forscience.sciencemag.org/highwire/filestream/685969/... · Supplemental Tables Pathway with enzymes ay reactions reactions enzymes enzymes enzymes of enzymes

#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

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ase

(+)U

TP

02

Not

test

edC

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eto

Cis

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Gam

ma-

lyas

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)Ala

nine

02

Not

test

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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

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alic

Enz

yme

(NA

D)

(-)A

MP

04

Not

test

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osph

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ctok

inas

e(+

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6-bi

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phog

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ydro

gena

se(+

)Met

hion

ine

01

Not

test

edPh

osph

ogly

cera

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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

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(+)F

6P0

1N

otte

sted

Treh

alos

e-ph

osph

atas

e(+

)Tre

halo

se0

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otte

sted

UT

PG

1PU

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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

Page 4: Supplementary Materials forscience.sciencemag.org/highwire/filestream/685969/... · Supplemental Tables Pathway with enzymes ay reactions reactions enzymes enzymes enzymes of enzymes

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

Page 5: Supplementary Materials forscience.sciencemag.org/highwire/filestream/685969/... · Supplemental Tables Pathway with enzymes ay reactions reactions enzymes enzymes enzymes of enzymes

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

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)L

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ne,(

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ephe

nate

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L-T

yros

ine,

(-)L

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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

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line

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4G

luta

mat

ede

hydr

ogen

ase

(NA

DP)

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DH

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uino

linat

e0.

7649

Gly

cera

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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

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icen

zym

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AD

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5932

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ithin

eca

rbam

oyltr

ansf

eras

eO

TC

ase

(-)L

-Ala

nine

0.71

27O

rota

teph

osph

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sfer

ase

OPR

Tase

none

0.72

16

Oro

tidin

e-5’

-pho

spha

tede

carb

oxyl

ase

OD

Cas

eno

ne0.

516

61

Page 6: Supplementary Materials forscience.sciencemag.org/highwire/filestream/685969/... · Supplemental Tables Pathway with enzymes ay reactions reactions enzymes enzymes enzymes of enzymes

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

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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

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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

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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

Page 10: Supplementary Materials forscience.sciencemag.org/highwire/filestream/685969/... · Supplemental Tables Pathway with enzymes ay reactions reactions enzymes enzymes enzymes of enzymes

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

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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

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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.

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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.

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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.

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−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.

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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.

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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

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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.

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−8

−4

0

4

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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●●

●●

●●

● ●

● ●

● ●

●●

● ●

● ●

●●

● ●

●●

● ●

● ●

● ●●

−8

−4

0

−5 −4 −3 −2

Fitt

ed lo

g 10 a

�ni

ty b

ased

on

cellu

lar d

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

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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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●●●

●●●

0%

25%

50%

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.

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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

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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.

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Substrates &Products

Enzymes

Allostery100% 50%:50% 100%

50%

:50%

100%

50%:50%

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●●

●●●●●●●●●●●●

●●

●●

●●

●●

●●

●●●●●●●●●●●●

HCS

beta−GS

PPAT

ACO

ALS

GLDH

TREP

ATCase

G6PD

ME

PHGDH

ALD

G5K

DAHP Synth

PyK

TPS

6PGD

PGM

ATP−PRTase

PFK

OTCase

S7P PFK

PDC

PGKODCase

CBL

ASS

FUM

GAPDH

CBS

ADKAspK

Substrates &Products

EnzymesAllostery

Substrates &Products

Enzymes

Allostery100% 50%:50% 100%

50%

:50%

100%

50%:50%

●●

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C

B

A

Substrates &Products

Enzymes

Allostery100% 50%:50% 100%

50%

:50%

100%

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Enzymes

Allostery100% 50%:50% 100%

50%

:50%

100%

50%:50%

Reversible Net Forward Strongly Forward

Enzymes Enzymes

EnzymesProducts

Products

Products

Substrates Substrates

Substrates

Regulators

Regulators

Regulators

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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Substrates &Products

Enzymes

Allostery100% 50%:50% 100%

50%

:50%

100%

50%:50%

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:50%

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Reversible Net Forward Strongly Forward

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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