new tools for metabolic engineering of bacteria · metabolic engineering of microbes ... the...
TRANSCRIPT
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Metabolic Engineering of Microbes for Production of Terpenoid Drugs
Jay D. Keasling
Synthetic Biology Dept., LBNLChemical Engineering Dept., UC
Berkeley, CA 94720
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Malaria• Caused by
Plasmodium, a single-cell protozoan
–Transmitted by Anopheles mosquito
–Destroys red blood cells
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Malaria
• Economists have proposed that malaria decreases the GDP of affected countries by as much as 50%.
• 1.5-2.7 million people die of malaria every year–90% of the victims
are children–40% of the world’s
population is at risk
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Chloroquine-based drugs
• Most widely-used drugs to treat malaria
• Plasmodium in South America and Southeast Asia is largely resistant to chloroquine
NCl
NHH3C
H3C
H3C
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Artemisia annuaArtemisinin
OO O
O
O
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ChemotherapeuticsTerpenoids> 50,000 known molecules
Essential oils
OHMenthol
C-10 Monoterpene
Carotenoids
LycopeneC-40 Tetraterpene
TaxolC-20 Diterpene
EleutherobinC-20 Diterpene
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Terpenoid metabolic pathwaysOPO(OH)OP(OH)2O OPO(OH)OP(OH)2O
OPO(OH)OP(OH)2O
OPO(OH)OP(OH)2OFarnesyl pyrophosphate(FPP)
Geranyl pyrophosphate(GPP)
Dimethylallyl pyrophosphate(DMAPP)
Isopentenyl pyrophosphate(IPP)
OPO(OH)OP(OH)2OGeranyl pyrophosphate
(GGPP)
Monoterpenes
Sesquiterpenes
DiterpenesCarotenoids
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Artemisinin metabolic pathwaysOPO(OH)OP(OH)2O OPO(OH)OP(OH)2O
OPO(OH)OP(OH)2O
OPO(OH)OP(OH)2O
OO O
OO
Farnesyl pyrophosphate(FPP)
Geranyl pyrophosphate(GPP)
Dimethylallyl pyrophosphate(DMAPP)
Isopentenyl pyrophosphate(IPP)
Amorphadiene
Artemisinin
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Pathways for terpenoid precursor biosynthesis
G6P
FDP
G3P
PYR
AcCoA
OAA
MAL
CIT
IPP DMAPP
GPP
DHAP
PEP
FPP
MEV
Mevalonate pathway
DXP
Non-mevalonate (DXP) pathway
Monoterpenes
Sesquiterpenes
GGPP
TCACycle
Gly
coly
sis
Diterpenes
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Artemisinin-based drugs• The current cost for an artemisinin-
based drug is approximately $2.25.–Artemisinin generally adds $1.00-
1.50 to the cost for drugs–Most developing countries spend
less than $4/person/year on health care
• As many as 10-12 treatments are needed for each person annually
• World Health Organization estimates that 700 tons will be needed annually
OO O
O
O
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GoalReduce the cost of artemisinin-based anti-malarial drugs by an order of magnitude.
ApproachEngineer a bacterium to produce artemisinin from an inexpensive, renewable resource. E. coli
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Microbial production of artemisininAdvantages
• Microbial fermentations are relatively simple to scale up
• Inexpensive starting materials can be used• Production not affected by weather
conditions• Pure product can be made (free of other
contaminating terpenes)
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Microbial production of artemisinin
• Need the genes for all of the enzymes in the pathway
• Not always simple to express in microbes the genes from very different organisms
• Need to balance metabolic pathways to optimize production
• Need a good “platform organism” with appropriate gene expression tools
Challenges
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Synthesis of artemisinin in E. coli
Identify the enzymes
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Amorphadiene and artemisinin biosynthetic pathway
O PP
Farnesyl diphosphate(FPP)
AmorphadieneSynthase
H O
O
Artem isinic Acid
Am orphadieneCytochrom eP450
OO O
O
O
Artem isinin
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Synthesis of artemisinin in E. coli
Clone the genes
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0
0.4
0.8
1.2
1.6
0 10 20 30 40 50
Time (hrs)
Cel
l gro
wth
(OD6
00nm
)
0
1
2
3
4
5
6
7
Vetis
pira
dien
e (µ
g/l)
0
0.4
0.8
1.2
1.6
Cel
l gro
wth
(OD6
00nm
)
0
2
4
6
8
10
12
Cadi
nene
(µg/
l)
0
0.4
0.8
1.2
1.6
2
Cel
l gro
wth
(OD
600n
m)
0
0.04
0.08
0.12
0.16
5-ep
i-aris
tolo
chen
e (µ
g/l)
Poor performance of plant sesquiterpene cyclases
Low yields: 0.05 to 0.7 ng/mL/OD
Expression of rare E. coli codon tRNA did not much help
5-epi-aristolochene
Cadinene
Vetispiradiene
Martin et al., Biotech. Bioeng. 2001
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Amorphadiene and artemisinin biosynthetic pathway
O PP
Farnesyl diphosphate(FPP)
AmorphadieneSynthase
H O
O
Artem isinic Acid
Am orphadieneCytochrom eP450
OO O
O
O
Artem isinin
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Assembly of rcAmorphadiene cyclase• Take gene sequence from patent• Optimize sequence for expression in desired
host• Synthesize 84 oligonucleotides of ~40
basepairs each• Assemble into complete gene using the
polymerase chain reaction (PCR)
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Terpene cyclase gene assembly84 primers ~40-mers
Screen, sequence and fix
1st round of PCR
2nd round of PCR
with end primers1.7 kb
Rescue gene
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Amorphadiene production by the synthetic amorphadiene cyclase
0
0.02
0.04
0.06
0.08
0.1
0 2 4 6 8 10 12 14
Time (Hours)
Amor
phad
iene
pro
duct
ion
(ug/
ml/O
D60
0)
142-fold improvement over other native cyclases(100 ng/mL/OD)
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Synthesis of artemisinin in cells
Supply of intracellular precursors
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DXP PathwayG6P
FDP
G3P
PYR
AcCoA
OAA
MAL
CIT
DHAP
PEP DXP
Non-mevalonate (DXP) pathway
IPP DMAPP
GPP
FPP Amorphadiene
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Eliminating bottlenecks in the DXP pathway
G6P
FDP
G3P
PYR
AcCoA
OAA
MAL
CIT
IPP
DHAP
PEP
DMAPP
GPP
FPP
DXP
DXS
IdI
IspA
Amorphadiene
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Amorphadiene production by the synthetic amorphadiene cyclase
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0 2 4 6 8 10 12 14
Time (Hours)
Am
orph
adie
ne p
rodu
ctio
n(u
g/m
l/OD
600)
Native DXP pathway
Engineered DXPpathway
Additional 3-fold(300 ng/mL/OD)
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Enhancing all of the steps in the DXP pathway
G6P
FDP
G3P
PYR
AcCoA
OAA
MAL
CIT
IPP
DHAP
PEP
DMAPP
GPP
FPP
DXP
pyridoxinethiamine
IdI
IspA
Amorphadiene
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Introducing an entirely new pathway
G6P
FDP
G3P
PYR
AcCoA
OAA
MAL
CIT
IPP
DHAP
PEP
DMAPP
GPP
FPP
DXP
MEVMevalonate pathway
Amorphadiene
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(1.2kb) (1.5kb) (1.6kb)Acetyl-CoA
Construction of synthetic mevalonate pathway operons
P
HMGS tHMGRatoBMevTMevalonate
IPPDMAPP
MBI(1.3kb) (1.3kb)(1.2kb)P
PMK MPDMK idi(0.5kb)
Mevalonate
E. coli genes Yeast genes
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Amorphadiene from the full mevalonate pathway
30-fold improvement(3 mg/L/OD)
Mevalonate pathway
DXP pathway
0
0.5
1
1.5
2
2.5
3
3.5
0 2 4 6 8 10 12 14Time (Hours)
Am
orph
adie
ne p
rodu
ctio
n(u
g/m
l/OD
600)
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(1.2kb) (1.5kb) (1.6kb)
Construction of synthetic mevalonate pathway operons
Acetyl-CoAP
HMGS tHMGRatoB
Mevalonate
MevT
MBIIPP
DMAPP(1.3kb) (1.3kb)(1.2kb)P
PMK MPDMK idi(0.5kb)
Mevalonate
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Optimizing the MEV pathwayG6P
FDP
G3P
PYR
AcCoA
OAA
MAL
CIT
IPP
DHAP
PEP
DMAPP
GPP
FPP
DXP
MEV
Exogenous MEV
Amorphadiene
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Optimizing the MEV pathwayG6P
FDP
G3P
PYR
AcCoA
OAA
MAL
CIT
IPP
DHAP
PEP
DMAPP
GPP
FPP
DXP
MEV
Exogenous MEV
Amorphadiene
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Increasing concentrations of mevalonate inhibit growth
5 mM10 mM20 mM40 mM
[Mevalonate]
0
0.2
0.4
0.6
0.8
1
1.2
0 2 4 6 8 10 12
Time (hours)
Cel
l Gro
wth
(OD
600)
Martin et al. 2003. Nat. Biotechnol. 21:796-802.
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Optimizing the MEV pathwayG6P
FDP
G3P
PYR
AcCoA
OAA
MAL
CIT
IPP
DHAP
PEP
DMAPP
GPP
FPP
DXP
MEV
Exogenous MEV
Amorphadiene
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Co-expression of sesquiterpene cyclase alleviates growth inhibition
FPPAmorphadiene
AmorphadieneCyclase (ADS)
OP P
No ADS With ADS
5 mM
10 mM20 mM40 mM
[Mevalonate]
0
0.2
0.4
0.6
0.8
1
1.2
0 2 4 6 8 10 12
Time (hours)
Cel
l Gro
wth
(OD
600)
0
0.2
0.4
0.6
0.8
1
1.2
0 2 4 6 8 10 12
Time (hours)
Cel
l Gro
wth
(OD
600)
Martin et al. 2003. Nat. Biotechnol. 21:796-802.
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0
200
400
600
800
1,000
1,200
1,400
0 2 4 6 8
FPP
01,0002,0003,0004,0005,0006,0007,0008,000
0 2 4 6 8
Cou
nts
(CPM
)
IPP
DMAPPFPP
IPP / DMAPP
Time (hrs)Time (hrs)
Intracellular prenyl pyrophosphates in MevB-supplemented strains
MevalonateMevB
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Intracellular prenyl pyrophosphates in MevB-supplemented strains
0
200
400
600
800
1,000
1,200
1,400
0 2 4 6 8
FPP
0
1,000
2,000
3,000
4,000
5,000
6,000
7,000
8,000
0 2 4 6 8
Cou
nts
(CPM
)
IPP
DMAPPFPP
ispAidi
MevB
IPP / DMAPP
Time (hrs)Time (hrs)
Mevalonate
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Intracellular prenyl pyrophosphates in MevB-supplemented strains
0
200
400
600
800
1,000
1,200
1,400
0 2 4 6 8
FPP
0
1,000
2,000
3,000
4,000
5,000
6,000
7,000
8,000
0 2 4 6 8
Cou
nts
(CPM
)
IPP
DMAPPFPP
ispAidi
MevB
IPP / DMAPP
Time (hrs)Time (hrs)
Mevalonate
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Intracellular prenyl pyrophosphates in MevB-supplemented strains
0
200
400
600
800
1,000
1,200
1,400
0 2 4 6 8
FPP
0
1,000
2,000
3,000
4,000
5,000
6,000
7,000
8,000
0 2 4 6 8
Cou
nts
(CPM
)
IPP
DMAPPFPP
ispAidi
MevB
IPP / DMAPP
AmorphadieneADS
Time (hrs)Time (hrs)
Mevalonate
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Accumulation of IPP/DMAPP inhibits growth
G6P
FDP
G3P
PYR
AcCoA
OAA
MAL
CIT
IPP DMAPP
DHAP
PEP
GPP
FPP
DXP
MEV
Exogenous MEV
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Accumulation of FPP also inhibits growth
G6P
FDP
G3P
PYR
AcCoA
OAA
MAL
CIT
IPP DMAPP
DHAP
PEP
GPP
FPP
DXP
MEV
Exogenous MEV Amorphadiene
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Production of amorphadiene relieves inhibition
G6P
FDP
G3P
PYR
AcCoA
OAA
MAL
CIT
IPP DMAPP
DHAP
PEP
GPP
FPP
DXP
MEV
Exogenous MEV Amorph
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(1.2kb) (1.5kb) (1.6kb)
Construction of synthetic mevalonate pathway operons
Acetyl-CoAP
HMGS tHMGRatoB
Mevalonate
MevT
MBIIPP
DMAPP(1.3kb) (1.3kb)(1.2kb)P
PMK MPDMK idi(0.5kb)
Mevalonate
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Balancing enzymatic reactions in the cell
atoB hmgS gene 2
Ac-CoA AcAc-CoA HMG-CoA MevAtoB HmgS HmgR
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Balancing enzymatic reactions in the cell
atoB hmgS gene 2
Ac-CoA AcAc-CoA HMG-CoA Mev
AtoB HmgS HmgR
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Using individual promoters of different strengths to balance a pathway
P1
atoB
P3
hmgS
P2
hmgR
Ac-CoA AcAc-CoA HMG-CoA MevAtoB HmgS HmgR
mRNA
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Synthetic operons
atoB hmgS
P
hmgRDNA
mRNA
Ac-CoA AcAc-CoA HMG-CoA Mev
AtoB HmgS HmgR
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Balancing reaction using ribosome binding site (RBS) strength
weakRBS
weakRBS
StrongRBS
Ribosomes Proteins
Ac-CoA AcAc-CoA HMG-CoA MevAtoB HmgS HmgR
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Balancing reaction using mRNA stability
Ac-CoA AcAc-CoA HMG-CoA MevAtoB HmgS HmgR
RNase mRNA
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A family of synthetic hairpins
acgucgacagguaccguauuuut1/2 = 2.6 min
pTC40
cc
c
c
uu
uu
g
g
g
a
a
u
gaccu
gggau
u
a
ga
gguaccguauuuut1/2 = 4.9 min
pHP14
cc
c
c
auu
g
g
gau
u
a
ga
gguaccguauuuu
u
uua
g
cc
a
a
g
u
cu u
a
u a
t1/2 = 2.1 min
pHP15t1/2 = 6.1 min
c
c
c
c
c
c
u
uuu
uu
g
gg
g
g
a
aaa
a
uu
u
u
a
ga
ggau
gguaccguauuuu
c gu a
pHP8
c
c
c
c
c
c
u
uauu
g
g ggg
g
g
a
a
aaa
a
uu
u
u
u
a
ga
gguaccguauuuut1/2 = 6.8 min
pHP10
cc
c
c
uauu
g
g
g
a
a
u
ccu
u
g ggg
a
a
aau
u
u
u
a
ga
gguaccguauuuut1/2 = 5.5 min
pHP9
c
c
c
c
c
c
u
uuu
uu
g
g ggg
g
g
a
a
aaa
a
uu
u
u
u
a
ga
gguaccguauuuut1/2 = 8.3 min
pHP4
t1/2 = 19.8 min
pHP17
acc
c
c
a
uu
g
g
gau
u
ga
gguaccguauuuu
g
cga
g
au
c
c
a
a
ug u
a
a t
uuu
uau acu ga
gc
t1/2 = 12.5 min
pHP16
c
c
c
c
c
c
u
uuu
uu
g
gg
g
g
g
g
a
a
a
aa
a
uu
c
u
u
a
ga
gguaccguauuuu
cuucu
g cagag
uu
u
a
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Synthetic operons
atoB hmgS
P
hmgRDNA
mRNA
Ac-CoA AcAc-CoA HMG-CoA Mev
AtoB HmgS HmgR
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Increasing flux into the mevalonate pathway
G6P
FDP
G3PDHAP
PEP
PYR
AcCoA
OAA
MAL
CIT
IPP DMAPP
GPP
FPP
DXP
MEVMevalonate pathway
Glucose
Amorphadiene
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Increasing flux into the mevalonate pathway
0
2
4
6
8
10
12
0 2 4 6 8 10 12 14
Time (Hours)
Am
orph
adie
ne p
rodu
ctio
n (u
g/m
l/OD
600)
LBLB + GlycerolTB
~3-fold improvement(10 mg/L/OD)
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Amorphadiene is lost from bioreactors O2
0
0.5
1
1.5
2
2.5
3
3.5
0 2 4 6 8 10 12 14Time (hr)
Am
orph
adie
ne p
rodu
ctio
n(u
g/m
l/OD
600)
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Amorphadiene is lost from bioreactors O2
Condenser
Amorphadiene
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Amorphadiene production in a two-phase fermentation O2
0
100
200
300
400
500
0 12 24 36 48 60 72
Time (hr)
Am
orph
adie
ne
conc
entr
atio
n (m
g/L)
1 million fold improved production!
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What’s left?
Amorphadiene
ArtemisinicAcid
H
H
H
H
O
HO
Artesunate(Artesunic Acid)
O
O
H
H HH
OO
OH
O
OH
O
HO
O
H
H
H
ArtemisinicAcid
O
O
H
H HH
OO
OMeH
O
O
H
H HH
OO
OEtH
O
O
H
H HH
OO
OH
O
OH
Artelinate(Artelinic Acid)ArteetherArtemether
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Artemisinin costs
Artesunate treatment
SP + Artesunate treatment
Current cost of drug $2.25-2.50
Cost with new process, including capital costs, yield 50 g/L
$.17/.10 $.27/.15
Cost with new process, with donated capital, yield 50 g/L
$.11/.07 $.21/.12
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Enzyme engineering to produce mono- and diterpenes
G6P
FDP
G3P
PYR
AcCoA
OAA
MAL
CIT
IPP
GPP
DHAP
PEP
DMAPP
FPP
MEV
DXP
Monoterpenes
Sesquiterpenes
GGPP Diterpenes
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Design of GPP and GGPP synthases
WT ipsA 75-ECIHAYSLIHDDLPAMDDDDLRRGLP-100Y80D 75-ECIHADSLIHDDLPAMDDDDLRRGLP-100S81F 75-ECIHAYFLIHDDLPAMDDDDLRRGLP-100
-- XXxxxDDxxxxD -- – Type 25th 4th
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Production of monoterpenes in Escherichia coli
G6P
FDP
G3P
PYR
AcCoA
OAA
MAL
CIT
IPP
GPP
DHAP
PEP
DMAPPMEV
DXP
MyrceneArabidopsis thaliana
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Production of diterpenes in Escherichia coli
G6P
FDP
G3P
PYR
AcCoA
OAA
MAL
CIT
IPP
DHAP
PEP
DMAPPMEV
DXP
GGPP
ent-Kaurenefungi
CasbeneCastor bean
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Taxol from the Pacific Yew
HOO
OHCH3
O
O O
CH3 CH3O
O
CH3
OCH3O
H3CHN
O
HO
O
12
34 5
614
13
1211
109
8 715 16
17
18
19
20
O
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Eleutherobin from marine coral
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Prostratin• Protein kinase C activator• Isolated from the stems of the
small Samoan tree Homalanthus nutans
• Inhibits human immunodeficiency virus type 1 (HIV-1) infection yet up-regulates viral expression from latent proviruses
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AcknowledgementsGraduate StudentsTrent A. CarrierKristala JonesChristina SmolkeDoug PiteraSydnor WithersBrian PflegerYasuo Yoshikuni
Post-docsArtem KhlebnikovSeon-Won KimVincent MartinJack NewmanKinkead Reiling
FundingNational Science FoundationOffice of Naval ResearchMaxygenDiversaUniversity of California Discovery Grant