Metabolic Engineering of Microbes for Production of Terpenoid Drugs

Jay D. Keasling

Synthetic Biology Dept., LBNLChemical Engineering Dept., UC

Berkeley, CA 94720

Malaria• Caused by

Plasmodium, a single-cell protozoan

–Transmitted by Anopheles mosquito

–Destroys red blood cells

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

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

Artemisia annuaArtemisinin

OO O

O

O

ChemotherapeuticsTerpenoids> 50,000 known molecules

Essential oils

OHMenthol

C-10 Monoterpene

Carotenoids

LycopeneC-40 Tetraterpene

TaxolC-20 Diterpene

EleutherobinC-20 Diterpene

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

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

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

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

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

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)

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

Synthesis of artemisinin in E. coli

Identify the enzymes

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

Synthesis of artemisinin in E. coli

Clone the genes

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

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

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)

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

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)

Synthesis of artemisinin in cells

Supply of intracellular precursors

DXP PathwayG6P

FDP

G3P

PYR

AcCoA

OAA

MAL

CIT

DHAP

PEP DXP

Non-mevalonate (DXP) pathway

IPP DMAPP

GPP

FPP Amorphadiene

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

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)

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

Introducing an entirely new pathway

G6P

FDP

G3P

PYR

AcCoA

OAA

MAL

CIT

IPP

DHAP

PEP

DMAPP

GPP

FPP

DXP

MEVMevalonate pathway

Amorphadiene

(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

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)

(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

Optimizing the MEV pathwayG6P

FDP

G3P

PYR

AcCoA

OAA

MAL

CIT

IPP

DHAP

PEP

DMAPP

GPP

FPP

DXP

MEV

Exogenous MEV

Amorphadiene

Optimizing the MEV pathwayG6P

FDP

G3P

PYR

AcCoA

OAA

MAL

CIT

IPP

DHAP

PEP

DMAPP

GPP

FPP

DXP

MEV

Exogenous MEV

Amorphadiene

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.

Optimizing the MEV pathwayG6P

FDP

G3P

PYR

AcCoA

OAA

MAL

CIT

IPP

DHAP

PEP

DMAPP

GPP

FPP

DXP

MEV

Exogenous MEV

Amorphadiene

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.

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

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

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

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

Accumulation of IPP/DMAPP inhibits growth

G6P

FDP

G3P

PYR

AcCoA

OAA

MAL

CIT

IPP DMAPP

DHAP

PEP

GPP

FPP

DXP

MEV

Exogenous MEV

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

Production of amorphadiene relieves inhibition

G6P

FDP

G3P

PYR

AcCoA

OAA

MAL

CIT

IPP DMAPP

DHAP

PEP

GPP

FPP

DXP

MEV

Exogenous MEV Amorph

(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

Balancing enzymatic reactions in the cell

atoB hmgS gene 2

Ac-CoA AcAc-CoA HMG-CoA MevAtoB HmgS HmgR

Balancing enzymatic reactions in the cell

atoB hmgS gene 2

Ac-CoA AcAc-CoA HMG-CoA Mev

AtoB HmgS HmgR

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

Synthetic operons

atoB hmgS

P

hmgRDNA

mRNA

Ac-CoA AcAc-CoA HMG-CoA Mev

AtoB HmgS HmgR

Balancing reaction using ribosome binding site (RBS) strength

weakRBS

weakRBS

StrongRBS

Ribosomes Proteins

Ac-CoA AcAc-CoA HMG-CoA MevAtoB HmgS HmgR

Balancing reaction using mRNA stability

Ac-CoA AcAc-CoA HMG-CoA MevAtoB HmgS HmgR

RNase mRNA

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

Synthetic operons

atoB hmgS

P

hmgRDNA

mRNA

Ac-CoA AcAc-CoA HMG-CoA Mev

AtoB HmgS HmgR

Increasing flux into the mevalonate pathway

G6P

FDP

G3PDHAP

PEP

PYR

AcCoA

OAA

MAL

CIT

IPP DMAPP

GPP

FPP

DXP

MEVMevalonate pathway

Glucose

Amorphadiene

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)

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)

Amorphadiene is lost from bioreactors O2

Condenser

Amorphadiene

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!

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

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

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

Design of GPP and GGPP synthases

WT ipsA 75-ECIHAYSLIHDDLPAMDDDDLRRGLP-100Y80D 75-ECIHADSLIHDDLPAMDDDDLRRGLP-100S81F 75-ECIHAYFLIHDDLPAMDDDDLRRGLP-100

-- XXxxxDDxxxxD -- – Type 25th 4th

Production of monoterpenes in Escherichia coli

G6P

FDP

G3P

PYR

AcCoA

OAA

MAL

CIT

IPP

GPP

DHAP

PEP

DMAPPMEV

DXP

MyrceneArabidopsis thaliana

Production of diterpenes in Escherichia coli

G6P

FDP

G3P

PYR

AcCoA

OAA

MAL

CIT

IPP

DHAP

PEP

DMAPPMEV

DXP

GGPP

ent-Kaurenefungi

CasbeneCastor bean

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

Eleutherobin from marine coral

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

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