supporting information reveals variability in evolutionary ... · supporting information systems...
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Supporting Information
Systems level analysis of the Chlamydomonas reinhardtii metabolic network
reveals variability in evolutionary co-conservation
Amphun Chaiboonchoea†, Lila Ghamsarib†, Bushra Dohaia†, Patrick Ngc†, Basel Khraiwesha, Ashish
Jaiswala, Kenan Jijaklia, Joseph Koussaa, David R. Nelsona, Hong Caia§, Xinping Yangb, Roger L.
Changd, Jason Papine*, Haiyuan Yuc*, Santhanam Balajia,b,f*, Kourosh Salehi-Ashtiania,b*
aDivision of Science and Math, New York University Abu Dhabi and Center for Genomics and
Systems Biology (CGSB), New York University Abu Dhabi Institute, Abu Dhabi, UAE bCenter for Cancer Systems Biology (CCSB) and Department of Cancer Biology, Dana-Farber
Cancer Institute, and Department of Genetics, Harvard Medical School, Boston, MA, USAcDepartment of Biological Statistics and Computational Biology and Weill
Institute for Cell and Molecular Biology, Cornell University, Ithaca, NY, USAdDepartment of Systems Biology, Harvard Medical School, Boston, MA, USAeDepartment of Biomedical Engineering, University of Virginia, Charlottesville, VA, USAfMRC Laboratory of Molecular Biology, Cambridge, UK.
*Correspondence to: [email protected], [email protected], [email protected],
†These authors contributed equally to this work §Present address: BGI-Shenzhen, Shenzhen 518083, China
1
Electronic Supplementary Material (ESI) for Molecular BioSystems.This journal is © The Royal Society of Chemistry 2016
Supplementary Figures: S1-S17
Fig. S1 Transformation of the Chlamydomonas Metabolic network to a gene-centric network. Conceptual transformation of a metabolic pathway (where nodes are metabolites) to a protein centric pathway (where nodes are gene products) is shown in (a), herein referred to as “type A transformation” where edges (typically enzymes) are transformed to nodes. In the resulting pathway, edges are connected to each other through metabolites. Following type A transformation of the Chlamydomonas metabolic network 1, 2, the number of connections between genes (degree) and the number of genes with the relevant degree is plotted for the resulting network in (b). The distribution suggests a non-linear trend with many genes with “few” connections and few genes with “many” connections as observed in most other biological networks like protein-protein interaction or gene regulatory networks. Currency metabolites (see Network Transformation) were excluded from the transformation.
2
Fig. S2 The transformed, protein-centric metabolic network of C. reinhardtii. A graphical representation of the transformed C. reinhardtii metabolic network. Following removal of currency metabolites, the gene-reaction-metabolite associations described in the network were used to transform the genome-scale metabolic network of C. reinhardtii to a gene-centric network. There are altogether 1,081 gene products and 11,094 edges (links). This network displays a scale-free non-linear distribution between the number of genes and their connections (see Fig. S1). The colors of the nodes represent their connectivity degree (blue highest, dark-red lowest); the size of the nodes corresponds to the clustering co-efficient of the nodes; the size and color of the edges correspond to edge-betweenness (short or blue, low; long or red, high).
3
Fig. S3 GO term enrichment analyses of dynamically and statically co-conserved pairs. Statistically significant GO terms for Biological Process ontology with enrichment values of p< 0.1 are shown for static (red) or dynamic (green) pairs (a). Statistically significant GO terms for Molecular Function ontology with enrichment values of p< 0.1 are shown in (b) for static (red) or dynamic (green) pairs. The bars represent the enrichment score (y-axis) of the dynamic and static in different biological functions (x-axis).
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4
Fig. S4 The largest five co-conserved hubs in the network. The hubs in the network were defined as nodes forming the top 20% of highly connected genes. The five most connected hubs in the network are shown: Hubs 1-4 encode ferredoxins, hub 5 encodes an acyl-carrier protein (ACP2). Green edges are between nodes showing evidence of dynamic co-conservation, red edges are between statically co-conserved pairs.
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Fig. S5 Gene ontology (GO) and interolog analysis of C. reinhardtii with S.cerevisiae and A.thaliana. GO terms (Biological process) of dynamically co-conserved interologs of C. reinhardtii/A. thaliana, and interologs of C. reinhardtii/S. cerevisiae are shown in (a) and (b) respectively; GO terms of statically co-conserved interlogs of C. reinhardtii/A. thaliana, and interologs of C.reinhardtii/S.cerevisiae are shown in (c) and (d). BinGO (a biological network gene ontology tool) was used to visualize the GO terms that were statistically overrepresented (hypergeometric test) in the gene sets. Nodes highlighted in yellow correspond to GO terms with a p-value of 0.05 or smaller.
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eosi
de p
hosp
hate
met
abol
ic
proc
ess
cellu
lar c
arbo
hydr
ate
cata
bolic
pr
oces
s
cGM
P bi
osyn
thet
ic p
roce
ss
a
glyc
osyl
atio
n
glyc
erol
met
abol
ic p
roce
ss
carb
ohyd
rate
met
abol
ic p
roce
ss
prot
ein
met
abol
ic p
roce
ss
gene
exp
ress
ion
prim
ary
met
abol
ic p
roce
sstr
ansl
atio
n
tran
spor
t
loca
lizat
ion
gluc
ose
met
abol
ic p
roce
ss
hexo
se m
etab
olic
pro
cess
esta
blis
hmen
t of l
ocal
izat
ion
cellu
lar p
rote
in m
etab
olic
pro
cess
aldi
tol m
etab
olic
pro
cess
mac
rom
olec
ule
mod
ifica
tion
poly
ol m
etab
olic
pro
cess
prot
ein
amin
o ac
id g
lyco
syla
tion
mac
rom
olec
ule
glyc
osyl
atio
n
prot
ein
mod
ifica
tion
proc
ess
glyc
opro
tein
met
abol
ic p
roce
ss
bios
ynth
etic
pro
cess
cellu
lar m
etab
olic
pro
cess
smal
l mol
ecul
e m
etab
olic
pro
cess
cellu
lar p
roce
ss
oxid
atio
n re
duct
ion
biol
ogic
al_p
roce
ss
met
abol
ic p
roce
ss
tran
smem
bran
e tr
ansp
ort
oxoa
cid
met
abol
ic p
roce
ss
smal
l mol
ecul
e bi
osyn
thet
ic p
roce
ss
cellu
lar a
min
e m
etab
olic
pro
cess
amin
e bi
osyn
thet
ic p
roce
ss
cellu
lar a
min
o ac
id b
iosy
nthe
tic
proc
ess
cellu
lar k
eton
e m
etab
olic
pro
cess
orga
nic
acid
met
abol
ic p
roce
ss
orga
nic
acid
bio
synt
hetic
pro
cess
amin
e m
etab
olic
pro
cess
cellu
lar n
itrog
en c
ompo
und
bios
ynth
etic
pro
cess
nitr
ogen
com
poun
d m
etab
olic
pr
oces
s
cellu
lar n
itrog
en c
ompo
und
met
abol
ic p
roce
ss
cellu
lar b
iosy
nthe
tic p
roce
ss
mac
rom
olec
ule
bios
ynth
etic
pro
cess
cellu
lar a
min
o ac
id a
nd d
eriv
ativ
e m
etab
olic
pro
cess
prot
ein
amin
o ac
id N
-lin
ked
glyc
osyl
atio
n vi
a as
para
gine
pept
idyl
-asp
arag
ine
mod
ifica
tion
pept
idyl
-am
ino
acid
mod
ifica
tion
prot
ein
amin
o ac
id N
-lin
ked
glyc
osyl
atio
n
cellu
lar a
min
o ac
id m
etab
olic
pr
oces
s
carb
oxyl
ic a
cid
met
abol
ic p
roce
ss
bran
ched
cha
in fa
mily
am
ino
acid
m
etab
olic
pro
cess
carb
oxyl
ic a
cid
bios
ynth
etic
pro
cess
bran
ched
cha
in fa
mily
am
ino
acid
bi
osyn
thet
ic p
roce
ss
cellu
lar m
acro
mol
ecul
e bi
osyn
thet
ic
proc
ess
mon
osac
char
ide
met
abol
ic p
roce
ss
glyc
opro
tein
bio
synt
hetic
pro
cess
cellu
lar c
arbo
hydr
ate
met
abol
ic
proc
ess
cellu
lar m
acro
mol
ecul
e m
etab
olic
pr
oces
s
alco
hol m
etab
olic
pro
cess
mac
rom
olec
ule
met
abol
ic p
roce
ss
b
7
hist
idin
e bi
osyn
thet
ic p
roce
ss
amin
e bi
osyn
thet
ic p
roce
ss
cellu
lar a
min
e m
etab
olic
pro
cess
coen
zym
e ca
tabo
lic p
roce
ss
arom
atic
com
poun
d bi
osyn
thet
ic
proc
ess
chor
ism
ate
met
abol
ic p
roce
ss
bran
ched
cha
in fa
mily
am
ino
acid
m
etab
olic
pro
cess
cyst
eine
met
abol
ic p
roce
ss
tric
arbo
xylic
aci
d cy
cle
bran
ched
cha
in fa
mily
am
ino
acid
bi
osyn
thet
ic p
roce
ss
serin
e fa
mily
am
ino
acid
bi
osyn
thet
ic p
roce
ss
L-se
rine
met
abol
ic p
roce
ss
argi
nine
met
abol
ic p
roce
ss
ener
gy d
eriv
atio
n by
oxi
datio
n of
or
gani
c co
mpo
unds
isol
euci
ne b
iosy
nthe
tic p
roce
ss
cyst
eine
bio
synt
hetic
pro
cess
from
se
rine
isol
euci
ne m
etab
olic
pro
cess
cyst
eine
bio
synt
hetic
pro
cess
cellu
lar r
espi
ratio
n
aero
bic
resp
irat
ion
tran
smem
bran
e tr
ansp
ort
gene
ratio
n of
pre
curs
or m
etab
olite
s an
d en
ergy
is
opre
noid
bio
synt
hetic
pro
cess
phos
phol
ipid
bio
synt
hetic
pro
cess
cellu
lar m
etab
olic
pro
cess
lipid
bio
synt
hetic
pro
cess
oxoa
cid
met
abol
ic p
roce
ss
fatty
aci
d bi
osyn
thet
ic p
roce
ss
cellu
lar l
ipid
met
abol
ic p
roce
ss
cellu
lar k
eton
e m
etab
olic
pro
cess
cellu
lar c
atab
olic
pro
cess
prim
ary
met
abol
ic p
roce
ss
smal
l mol
ecul
e m
etab
olic
pro
cess
cellu
lar c
arbo
hydr
ate
bios
ynth
etic
pr
oces
s
cellu
lar m
acro
mol
ecul
e bi
osyn
thet
ic
proc
ess
cellu
lar p
rote
in m
etab
olic
pro
cess
cellu
lar c
arbo
hydr
ate
met
abol
ic
proc
ess
carb
ohyd
rate
bio
synt
hetic
pro
cess
smal
l mol
ecul
e bi
osyn
thet
ic p
roce
ss
orga
nic
acid
met
abol
ic p
roce
ss
glyc
opro
tein
bio
synt
hetic
pro
cess
carb
on u
tiliz
atio
ntr
ansl
atio
n
orga
nic
subs
tanc
e m
etab
olic
pro
cess
prot
ein
amin
o ac
id g
lyco
syla
tion
mac
rom
olec
ule
met
abol
ic p
roce
ssca
rboh
ydra
te c
atab
olic
pro
cess
esta
blis
hmen
t of l
ocal
izat
ion
biol
ogic
al_p
roce
ss
mac
rom
olec
ule
glyc
osyl
atio
nm
acro
mol
ecul
e m
odifi
catio
n
prot
ein
mod
ifica
tion
proc
ess
gene
exp
ress
ion
carb
on fi
xatio
n
glyc
opro
tein
met
abol
ic p
roce
ss
phot
osyn
thes
is
lipid
met
abol
ic p
roce
ss
orga
noph
osph
ate
met
abol
ic p
roce
ss
met
abol
ic p
roce
ss oxid
atio
n re
duct
ion
prot
ein
met
abol
ic p
roce
ss
cellu
lar m
acro
mol
ecul
e m
etab
olic
pr
oces
s
pyri
mid
ine
nucl
eotid
e m
etab
olic
pr
oces
s
hexo
se b
iosy
nthe
tic p
roce
ss
nucl
eosi
de m
etab
olic
pro
cess
gluc
oneo
gene
sis
NA
D m
etab
olic
pro
cess
glyc
erol
eth
er m
etab
olic
pro
cess
prot
ein
amin
o ac
id N
-lin
ked
glyc
osyl
atio
n vi
a as
para
gine
hexo
se m
etab
olic
pro
cess
NA
D b
iosy
nthe
tic p
roce
ss gluc
ose
met
abol
ic p
roce
ss
pept
idyl
-asp
arag
ine
mod
ifica
tion
hexo
se c
atab
olic
pro
cess
NA
DP
met
abol
ic p
roce
ss
prot
ein
amin
o ac
id N
-lin
ked
glyc
osyl
atio
n
nico
tinam
ide
nucl
eotid
e m
etab
olic
pr
oces
s
pept
idyl
-am
ino
acid
mod
ifica
tion
glyc
osyl
atio
n
wat
er-s
olub
le v
itam
in m
etab
olic
pr
oces
s
mon
osac
char
ide
bios
ynth
etic
pr
oces
s
mon
osac
char
ide
met
abol
ic p
roce
ss
mon
osac
char
ide
cata
bolic
pro
cess
alco
hol b
iosy
nthe
tic p
roce
ss
alco
hol m
etab
olic
pro
cess
alco
hol c
atab
olic
pro
cess
cellu
lar c
arbo
hydr
ate
cata
bolic
pr
oces
s
orga
nic
ethe
r met
abol
ic p
roce
ss
orga
nic
acid
bio
synt
hetic
pro
cess
cellu
lar a
min
o ac
id a
nd d
eriv
ativ
e m
etab
olic
pro
cess
pter
idin
e an
d de
riva
tive
met
abol
ic
proc
ess
mon
ocar
boxy
lic a
cid
met
abol
ic
proc
ess
mol
ybdo
pter
in c
ofac
tor m
etab
olic
pr
oces
s
coen
zym
e m
etab
olic
pro
cess
folic
aci
d an
d de
riva
tive
met
abol
ic
proc
ess
cofa
ctor
bio
synt
hetic
pro
cess
hete
rocy
cle
met
abol
ic p
roce
ss
Mo-
mol
ybdo
pter
in c
ofac
tor
met
abol
ic p
roce
ss
cellu
lar n
itrog
en c
ompo
und
bios
ynth
etic
pro
cess
carb
ohyd
rate
met
abol
ic p
roce
ss
mac
rom
olec
ule
bios
ynth
etic
pro
cess
cata
bolic
pro
cess
bios
ynth
etic
pro
cess
smal
l mol
ecul
e ca
tabo
lic p
roce
ss
purin
e nu
cleo
side
met
abol
ic p
roce
ssri
bonu
cleo
side
met
abol
ic p
roce
ssnu
cleo
tide
bios
ynth
etic
pro
cess
ribo
flavi
n an
d de
riva
tive
bios
ynth
etic
pro
cess
py
rim
idin
e nu
cleo
tide
bios
ynth
etic
pr
oces
s
pyri
mid
ine
deox
yrib
onuc
leot
ide
bios
ynth
etic
pro
cess
2'-d
eoxy
ribo
nucl
eotid
e m
etab
olic
pr
oces
s
folic
aci
d an
d de
rivat
ive
bios
ynth
etic
pr
oces
s gr
oup
tran
sfer
coe
nzym
e m
etab
olic
pr
oces
s co
enzy
me
bios
ynth
etic
pro
cess
Mo-
mol
ybdo
pter
in c
ofac
tor
bios
ynth
etic
pro
cess
ox
idor
educ
tion
coen
zym
e m
etab
olic
pr
oces
s
coen
zym
e A
bios
ynth
etic
pro
cess
sulfu
r am
ino
acid
met
abol
ic p
roce
ss
arom
atic
am
ino
acid
fam
ily
bios
ynth
etic
pro
cess
sulfu
r am
ino
acid
bio
synt
hetic
pr
oces
s ac
etyl
-CoA
cat
abol
ic p
roce
ss
cellu
lar a
min
o ac
id m
etab
olic
pr
oces
s
sulfu
r com
poun
d bi
osyn
thet
ic
proc
ess
arom
atic
am
ino
acid
fam
ily
met
abol
ic p
roce
ss
dica
rbox
ylic
aci
d m
etab
olic
pro
cess
pyru
vate
met
abol
ic p
roce
ssvi
tam
in b
iosy
nthe
tic p
roce
ss
nucl
eoba
se, n
ucle
osid
e, n
ucle
otid
e an
d nu
clei
c ac
id b
iosy
nthe
tic
proc
ess
porp
hyri
n bi
osyn
thet
ic p
roce
ss
ribo
flavi
n bi
osyn
thet
ic p
roce
ss
ribo
flavi
n m
etab
olic
pro
cess
wat
er-s
olub
le v
itam
in b
iosy
nthe
tic
proc
ess
ribo
flavi
n an
d de
riva
tive
met
abol
ic
proc
ess
nucl
eoba
se, n
ucle
osid
e an
d nu
cleo
tide
bios
ynth
etic
pro
cess
nucl
eoba
se, n
ucle
osid
e an
d nu
cleo
tide
met
abol
ic p
roce
ss
pent
ose-
phos
phat
e sh
unt
nucl
eotid
e m
etab
olic
pro
cess
nucl
eosi
de d
ipho
spha
te b
iosy
nthe
tic
proc
ess
nucl
eosi
de d
ipho
spha
te m
etab
olic
pr
oces
s
deox
yrib
onuc
leot
ide
bios
ynth
etic
pr
oces
s
deox
yrib
onuc
leot
ide
met
abol
ic
proc
ess
pyri
mid
ine
nucl
eosi
de d
ipho
spha
te
bios
ynth
etic
pro
cess
deox
yrib
onuc
leos
ide
diph
osph
ate
bios
ynth
etic
pro
cess
deox
yrib
onuc
leos
ide
diph
osph
ate
met
abol
ic p
roce
ss
pyri
mid
ine
deox
yrib
onuc
leos
ide
diph
osph
ate
bios
ynth
etic
pro
cess
2'-d
eoxy
ribo
nucl
eotid
e bi
osyn
thet
ic
proc
ess
dTD
P b
iosy
nthe
tic p
roce
ss
vita
min
met
abol
ic p
roce
ssnu
cleo
base
, nuc
leos
ide,
nuc
leot
ide
and
nucl
eic
acid
met
abol
ic p
roce
ss
gluc
ose
cata
bolic
pro
cess
pros
thet
ic g
roup
met
abol
ic p
roce
ss
nitr
ogen
com
poun
d m
etab
olic
pr
oces
s
pter
idin
e an
d de
riva
tive
bios
ynth
etic
pr
oces
s
amin
e m
etab
olic
pro
cess
porp
hyri
n m
etab
olic
pro
cess
tetr
apyr
role
bio
synt
hetic
pro
cess
hist
idin
e m
etab
olic
pro
cess
hete
rocy
cle
bios
ynth
etic
pro
cess
tetr
apyr
role
met
abol
ic p
roce
ssco
fact
or m
etab
olic
pro
cess
mol
ybdo
pter
in c
ofac
tor b
iosy
nthe
tic
proc
ess
hist
idin
e fa
mily
am
ino
acid
bi
osyn
thet
ic p
roce
ss
hist
idin
e fa
mily
am
ino
acid
m
etab
olic
pro
cess
acet
yl-C
oA m
etab
olic
pro
cess
glut
amin
e fa
mily
am
ino
acid
bi
osyn
thet
ic p
roce
ss
serin
e fa
mily
am
ino
acid
met
abol
ic
proc
ess
cellu
lar a
min
o ac
id b
iosy
nthe
tic
proc
ess ar
gini
ne b
iosy
nthe
tic p
roce
ss
glut
amin
e fa
mily
am
ino
acid
m
etab
olic
pro
cess
nico
tinam
ide
nucl
eotid
e bi
osyn
thet
ic p
roce
ss
nucl
eosi
de b
isph
osph
ate
met
abol
ic
proc
ess py
ridi
ne n
ucle
otid
e m
etab
olic
pr
oces
s
puri
ne r
ibon
ucle
osid
e m
etab
olic
pr
oces
s
coen
zym
e A
met
abol
ic p
roce
ssN
AD
PH
reg
ener
atio
n
pyri
dine
nuc
leot
ide
bios
ynth
etic
pr
oces
s
carb
oxyl
ic a
cid
met
abol
ic p
roce
ss
sulfu
r met
abol
ic p
roce
ss
fatty
aci
d m
etab
olic
pro
cess
cofa
ctor
cat
abol
ic p
roce
ss
cellu
lar n
itrog
en c
ompo
und
met
abol
ic p
roce
ss
cellu
lar b
iosy
nthe
tic p
roce
ss
glyc
olys
is
cellu
lar a
rom
atic
com
poun
d m
etab
olic
pro
cess
ca
rbox
ylic
aci
d bi
osyn
thet
ic p
roce
ss
dTD
P m
etab
olic
pro
cesspy
rim
idin
e de
oxyr
ibon
ucle
osid
e di
phos
phat
e m
etab
olic
pro
cess
pyri
mid
ine
deox
yrib
onuc
leot
ide
met
abol
ic p
roce
ss
pyri
mid
ine
nucl
eosi
de d
ipho
spha
te
met
abol
ic p
roce
ss
nucl
eosi
de p
hosp
hate
met
abol
ic
proc
ess
catio
n tr
ansp
ort
mon
oval
ent i
norg
anic
cat
ion
tran
spor
t
ion
tran
spor
t
anio
n tr
ansp
ort
prot
on tr
ansp
ort
hydr
ogen
tran
spor
t
tran
spor
t
inor
gani
c an
ion
tran
spor
t
sodi
um io
n tr
ansp
ort
met
al io
n tr
ansp
ort
phos
phat
e tr
ansp
ort
cellu
lar h
omeo
stas
is
cell
redo
x ho
meo
stas
is
sign
alin
g pr
oces
s
regu
latio
n of
cel
lula
r pro
cess
regu
latio
n of
bio
logi
cal p
roce
ss
sign
alin
g
biol
ogic
al r
egul
atio
n
phos
phol
ipid
met
abol
ic p
roce
ss
isop
reno
id m
etab
olic
pro
cess
cellu
lar p
roce
ss
sign
al tr
ansd
uctio
n
sign
al tr
ansm
issi
on
regu
latio
n of
bio
logi
cal q
ualit
y
hom
eost
atic
pro
cess
loca
lizat
ion
c
8
9
catio
n tr
ansp
ort
mon
oval
ent i
norg
anic
cat
ion
tran
spor
t
tran
spor
t
esta
blis
hmen
t of l
ocal
izat
ion
loca
lizat
ion
biol
ogic
al_p
roce
ss
prot
ein
amin
o ac
id N
-lin
ked
glyc
osyl
atio
n vi
a as
para
gine
prot
ein
amin
o ac
id N
-lin
ked
glyc
osyl
atio
n
cellu
lar a
ldeh
yde
met
abol
ic p
roce
ss
pept
idyl
-am
ino
acid
mod
ifica
tionpr
otei
n m
odifi
catio
n pr
oces
s
cellu
lar p
rote
in m
etab
olic
pro
cess
glyo
xyla
te c
ycle
pept
idyl
-asp
arag
ine
mod
ifica
tion
cellu
lar c
arbo
hydr
ate
met
abol
ic
proc
ess
prot
ein
amin
o ac
id g
lyco
syla
tion
cellu
lar n
itrog
en c
ompo
und
bios
ynth
etic
pro
cess
cellu
lar b
iosy
nthe
tic p
roce
ssni
trog
en c
ompo
und
met
abol
ic
proc
ess
smal
l mol
ecul
e bi
osyn
thet
ic p
roce
ss
ATP
syn
thes
is c
oupl
ed p
roto
n tr
ansp
ort
bios
ynth
etic
pro
cess
cellu
lar a
min
o ac
id a
nd d
eriv
ativ
e m
etab
olic
pro
cess
hete
rocy
cle
bios
ynth
etic
pro
cess
tetr
apyr
role
met
abol
ic p
roce
ss
smal
l mol
ecul
e m
etab
olic
pro
cess
nucl
eoba
se, n
ucle
osid
e an
d nu
cleo
tide
bios
ynth
etic
pro
cess
nucl
eosi
de b
isph
osph
ate
met
abol
ic
proc
ess
nucl
eosi
de m
etab
olic
pro
cess
tetr
apyr
role
bio
synt
hetic
pro
cess
pter
idin
e an
d de
rivat
ive
bios
ynth
etic
pr
oces
s
Mo-
mol
ybdo
pter
in c
ofac
tor
bios
ynth
etic
pro
cess
cofa
ctor
bio
synt
hetic
pro
cess
arom
atic
com
poun
d bi
osyn
thet
ic
proc
ess
pigm
ent b
iosy
nthe
tic p
roce
ss
mol
ybdo
pter
in c
ofac
tor m
etab
olic
pr
oces
s
cellu
lar a
rom
atic
com
poun
d m
etab
olic
pro
cess
cofa
ctor
met
abol
ic p
roce
ss
Mo-
mol
ybdo
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in c
ofac
tor
met
abol
ic p
roce
ss
pigm
ent m
etab
olic
pro
cess
pter
idin
e an
d de
riva
tive
met
abol
ic
proc
ess
mol
ybdo
pter
in c
ofac
tor b
iosy
nthe
tic
proc
ess
phos
phor
us m
etab
olic
pro
cess
gene
exp
ress
ion
mac
rom
olec
ule
met
abol
ic p
roce
ss
mac
rom
olec
ule
mod
ifica
tion
mon
ocar
boxy
lic a
cid
met
abol
ic
proc
ess
prim
ary
met
abol
ic p
roce
ss
gene
ratio
n of
pre
curs
or m
etab
olite
s an
d en
ergy
mac
rom
olec
ule
bios
ynth
etic
pro
cess
met
abol
ic p
roce
ss
cellu
lar k
eton
e m
etab
olic
pro
cess
cellu
lar m
acro
mol
ecul
e bi
osyn
thet
ic
proc
ess
phot
osyn
thes
is
mac
rom
olec
ule
glyc
osyl
atio
ngl
ycos
ylat
ion
glyc
opro
tein
bio
synt
hetic
pro
cess
glyo
xyla
te m
etab
olic
pro
cess
tran
slat
ion
cellu
lar m
acro
mol
ecul
e m
etab
olic
pr
oces
s
carb
ohyd
rate
met
abol
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roce
ssgl
ycop
rote
in m
etab
olic
pro
cess
pros
thet
ic g
roup
met
abol
ic p
roce
ss
cellu
lar p
roce
ss
ion
tran
smem
bran
e tr
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ort
hydr
ogen
tran
spor
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on tr
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smem
bran
e tr
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ener
gy c
oupl
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roto
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dow
n el
ectr
oche
mic
al g
radi
ent
oxid
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n re
duct
ion
ion
tran
spor
t
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zym
e bi
osyn
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roce
ss
coen
zym
e m
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cess
chlo
roph
yll m
etab
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pro
cess
grou
p tr
ansf
er c
oenz
yme
met
abol
ic
proc
ess
coen
zym
e A
bios
ynth
etic
pro
cess
purin
e rib
onuc
leos
ide
met
abol
ic
proc
ess
coen
zym
e A
met
abol
ic p
roce
ss
ribon
ucle
osid
e m
etab
olic
pro
cess
puri
ne n
ucle
osid
e m
etab
olic
pro
cess
porp
hyri
n bi
osyn
thet
ic p
roce
ss
chlo
roph
yll b
iosy
nthe
tic p
roce
sspo
rphy
rin
met
abol
ic p
roce
ss
cellu
lar m
etab
olic
pro
cess
nucl
eoba
se, n
ucle
osid
e, n
ucle
otid
e an
d nu
clei
c ac
id m
etab
olic
pro
cess
nu
cleo
base
, nuc
leos
ide,
nuc
leot
ide
and
nucl
eic
acid
bio
synt
hetic
pr
oces
s
nucl
eoba
se, n
ucle
osid
e an
d nu
cleo
tide
met
abol
ic p
roce
ss
hete
rocy
cle
met
abol
ic p
roce
ss
prot
ein
met
abol
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roce
ss
phos
phat
e m
etab
olic
pro
cess
phos
phor
ylat
ion
oxoa
cid
met
abol
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roce
ssox
idat
ive
phos
phor
ylat
ion
carb
oxyl
ic a
cid
met
abol
ic p
roce
ss
cellu
lar n
itrog
en c
ompo
und
met
abol
ic p
roce
ss
amin
e m
etab
olic
pro
cess
orga
nic
acid
bio
synt
hetic
pro
cess
amin
e bi
osyn
thet
ic p
roce
ss
cellu
lar a
min
e m
etab
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pro
cess
hist
idin
e fa
mily
am
ino
acid
m
etab
olic
pro
cess
carb
oxyl
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bios
ynth
etic
pro
cess
cellu
lar a
min
o ac
id m
etab
olic
pr
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s ce
llula
r am
ino
acid
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synt
hetic
pr
oces
s
orga
nic
acid
met
abol
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roce
ss
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idin
e bi
osyn
thet
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roce
ss
ATP
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ynth
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cess
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idin
e m
etab
olic
pro
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idin
e fa
mily
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ino
acid
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roce
ss
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ne b
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roce
ss
bran
ched
cha
in fa
mily
am
ino
acid
bi
osyn
thet
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roce
ss
isol
euci
ne m
etab
olic
pro
cess
bran
ched
cha
in fa
mily
am
ino
acid
m
etab
olic
pro
cess
nucl
eosi
de tr
ipho
spha
te m
etab
olic
pr
oces
s pu
rine
nuc
leot
ide
bios
ynth
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pr
oces
s pu
rine
rib
onuc
leot
ide
bios
ynth
etic
pr
oces
s
puri
ne n
ucle
otid
e m
etab
olic
pro
cess
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ne n
ucle
osid
e tr
ipho
spha
te
met
abol
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roce
ss
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ucle
otid
e m
etab
olic
pro
cess
puri
ne r
ibon
ucle
otid
e m
etab
olic
pr
oces
s nu
cleo
side
pho
spha
te m
etab
olic
pr
oces
s
puri
ne r
ibon
ucle
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e tr
ipho
spha
te
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ynth
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pro
cess
purin
e ri
bonu
cleo
side
trip
hosp
hate
m
etab
olic
pro
cess
nucl
eotid
e m
etab
olic
pro
cess
nucl
eosi
de tr
ipho
spha
te
bios
ynth
etic
pro
cess
ribo
nucl
eotid
e bi
osyn
thet
ic p
roce
ss
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nucl
eosi
de tr
ipho
spha
te
met
abol
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ss
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ne n
ucle
osid
e tr
ipho
spha
te
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ynth
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pro
cess
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eotid
e bi
osyn
thet
ic p
roce
ss
deox
yrib
onuc
leot
ide
met
abol
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proc
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pyri
mid
ine
nucl
eotid
e m
etab
olic
pr
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s
ribo
nucl
eosi
de tr
ipho
spha
te
bios
ynth
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pro
cess
AT
P m
etab
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pro
cess
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de d
ipho
spha
te m
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iosy
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tic
proc
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dTD
P bi
osyn
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ss
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ide
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osph
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osph
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osph
ate
bios
ynth
etic
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cess
2'-d
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etab
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pr
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deox
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ynth
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cleo
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hate
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osyn
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ic p
roce
ss
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ine
deox
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onuc
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ide
diph
osph
ate
met
abol
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roce
ss
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mid
ine
deox
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onuc
leot
ide
bios
ynth
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pro
cess
2'-d
eoxy
ribo
nucl
eotid
e bi
osyn
thet
ic
proc
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dTD
P m
etab
olic
pro
cess
d
Fig. S6 Network module analysis using MCODE. The top 5 modules identified based on MCODE scores are represented from (a) 21 total modules for statically co-conserved gene pairs and (b) 41 total modules of dynamically co-conserved gene pairs. The regions of the dynamic and static sub networks where these modules were detected (Fig. 2 in the main text) can be found based on the numbers associated with each module.
10
21 3
4 5
a
b
1
2
3 4 5
1
Fig. S7 Light and dark expressed genes (a) and interactions (b). Dark and light expressed genes have differential topological and functional characteristics in the network.
Fig. S8 Predicted flux values from the in silico Chlamydomonas model. Flux balance analyses were carried out for maximization of biomass under dark with acetate (blue bars) and light without acetate (red bars) conditions. Biomass yield is given on the y-axis.
1ACP
Cth
COAt
hGL
YC3P
thN
ADPt
hTS
ULth
H2t
iPE
1829
Z12Z
1835
Z9Z1
2Zt
NAD
PHtf
ALAN
A1tx
5MTA
tmDG
TPtm
MAL
AKGt
mPT
RCtm
iN
H4t
nAS
POm
G5DH
xDH
DHGP
PSEC
OAH
8AL
AAT
LUTH
CYSD
O3H
AD10
0AC
P161
9ZD9
DSFA
80AC
PHi
AACP
S1FA
COAL
160
FOLD
3GR
GTPR
DPAR
hAC
OADA
GAT1
8018
19Z1
8111
...AC
PT16
0181
11Z
ASQD
PADS
1829
Z12Z
160
DGDG
D4DS
1839
Z12Z
15Z1
64...
DGDG
W3D
S183
9Z12
Z15Z
164.
..DG
TSDS
1839
Z12Z
15Z1
835Z
...M
GDGS
1819
Z161
9ZPA
PA18
111Z
1819
ZhPL
DAGA
T160
1819
Z181
11Z1
PLDA
GAT1
8111
Z181
9Z18
11...
SQDG
S160
TAGA
H18
111Z
1811
1Z18
45Z.
..1A
GPEA
T180
1835
Z9Z1
2ZLP
LPS1
AGPE
1829
Z12Z
PLPS
A218
29Z1
2Z18
35Z9
Z1...
HSK
GAPD
Hf
PGK
GDPM
TIB
PHDH
DPS
AMCL
MM
2N
ODOy
PROA
KGOX
1AP
CPT
RBK
TRPS
2hCH
LASG
MPM
ECY3
HPH
LmAG
POP
ATPP
Hm
GTPO
PmPR
FGS
ATUD
mDU
RIK1
UTUP
PFLA
CTm
AUN
OR3D
SPH
RST
ARCH
300D
EGRA
ICDH
hrIM
DHT(
3c2h
mp)
MH
GS
-15-10
-505
101520253035
mill
imol
es g
ram
[dry
wei
ght]
-1 h
our
-1
11
Fig. S9 Metabolic locations for correlated downregulation (a) and upregulation (b) of flux and gene expression levels. Numbers correspond to the number of genes expressed in each of the subcellular compartments for each set.
b
a
1
1
8
13
3470
115
1
25
8
26
28
5
55
1
1
12
Fig. S10 Distribution of synthetic interactions with respect to their severity and growth condition. Synthetic interactions were identified under growth in dark with acetate (DA) and light with no acetate (LNA), the identified interactions were then binned based on the extent that biomass is reduced (in comparison to the wild-type model).
Lethal (100-80)% (60-80)% (40-20)% (20-0)%0
100
200
300
400
500
600 DA LNA
Synthetic Interactions
Num
ber
of In
tera
ctio
ns
13
Fig. S11 Subnetworks of synthetic lethal interactions under dark with acetate (a) and light with out acetate (b). Synthetic lethal pairs were used to generate the shown subnetworks. The blue box represents Carbohydrate metabolic process (GO: biological process) and N-Glycan Biosynthesis (KEGG pathway). The green circle represents Electron transport (GO: biological process) and Valine, leucine and isoleucine degradation (KEGG pathway).
14
Fig. S12 KEGG term enrichment between two conditions (light and dark) for synthetic lethal interactions. The bars represent the enrichment score (y-axis) of the dark with acetate (red) and light with no acetate (blue) in different KEGG pathways (x-axis).
Valin
e, le
ucin
e an
dis
oleu
cine
deg
rada
tion
Prop
anoa
te m
etab
olis
mBi
osyn
thes
is o
fal
kalo
ids d
eriv
ed fr
omor
nith
ine,
lysi
ne a
ndni
cotin
ic a
cid
Bios
ynth
esis
of
alka
loid
s der
ived
from
shik
imat
e pa
thw
ayBi
osyn
thes
is o
fal
kalo
ids d
eriv
ed fr
omhi
stid
ine
and
puri
neBi
osyn
thes
is o
f pla
ntho
rmon
esN
-Gly
can
bios
ynth
esis
Bios
ynth
esis
of
terp
enoi
ds a
nd st
eroi
dsBi
osyn
thes
is o
fal
kalo
ids d
eriv
ed fr
omte
rpen
oid
and
poly
ketid
eBi
osyn
thes
is o
fph
enyl
prop
anoi
dsPy
ruva
te m
etab
olis
mBe
nzoa
te d
egra
datio
nvi
a Co
A lig
atio
nGl
ycin
e, se
rine
and
thre
onin
e m
etab
olis
mFa
tty
acid
met
abol
ism
Buta
noat
e m
etab
olis
mTr
ypto
phan
met
abol
ism
Capr
olac
tam
degr
adat
ion
Amin
o su
gar a
ndnu
cleo
tide
suga
rm
etab
olis
mGl
utat
hion
e m
etab
olis
mPe
ntos
e an
dgl
ucur
onat
ein
terc
onve
rsio
nsGl
ycol
ysis
/Gl
ucon
eoge
nesi
sLy
sine
deg
rada
tion
Asco
rbat
e an
d al
dara
tem
etab
olis
mCy
stei
ne a
ndm
ethi
onin
e m
etab
olis
mPo
rphy
rin
and
chlo
roph
yll m
etab
olis
mDr
ug m
etab
olis
m -
othe
r enz
ymes
Pent
ose
phos
phat
epa
thw
ayPy
rim
idin
e m
etab
olis
mLi
popo
lysa
ccha
ride
bios
ynth
esis
Carb
on fi
xatio
n in
phot
osyn
thet
icor
gani
sms
Sulfu
r met
abol
ism
0.00E+00
2.00E-02
4.00E-02
6.00E-02
8.00E-02
Light with no acetate Dark with acetate
Enri
chm
ent s
core
s (L
og s
cale
)
Fig. S13 The pathways of the reactions of co-set1 in set 4 that has zero average profile distance (Table S18) in purine metabolism. The figure shows the purine metabolism as obtained from KEGG. The reactions XNDH (via xanthine dehydrogenase) and URO (via urate oxidase) are highlighted in red rectangular indicated with numbers 1 and 2, respectively. In XNDH xanthine dehydrogenase dehydrogenases the xanthine into Urate, in which the latter is oxidized into 5-Hydroxyisourate in the reaction URO.
1 2
1 XNDH 2 URO
Purine metabolism
15
Fig. S14 The pathways in the n-glycan biosynthesis from KEGG of reactions in co-set5 in set 2, reactions of co-set4 set 1, and the reactions associated with lethal gene pair CRv4_Au5.s16.g6321.t1 and CRv4_Au5.s12.g3687.t1 with average profile distance 3.6 with average profile distance of 3.2,1.5 and 3.6, respectively. In this figure the red rectangles, indicated with number 2,6, and 7, correspond to reactions of co-set5 in set 2 (Suppl. Data Table S18), where the reaction DOLPTG1 is catalyzed via glycotransferase to activate a nucleotide sugar and converts it into a nucleophillic glycosyl acceptor. The reactions of GLNACT (via N-acetylglucosaminyltransferase) and G12MT2 (via ALG11 mannosyltransferase with a DXD motif) produce the sequon Asn-X-Ser/Thr through DOLPTG1 (via glycosyltransferase). The blue rectangles, indicated with numbers 2,4, and 5, correspond to reactions of co-set4 in set 1 (Suppl. Data Table S17), where both GLNACT and DOLPMT (via dolichyl-phosphate beta-D-mannosyltransferase) are needed as precursor reactions for the production of the Asn-X-Ser/Thr sequon through DOLPMT2 (via dolichyl-phosphate-mannose-glycolipid alpha-mannosyltransferase).The pink rectangles, indicated with numbers 1 and 3, correspond to the reactions associated with the lethal gene pair CRv4_Au5.s16.g6321.t1 and CRv4_Au5.s12.g3687 (Suppl. Data Table S12).CRv4_Au5.s16.g6321.t1 associates the reaction GLNACPT (via UDP-GlcNAc:dolichyl-phosphate N-acetylglucosaminephosphotransferase) in purine metabolism) and CRv4_Au5.s12.g3687.t1 associates the reaction AGAKI (via D-xylose isomerase in the fructose and mannose metabolism). The metabolisms in which both genes are involved are related through the production of mannose-GDP.
12
3 4
5
6
7
1 GLCNACPT 2 GLNACT 3 AGAKI 4 DOLPMT 5 DOLPMT2 6 G12MT2 7 DOLPGT1
N-Glycan biosynthesis
16
Fig. S15 The pathways of the reactions of co-set1 in set 6 that has 3.2 average profile distance (Table S17) in fatty acid biosynthesis. The figure shows the pathways of the fatty acid biosynthesis as obtained from KEGG. The red rectangles correspond to the reaction 3OAR40 and the reaction 3HAD120, indicated with numbers 1 and 2, respectively. In this biosynthesis, 3HAD120 produces dodecanoic acid through the enzyme (3R)-3-Hydroxybutanoyl-[acyl-carrier-protein] hydro-lyase, and 3OAR40 produces the Butyryl-acp through 3-oxoacyl-[acyl-carrier-protein] reductase (n-C4:0).
17
Fatty acid biosynthesis
1
2
1 3OAR40 2 3HAD120
Fig. S16 The pathways of the reactions associated with lethal gene pair CRv4_Au5.s16.g6244.t1 and CRv4_Au5.s17.g7556.t1 (Table S12), that has zero average profile distance. In this figure, the red rectangles show the reactions associated with the genes CRv4_Au5.s16.g6244 (indicated with number 1) and CRv4_Au5.s17.g7556.t1 (indicated with number 2) in steroid biosynthesis and pentose phosphate pathway, respectively, as obtained from KEGG. The gene CRv4_Au5.s17.g7556.t1 is used to catalyze the reaction glucose-6-phosphate-1-dehydrogenase (g6p-A) which converts D-Glucose 6-phophate into D-Glucono-1,5-lactone 6-phosphate in the pentose phosphate pathway. The other gene, CRv4_Au5.s16.g6244.t1, is used in steroids biosynthesis.
18
1
Steroid biosynthesis
1 CRv4_Au5.s16.g6244.t1 2 CRv4_Au5.s17.g7556.t1
2
Pentose Phosphate pathway
Fig. S17 The pathways of the reactions associated with lethal gene pair CRv4_Au5.s16.g6244.t1 and CRv4_Au5.s17.g7556.t1 (Table S13) that has 3.4 average profile distance. In this figure the red rectangles show reactions associated with lethal gene CRv4_Au5.s9.g15712.t1 (indicated with number 1) and CRv4_Au5.s10.g816.t1 (indicated with number 2) that are utilized in the Glycine, Serine and Threonine metabolism and Glyoxylate and Dicarboxylate metabolism, respectively obtained from KEGG. In the glycine, serine and threonine metabolism, the glycine is converted into L-serine by the reaction glycine hydroxymethyltransferase, specifically associated with CRv4_Au5.s9.g15712.t1 gene. CRv4_Au5.s10.g816.t1 is associated with glycine synthase for the conversion of glycine into ammonia in the glyoxylate and dicarboxylate metabolism
19
1
Glycine, Serine, and Threonine metabolism Glyoxylate and Dicarboxylate metabolism
2
1 CRv4_Au5.s9.g15712.t1 2 CRv4_Au5.s10.g816.t1
Supplementary Methods: S1-S6
Method S1: Evolutionary affinity assignments. BLASTP searches were done for each protein
sequence of each gene in the network against proteomes of available and completely sequenced
eukaryotic genomes. These proteome sequences were obtained from SUPERFAMILY database
(version: genome_sequence_29-Aug-2010.sql). The top ten hits for each genome (e-value cutoff 0.001)
were identified and BLASTCLUST
(http://www.ncbi.nlm.nih.gov/IEB/ToolBox/C_DOC/lxr/source/doc/blast/blastclust.html) was done on
these hits to identify homology clusters (sequence identity of 30% and length cut-off of 70%) 3. Each
genome was further categorized into one of the thirteen eukaryotic lineages. Evolutionary conservation
of C. reinhardtii genes was defined on the basis of lineages present in each cluster was identified by
BLASTCLUST. Using this approach, each metabolic gene in the network was determined to be
conserved/non-conserved in each of the major eukaryotic lineages e.g., fungi, animals, plants, etc.
Method S2: Co-conservation analyses. Mutual information index (MI) and evolutionary profile
distance (PD) calculations – Mutual information index 4 for neighboring gene pairs X, Y, were
calculated as follows:
MI (X, Y) = H(X)+H(Y)-H(X,Y)
Where H(N) = Σ(-(frequency of affinity index)*log(frequency of affinity index)); H(X,Y) is the frequency
of co-occurrence affinity index and affinity index values are 0 or 1.
Evolutionary profile distance (PD) was obtained by measuring Euclidean distance between each
neighboring gene in the real or randomized network as follows:
PD (X, Y) = √ (Σ over N’s (value of lineage N of Gene1 – Value of Lineage N of Gene 2)^2), where N =
1 to 13). This definition is a variant of profile similarity used for gene expression clustering and
analysis5.
Profile randomization – For the randomization processes, the phylogenetic profile for each gene is
randomized such that the binary affinity values of “1” and “0” were retained but their positional
occurrences were varied. Thus the generated randomized phylogenetic profiles evaluated MI and PD
values for each pair of genes in the network. We note that for these analyses, the positions of genes in
the network were not randomized to evaluate the significance as evolution information content comes
from phylogenetic profiles.
Network randomization and p-value calculations - For the MI and PD calculations, significant p-values
were estimated by using random networks. These random networks were generated from the original
network by maintaining the number of partners per gene while rewiring randomly their partners. We
generated 1,000 such random networks and for every pair of genes in each network we calculated MI
and PD values then performed randomization of profiles as described (above) and evaluated how often
20
the difference in the trend between randomized profiles and original profiles for these random networks
was found to be equal or higher for the real network.
Method S3: Statistical analysis of synthetic interactions-Kolmogrov-Smirnov test. To check the
normality of the profile distance distribution within the synthetically interacting gene sets, we
performed a Kolmogorov-Smirnov (KS) test measuring the maximum absolute difference between our
data and standard normal distribution. The standard normal distribution was obtained from profile
distances between all the 1,086 genes in the network. The KS test determines if the null hypothesis, that
the data comes from a standard normal distribution, or the alternative, that it does not come from such a
distribution, applies. A result of h = 1 is returned if the test rejects the null hypothesis or 0 otherwise.
The decision is transformed into a significance level (p-value) directly compared to a chosen threshold,
5%; if p-value was less than 5%, then the difference is significant, otherwise it is not considered. As
illustrated in Table S11, the KS test revealed that the synthetic interactions distribution does not come
from a standard normal distribution. A significant difference was observed between the random pairs in
the network and the synthetic interactions profile distances at both conditions. These results show that
the evolutionary affinities of the genes involved in the synthetic interactions at both conditions LNA
and DA differ from overall pairwise distance distributions of the network.
Method S4: Statistical analysis of synthetic interactions and co-sets-Hypergeometric test. In our
analysis we performed a hypergeometric test on the profile distance data of the synthetic interactions
genes. Three success values were chosen; greater or equal to 1, greater or equal to 2, and greater or
equal to 3 for both conditions. The reason behind these choices is that most of the profile distance
values lie above 1. The test provides a statistical measure to examine if the synthetic interaction
distances are enriched for larger than random network values or not. As illustrated in Table S15,
probabilities at dark conditions were higher than those at light condition in most cases. Similarly,
statistically significant enrichment was observed in co-sets (see main text for details of these
enrichment analyses).
Method S5: Transformed metabolic networks and paired-ortholog analyses. To examine if the
found dynamic and static pairs occur in Arabidopsis and yeast networks. The latest version of a yeast
metabolic network (http://sourceforge.net/projects/yeast/files/yeast_7.11.zip) was used to generate a
gene-centric network. The yeast gene-centric network consisted of 117,559 edges (connections)
between 903 metabolic gene products, with an average connectivity of ~260, and clustering coefficient
of 0.83. The network has 2 connected components. We transformed the Arabidopsis metabolic network,
reported by Mintz-oron et al., 2011, which consisted of 1,363 unique reactions and 1,078 metabolites 6.
The Arabidopsis gene-centric network consisted of 369,479 edges (connections) between 1,792
metabolic gene products with an average connectivity of ~73, and clustering coefficient of 0.50. The
network has 5 connected components.
21
The ortholog annotation of genes from multiple genomes between C. reinhardtii, A. thalina and S.
cerevisiae were derived from the 11th version of OMA (Orthologus Matrix) Database7
(http://omabrawer.org). The Chlamydomonas IDs are from mapping of Augustus 5 (with BLAST) to
Phytozome version 8 (http://www.phytozome.net/).
Gene ontology analysis was used to identify enrichment, the conservation or rewiring of functional
categories of the orthologue genes that had a common ancestor between C. reinhardtii to A. thalina and
S. cerevisiae. The entire interologs static and dynamic pairs found between C. reinhardtii/S. cerevisiae
and C. reinhardtii/A. thalina were used to examine the functional categories. We identified the multiple
GO enriched functional categories that conserved and varied between the three model organisms as
shown in Figure 4 and Figure S5 A-D. Over-representation of GO terms in a set of genes was
determined by using the Biological Networks Gene Ontology tool (BiNGO)
(http://ww.psb.ugent.be/cbd/papers/BiNGO/) 8. BiNGO retrieved the relevant GO annotation then tested
for significance using the Hypergeometric test with no corrected for multiple testing. Statistically,
hypergeometric distribution is a measure of the probability that describes the number of successes in a
sequence of n draws from a finite population without replacement. GO categories that were
significantly over-represented among the differentially expressed proteins were identified.
Method S6: Correlated and anti-correlated analyses of expression and flux. The crosstalk between
metabolic reactions and gene regulation are essential and fundamental to the living cell. The cell adjusts
gene regulatory network and shifts metabolic phenotypic states according to environmental changes. In
this segment, differential expression of mRNA under two conditions (dark with acetate and light
without acetate) was compared with metabolic flux levels based on the reactions as shown in Figure S8.
The reactions were optimized for biomass production under the corresponding light (no acetate) and
dark with acetate growth conditions. We considered the collective expression of all genes in reactions
rather than individual genes; we computed the total expression levels of all genes from dark (with
acetate) to light (without acetate) conditions.
The expression patterns and regulation of a number of enzymes that catalyze the same reaction are often
different 9. To explore the agreement between changes of gene expression and predicted fluxes under
light or dark conditions, we compared these results to the reaction’s predicted fluxes by flux balance
analysis (FBA) to demonstrate the concordances of transcriptional and flux changes (Fig. S8). We
further characterized these agreements based on the compartments that the reactions occur (Fig. S9).
22
Supplementary Notes: S1 and S2
Note S1: Co-sets examples for dark and light conditions. Based on Figure 6, co-set1 in set 4 (Table
S18) can be extracted as an example of a co-set with an average profile distance of 0, which signifies
that the genes associated with the co-set have similar profile distances. The reactions in this co-set,
namely URO (via urate oxidase) and XNDH (via xanthine dehydrogenase) were found to be conserved
in Amoebazoa, Fungi, Metazoa, Stramenopiles, and Verdiplantae, and they are involved in purine
metabolism. Specifically, in XNDH, the xanthine dehydrogenase dehydrogenases the xanthine into
Urate, which is oxidized into 5-Hyroxyisourate by urate oxidase and the URO reaction. In C.
reinhardtii, a urate oxidase type II enzyme was found to be activated early in response to nitrogen
starvation 10. Generally however, URO involves the oxidation of uric acid to form allantoin 11, and
XNDH involves the oxidation of hypoxanthine to xanthine and xanthine to uric acid during purine
catabolism 12 (Fig. S13).
On the other hand, co-set 5 in set 2 (Table S18) serves as an example of a co-set with a long average
profile distance (average distance of co-set 5 is 3.2). Although the genes of this co-set differ
evolutionarily due to their long profile distance, their reactions share the same biological function;
which is N-glycan biosynthesis (DOLPGT1, GLCNACT, G12MT2) with the exception of
(DM_asnglcnacglcnacman-LPAREN-man-RPAREN-man) being a demand reaction. In the n-glycan
biosynthesis, the first step is the transferase reaction of Uridine diphosphate N-acetylglucosamine and
the lipid P-Dol (dolichol phosphate) to generate a protein bound N-glycan in the cytoplasmic side of
the endoplasmic reticulum’s (ER’s) membrane. This precursor is modified by a series of reactions in
the ER and the Golgi, starting with its catalysis by glycotransferase (DOLPTG1) to activate a
nucleotide sugar and converts it into a nucleophillic glycosyl acceptor. This ensures proper folding of
the nascent protein through enabling its interaction with ER-resident chaperones, such as calnexin and
calreticulin. The reactions of GLNACT (N-acetylglucosaminyltransferase) and G12MT2 (via ALG11
mannosyltransferase with a DXD motif) produce the sequon Asn-X-Ser/Thr through DOLPTG1
(glycosyltransferase). Found in many glycosyltransferases that utilize nucleotide sugars, the DXD
motif is thought to be involved in the binding of a manganese ion that is required for association of the
enzymes with nucleotide sugar substrates. GLCNACT is associated with the CRv4_Au5.s13.g4777.t1
or CRv4_Au5.s16.g6452.t1 genes, and G12MT2 is associated with CRv4_Au5.s14.g5384.t1 (glycosyl
transferase). DOLPGT1, and GLCNACT are conserved in all 13 linages except Aloveolata and
Choanoflgellida, while the G12MT2 is not conserved in any of the 13 lineages (Fig. S14).
The genes associated with coupled reactions of co-set4 of set 1 have a low profile distance (1.5), which
means that they differ slightly in terms of evolutionary affinities. Their genes were conserved in all 13
eukaryotic lineages except Amoebozoa and Euglenzoa. The reactions of this co-set are DOLPMT2
(dolichyl-phosphate-mannose-glycolipid-alpha-mannosyltransferase), GLCNACT, and DOLPMT, and
they are also involved in the N-Glycan biosynthesis pathway, the same pathway as the previous
23
example. Both GLNACT and DOLPMT are needed as precursor reactions for the production of the
Asn-X-Ser/Thr sequon through DOLPMT2 (Fig. S14).
An average profile distance of 3.2 was found between genes associated with reactions of co-set 1 in set
6 (Table S17). The coupled reactions 3HAD120 and 3OAR40 are involved in fatty acid biosynthesis
(Fig. S14). Where 3HAD120 produces dodecanoic acid through the enzyme (3R)-3-Hydroxybutanoyl-
[acyl-carrier-protein] hydro-lyase, and 3OAR40 produces the Butyryl-acp through 3-oxoacyl-[acyl-
carrier-protein] reductase (n-C4:0). The reaction 3HAD120 is associated with seven genes; five of
them are conserved in Ameobazoa, Heptophceae, and Viridiplantae. The 3OAR40 reaction is carried
out through the same seven genes with the addition of CRv4_Au5.s3.g10590.t1.
CRv4_Au5.s3.g10590.t1 is conserved in Rhodophyta and Stramenopiles and codes for an enzyme that
resembles an enoyl reductase, which catalyzes the second reduction step in fatty acid synthesis. This
enzyme is also predicted to belong to the SDRs family of diverse oxidoreductases that have a single
domain with structurally conserved Rossmann fold (alpha/beta folding pattern with a central beta-
sheet) NAD(P)(H) binding region and a structurally diverse C-terminal region.
Note S2: Synthetic interactions examples for light and dark conditions. From Fig. 6, the largest
profile distance was 3.6 and the shortest profile distance was zero. Both values exist frequently in lethal
interactions and synthetic sick interactions (80% to 100 % growth reduction).
The longest profile distance in a lethally interacting pair and under light condition was found to be 3.6.
This value is because one of the genes is conserved in all lineages while the other is not conserved in
any of the 13 eukaryotic lineages. For example the gene CRv4_Au5.s16.g6321.t1 is conserved in all
lineages while CRv4_Au5.s12.g3687.t1 is not conserved in any of the lineages. From biological aspect,
CRv4_Au5.s16.g6321.t1 is involved in the N-glycan metabolism through the UDP-GlcNAc:dolichyl-
phosphate N-acetylglucosaminephosphotransferase reaction, while the other gene
(CRv4_Au5.s12.g3687.t1) is utilized in the fructose and mannose metabolism as D-xylose isomerase.
The metabolisms in which both genes are involved are related through the production of mannose-GDP
(Fig. S14).
In contrast, the shortest profile distance among the lethal gene pairs was zero. When the profile
distance is zero, the evolutionary profile of the involved genes is the same. This means they are both
either conserved in same lineages or are both conserved in none of the thirteen eukaryotic lineages. For
example CRv4_Au5.s17.g7556.t1 (glucose-6-phosphate-1 dehydrogenase) and
CRv4_Au5.s16.g6244.t1 (cytochrome P450) are conserved in none of the thirteen lineages and are
utilized in the Pentose phosphate pathway and the Biosynthesis of steroids respectively. The gene
CRv4_Au5.s17.g7556.t1 is used to catalyze the reaction glucose-6-phosphate-1-dehydrogenase (g6p-
A) which converts D-Glucose 6-phophate into D-Glucono-1,5-lactone 6-phosphate in the pentose
phosphate pathway. The other gene, CRv4_Au5.s16.g6244.t1, is used in steroids biosynthesis
specifically for the oxidation of calcidiol into calcitriol (Fig. S16).
24
Under dark growth, the lethal gene pair, CRv4_Au5.s9.g15712.t1 and CRv4_Au5.s10.g816.t1, has a
large profile distance value of 3.4. The first gene is conserved in all lineages while other one is not
conserved in any other lineages. Biologically, CRv4_Au5.s9.g15712.t1 is involved in glycine, serine
and threonine metabolism, while CRv4_Au5.s10.g816.t1 is involved in Nitrogen metabolism. In the
glycine, serine and threonine metabolism, the glycine is converted into L-serine by the reaction glycine
hydroxymethyltransferase, specifically associated with C Rv4_Au5.s9.g15712.t1 gene.
CRv4_Au5.s10.g816.t1 is associated with glycine synthase for the conversion of glycine into ammonia
in the nitrogen metabolism (Fig. S17).
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