Engineering Microbial Metabolism for

Production of Anti-Malarial Drugs

Jay D. Keasling

Joint BioEnergy Institute

Lawrence Berkeley National Laboratory

University of California, Berkeley

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%.

• 1-3 million

people die of

malaria every year

–90% are children

• 300-500 million

people infectedSource: Roll Back Malaria

World Malaria Report 2005

• Most widely-

used drugs to

treat malaria

Quinine-based drugs

• Plasmodium in many parts of the world is largely resistant to chloroquine

Source: Roll Back Malaria

World Malaria Report 2005

Artemisinin

Artemisia annua

O

O

O

O

O

H

HH

A brief history of artemisinin

168 B.C. Recipes For 52 Kinds Of Diseases found

in the Mawangdui Han Dynasty tomb

Hemorrhoids

340 A.D. Zhou Hou Bei Ji Fang (Handbook of

Prescriptions for Emergency Treatments)

Fevers (malaria)

1972 Active ingredient (artemisinin) isolated

Artemisinin is produced in oil sacs

on Artemisia annua leaves

O

O

O

O

O

H

HH

Artemisinin... ...is produced by trichomes... ...found on

Artemisia annua leaves...

Current process

Artemisinin

O

O

O

O

O

H

HH

Plant synthesis

Purification

Artesunate

O

O

H

H HH

OO

OH

O

OH

OO

O

H

H HH

OO

OH

O

OH

ArtelinateArteetherArtemether

O

O

OMe

O

O

H

HHO

O

OEt

O

O

H

HH

Chemical

Conversions

Artemisinin demand analysisDemand predicted to grow significantly

Source: Boston Consulting Group

Predicted Demand(various scenarios)

Millions of treatments

50

100

150

200

250

300

350

03 04 05 06 07 08 09 10 11 12 13 14 15 16 17 18 19 20

Artemisinin

Treatments

delivered

Artemisinin price swingsFood is more profitable than artemisinin

Artemisinin prices

($/kg)Food prices

(indexed to Jan 2002)

Artemisininprices

Commodityfood prices

2002 2003 2004 2005 2006 2007 2008 2009

Source: Boston Consulting Group

Artemisinin supplyHuge shortfalls predicted 2010 and beyond

50

100

150

200

250

300

350

03 04 05 06 07 08 10 11 12 13 14 15 16 17 18 19 20

Tonnes of

Artemisinin

09

Source: Boston Consulting Group

Produced

Stockpiled Demand

Minimum

supply

Goal

Reduce the cost of artemisinin-based anti-

malarial drugs by an order of magnitude.

Approach

Engineer a microorganism

to produce artemisinin from

an inexpensive, renewable

resource.

Microbial process

Artemisinin

O

O

O

O

O

H

HH

Microbial synthesis

Purification

Artesunate

O

O

H

H HH

OO

OH

O

OH

OO

O

H

H HH

OO

OH

O

OH

ArtelinateArteetherArtemether

O

O

OMe

O

O

H

HHO

O

OEt

O

O

H

HH

Chemical

Conversions

Process design

ispHispG idi ispAdxrdxs ispD

DXP Pathway

OPP

FPPPyruvate

+ G3P

Microbial synthesis

Artemisinic

Acid

H

H

O

HO

Purification

Artemisinin

O

O

O

O

O

H

HHHO

O

H

H

HOO

HO

O

H

H

H

Dihydroartemisinic

Acid

Dihydroartemisinic

Acid Hydroperoxide

ReductionPeroxidationOxidation and

Ring-Closure

O

O

OH

O

O

H

HH

Dihydroartemisinin Artesunate

(Artesunic Acid)

O

O

H

H HH

OO

OH

O

OH

OO

O

H

H HH

OO

OH

O

OHArtelinate

(Artelinic Acid)ArteetherArtemether

O

O

OMe

O

O

H

HHO

O

OEt

O

O

H

HH

Chemical

Conversions

Reduction

Amorphadiene Artemisinic

Acid

ADS

H

H

p450

Synthase Hydroxylase Unit

CPR

H

H

O

HO

ispFispE

OPP

OPP

Central metabolism

OHC

OH

O P

O

O-

O-

H3C CO2H

O

Constructing an artemisinic acid-

producing microbe

ispHispG idi ispAdxrdxs ispD

DXP Pathway

OPP

FPPPyruvate

+ G3P

Microbial synthesis

Amorphadiene Artemisinic

Acid

ADS

H

H

p450

Synthase Hydroxylase Unit

CPR

H

H

O

HO

ispFispE

OPP

OPP

Central metabolism

OHC

OH

O P

O

O-

O-

H3C CO2H

O

E. coli as our chassis

Microbial synthesis

Quinones

ispHispG idi ispAdxrdxs ispD

DXP Pathway

OPP

FPPPyruvate

+ G3PispFispE

OPP

OPP

Central metabolism

OHC

OH

O P

O

O-

O-

H3C CO2H

O

Many of the parts are available only in

the native producer

ispHispG idi ispAdxrdxs ispD

DXP Pathway

OPP

FPPPyruvate

+ G3P

Microbial synthesis

Amorphadiene Artemisinic

Acid

ADS

H

H

p450

Synthase Hydroxylase Unit

CPR

H

H

O

HO

ispFispE

OPP

OPP

Central metabolism

OHC

OH

O P

O

O-

O-

H3C CO2H

O

Artemisia annua

Amorphadiene synthase from A. annua

ispHispG idi ispAdxrdxs ispD AmorphadieneADS

DXP Pathway

H

HOPP

Synthase

FPPPyruvate

+ G3P

Microbial synthesis

ispFispE

OPP

OPP

Central metabolism

OHC

OH

O P

O

O-

O-

H3C CO2H

O

Artemisia annua

5-epi-aristolochene synthase

as a model

ispHispG idi ispAdxrdxs ispD 5-epi-aristolocheneEAS

DXP Pathway

OPP

Synthase

FPPPyruvate

+ G3P

Microbial synthesis

ispFispE

OPP

OPP

Central metabolism

OHC

OH

O P

O

O-

O-

H3C CO2H

O

Tobacco

EAS

PPO

5-epi-aristolocheneFPP

Production of a model isoprenoid

in E. coli

Very low production of isoprenoid

resulted when using the native gene

0.0001

0.001

0.01

0.1

1

10

100

1000

Construct

Iso

pre

no

id (

mg

/L)

ispH ispG idi ispA dxr dxs ispD 5-epi-aristolochene EAS

DXP Pathway

OPP

Synthase

FPP Pyruvate

+ G3P ispF ispE

OPP

OPP

Central metabolism

OHC

OH

O P

O

O-

O-

H3C CO2H

O

Amorphadiene synthase by design

ispHispG idi ispAdxrdxs ispD AmorphadieneADS

DXP Pathway

H

HOPP

Synthase

FPPPyruvate

+ G3P

Microbial synthesis

ispFispE

OPP

OPP

Central metabolism

OHC

OH

O P

O

O-

O-

H3C CO2H

O

ADS

H

H

PPO

AmorphadieneFPP

Martin. 2003. Nat. Biotech. 21:796-802.

Gene synthesis improves amorphadiene

production

142-fold improved

production!

0.0001

0.001

0.01

0.1

1

10

100

1000

Construct

Iso

pre

no

id (

mg

/L)

ispH ispG idi ispA dxr dxs ispD Amorphadiene ADS

DXP Pathway

H

HOPP

Synthase

FPP Pyruvate

+ G3P

Microbial synthesis

ispF ispE

OPP

OPP

Central metabolism

OHC

OH

O P

O

O-

O-

H3C CO2H

O

Microbially-derived artemisinin

ispHispG idi ispAdxrdxs ispD AmorphadieneADS

DXP Pathway

H

HOPP

Synthase

FPPPyruvate

+ G3PispFispE

OPP

OPP

Central metabolism

OHC

OH

O P

O

O-

O-

H3C CO2H

O

The DXP pathway produces

quinones in E. coliG6P

FDP

G3PDHAP

PEP

PYR

AcCoA

OAA

MAL

CIT

DXP

DXP pathway

IPP DMAPP

FPP AmorphadieneQuinones

Pathways for isoprenoid precursor

biosynthesis

MEV

Mevalonate pathway

G6P

FDP

G3PDHAP

PEP

PYR

AcCoA

OAA

MAL

CIT

DXP

DXP pathway

IPP DMAPP

FPP AmorphadieneQuinones

Acetate

Acetyl-CoAP

HMGS tHMGRatoBMevT

Mevalonate

FPP

MBISP

PMK MPDMK idi

Mevalonate

ispA

Construction of synthetic mevalonate

pathway operons

Martin. 2003. Nat. Biotech. 21:796-802.

Constructing the mevalonate

pathway

FDP

G3PDHAP

PEP

PYR

AcCoA

OAA

MAL

CIT

IPP DMAPP

FPP

DXP

MEV

Exogenous

MEV

Quinones

X

The yeast mevalonate pathway improves

yields ~90-fold

0.0001

0.001

0.01

0.1

1

10

100

1000

Construct

Iso

pre

no

id (

mg

/L)

PMK MPD MK idi ispA HMGS atoB tHMGR Amorphadiene

Mevalonate pathway (TOP)

H

H

Mevalonate pathway

(BOTTOM)

OPP

Synthase

FPP Mevalonate

OH

OHCH3

HO2CH3C SCoA

O

Acetyl-

CoA

Martin. 2003. Nat. Biotech. 21:796-802.

Optimizing the mevalonate pathwayG3PDHAP

PEP

PYR

AcCoA

OAA

MAL

CIT

IPP DMAPP

FPP

DXP

Sesquiterpene

MEV

Exogenous

MEV

Optimizing the mevalonate pathwayG3PDHAP

PEP

PYR

AcCoA

OAA

MAL

CIT

IPP DMAPP

FPP

DXP

MEV

Exogenous

MEV

Sesquiterpene

Increasing concentrations of mevalonate

inhibit growth

5 mM

10 mM

20 mM

40 mM

[Mevalonate]

0

0.2

0.4

0.6

0.8

1

1.2

0 2 4 6 8 10 12

Time (hours)

Ce

ll G

row

th (

OD

60

0)

Martin. 2003. Nat. Biotech. 21:796-802.

Optimizing the mevalonate pathwayG3PDHAP

PEP

PYR

AcCoA

OAA

MAL

CIT

IPP DMAPP

FPP

DXP

Sesquiterpene

MEV

Exogenous

MEV

Co-expression of sesquiterpene

cyclase alleviates growth inhibition

FPP

AmorphadieneAmorphadiene

Cyclase (ADS)

OPP

No ADS With ADS

5 mM

10 mM

20 mM

40 mM

[Mevalonate]

0

0.2

0.4

0.6

0.8

1

1.2

0 2 4 6 8 10 12

Time (hours)

Ce

ll G

row

th (

OD

60

0)

0

0.2

0.4

0.6

0.8

1

1.2

0 2 4 6 8 10 12

Time (hours)

Ce

ll G

row

th (

OD

60

0)

Optimizing the mevalonate pathwayG3PDHAP

PEP

PYR

AcCoA

OAA

MAL

CIT

DXP

MEV

Exogenous

MEV

IPP DMAPP

FPP Sesquiterpene

Martin. 2003. Nat. Biotech. 21:796-802.

Optimizing the mevalonate pathwayG3PDHAP

PEP

PYR

AcCoA

OAA

MAL

CIT

DXP

MEV

Exogenous

MEV

IPP DMAPP

FPP Sesquiterpene

Martin. 2003. Nat. Biotech. 21:796-802.

High expression of amorphadiene synthase

relieves inhibitionG3PDHAP

PEP

PYR

AcCoA

OAA

MAL

CIT

IPP DMAPP

FPP

DXP

Sesquiterpene

MEV

Exogenous

MEV

Balancing expression is difficult with

inducible promoters

• Typically, we use inducible promoters to control

expression of genes in a metabolic pathway

• Inducers are expensive

• Tuning expression can be challenging

PMKMK PMD Idi IspA FPP ADSMev Terpene

IPTG Arabinose

Sensing toxic intermediates to balance

metabolic pathway fluxes

• Can we use the toxicity of FPP to regulate the

metabolic pathways that produce and consume it?

• Can we reduce accumulation of toxic

intermediates?

• How do we regulate the pathway dynamically … in

the face of changing environmental conditions?

PMKMK PMD Idi IspA FPP ADSMev Terpene

Identification of FPP-responsive

promoters

• No terpene synthase to consume

FPP

• Examine differences in gene

expression in the presence and

absence of mevalonate

PMKMK PMD Idi IspAMev

FPPPMKMK PMD Idi IspA

FPP Gene X

Mev

Two FPP-responsive promoters

HMGSatoB tHMGR PMKMK PMD Idi IspA FPP ADS

IPTG

PlacPlacPlac

HMGSatoB tHMGR PMKMK PMD Idi IspA FPP ADS

PrstAPgadEPgadE

Inducible system

Dynamically-controlled system

FPP-responsive promoters

improve terpene production

0300600900

120015001800

pA

DS

prs

tA-A

DS

pA

DS

prs

tA-A

DS

lacUV5-MevT-MBIS pgadE-MevT-MBIS

Am

orp

had

ien

e

Pro

du

cti

on

(m

g/L

)

-IPTG

+IPTG

FPP ADS

PrstA

FPP ADS

PrstA

ADS

IPTG

Plac

ADS

IPTG

Plac

HMGS atoB tHMGR PMK MK PMD Idi IspA

IPTG

Plac Plac

HMGS atoB tHMGR PMK MK PMD Idi IspA FPP

PgadE PgadE

High product titers

with inducer-free control

Constructing the mevalonate pathwayG3PDHAP

PEP

PYR

AcCoA

OAA

MAL

CIT

IPP DMAPP

FPP

DXP

Sesquiterpene

MEV

Growth inhibition by MevT pathway

MevalonatetHMGRAtoB

Acetyl-CoAAcetoacetyl

-CoAHMG-CoA

HMGS

pBad33

pHMGS

pHMGSR

pBad33MevT

pHMGS(C159A)

pMevT(C159A)

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

0 2 4 6 8 10 12

Time (hours)

Ce

ll G

row

th (

OD

60

0) pBad33

pHMGS

pHMGSR

pBad33MevT

pHMGS(C159A)

pMevT(C159A)

pBad33

pHMGS

pHMGSR

pBad33MevT

pHMGS(C159A)

pMevT(C159A)

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

0 2 4 6 8 10 12

Time (hours)

Ce

ll G

row

th (

OD

60

0)

atoB HMGS tHMGR

atoB HMGS tHMGR

atoB HMGS tHMGR

atoB HMGS tHMGR

Accumulation of the toxic intermediate

HMG-CoA

Ac-CoA AcAc-CoA HMG-CoA Mev

AtoB HmgS tHmgR

PMK MPDMK idi ispAHMGSatoB tHMGR AmorphadieneADS

H

HOPP

FPPMevalonate

OH

OHCH3

HO2CH3C SCoA

O

Acetyl-

CoA

HMG-CoA inhibits fatty acid production

PMK MPDMK idi ispAHMGSatoB tHMGR AmorphadieneADS

Mevalonate pathway

(TOP)

H

H

Mevalonate pathway

(BOTTOM)

OPP

Synthase

FPPMevalonate

OH

OHCH3

HO2CH3C SCoA

O

Acetyl-

CoA

HMG-CoA

Fatty acid

biosynthetic

pathway

HMG-CoA inhibits fatty acid production

PMK MPDMK idi ispAHMGSatoB tHMGR AmorphadieneADS

Mevalonate pathway

(TOP)

H

H

Mevalonate pathway

(BOTTOM)

OPP

Synthase

FPPMevalonate

OH

OHCH3

HO2CH3C SCoA

O

Acetyl-

CoA

HMG-CoA

Fatty acid

biosynthetic

pathway

Addition of fatty acids to the growth

medium restores growth

PMK MPDMK idi ispAHMGSatoB tHMGR AmorphadieneADS

Mevalonate pathway

(TOP)

H

H

Mevalonate pathway

(BOTTOM)

OPP

Synthase

FPPMevalonate

OH

OHCH3

HO2CH3C SCoA

O

Acetyl-

CoA

HMG-CoA

HO

OFatty acid addition to the

growth medium

Accumulation of the toxic intermediate

HMG-CoA

mRNA

Ac-CoA AcAc-CoA HMG-CoA Mev

AtoB HmgS tHmgR

P

HMGS tHMGRatoB

Balancing the mevalonate pathway by tuning

mRNA stability and translation efficiency

mRNA

Ac-CoA AcAc-CoA HMG-CoA Mev

AtoB HmgS tHmgR

P1

HMGS tHMGRatoB

RNase E

site

RNase E

site

RBS RBS RBS

Combinatorial design of intergenic

regionsD

CA

B

EE E E

atoB hmgS hmgR

E E

P1

HMGS tHMGRatoB

Pfleger. 2006. Nat. Biotech. 24:1027

How do we choose?

atoB hmgS hmgRE E

atoB hmgS hmgRE E

atoB hmgS hmgREE

Ac-CoA AcAc-CoA HMG-CoA MevAtoB HmgS tHmgR

Ac-CoA AcAc-CoA HMG-CoA Mev

AtoB HmgS tHmgR

Ac-CoA AcAc-CoA HMG-CoA MevAtoB HmgS tHmgR

A fluorescent screen for improved

mevalonate production

MEV

MEV

Ac-CoA

MEV

Biomass

E. coli

GFP-engineered

MEV auxotroph

E. coli

MEV Producer

Mutant

pathway

Pfleger. 2006. Nat. Biotech. 24:1027

B.)B.)

Best producers make about 7X more

mevalonate

Pfleger. 2006. Nat. Biotech. 24:1027

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

Down-regulation of the last

two genes relieves toxicity,

increases Mev production,

and increases Ac-CoA

concentrationRBS

RBSatoB hmgS hmgR

Pfleger. 2006. Nat. Biotech. 24:1027

Synthetic scaffolds:

another way to solve the problem

Ac-CoA AcAc-CoA HMG-CoA Mev

AtoB HmgS tHmgR

tHmgRHmgSAtoB

Connecting metabolic pipes with

synthetic scaffolds

Connecting the enzymes in some way might

reduce loss of intermediate to the bulk

Ac-CoA AcAc-CoA HMG-CoA Mev

AtoB HmgS tHmgR

tHmgRHmgSAtoB

Connecting metabolic pipes with

synthetic scaffolds

Adding additional copies of rate-limiting

enzymes might increase pathway flux

Ac-CoA AcAc-CoA HMG-CoA Mev

AtoB HmgS tHmgR

tHmgRHmgSAtoB

There are no standards

for connecting enzymes together

Ac-CoA AcAc-CoA HMG-CoA Mev

AtoB HmgS tHmgR

tHmgRHmgSAtoB

Synthetic scaffolds co-localize pathway

enzymes and reduce intermediate runoff

tHmgRHmgSAtoB

Ato

B

Hm

gR

Hm

gS

Dueber. 2009. Nat. Biotech. 27:753.

Synthetic scaffolds co-localize pathway

enzymes and reduce intermediate runoff

AtoB HmgS HmgR Scaffold

Ptet PBAD

Ato

B

Hm

gR

Hm

gS

Dueber. 2009. Nat. Biotech. 27:753.

Synthetic scaffolds can control relative

enzyme ratios and optimize flux

Ato

B

Hm

gS

Hm

gR

Hm

gR

Hm

gR

tHmgRHmgSAtoB

Dueber. 2009. Nat. Biotech. 27:753.

Variation in the number of

HmgS and HmgR on the scaffold

Ato

B

Hm

gS

Hm

gR

Hm

gR

Hm

gR

Hm

gR

Ato

B

Hm

gS

Hm

gR

Hm

gR

Ato

B

Hm

gS

Hm

gR

Ato

B

Hm

gS

Hm

gR

Hm

gS

Ato

B

Hm

gS

Hm

gR

Hm

gR

Hm

gS

Ato

B

Hm

gS

Hm

gS

Hm

gRH

mg

S

Hm

gS

Hm

gR

Hm

gR

Hm

gR

Ato

B

Hm

gS

Hm

gS

Hm

gR

Hm

gR

Hm

gR

Hm

gR

Ato

B

Hm

gS

Hm

gS

Hm

gRH

mg

S

Hm

gS

Hm

gR

Ato

B

Hm

gS

Hm

gS

Hm

gS

Hm

gS

Hm

gR

Hmg

R

HmgS

Dueber. 2009. Nat. Biotech. 27:753.

Synthetic scaffolds have a dramatic

effect on the mevalonate pathway

HmgS

HmgR

AtoB

n = 1

Dueber. 2009. Nat. Biotech. 27:753.

Increases in Yield are Scaffold Dependent

HmgR

x=1

y

Z

AtoB

HmgS

AtoB HmgS HmgR Scaffold

Ptet PBAD

A: Optimized scaffold

Ptet PBAD

AtoB HmgS HmgR Scaffold GFP

B: Scaffold competitor

AtoB HmgS HmgR Scaffold

Ptet PBAD

GFP

PBAD

C: More scaffold competitor

AtoB HmgS HmgR Scaffold

Ptet PBAD

D: Enzymes without peptide ligands

80

60

40

20

0

Fo

ld in

cre

ase

ov

er

no

scaff

old

A B C D

Dueber. 2009. Nat. Biotech. 27:753.

Synthetic scaffolds can control relative

enzyme ratios and optimize flux

tHmgR

Ato

B

Hm

gS

Hm

gR

Hm

gR

Hm

gS

tHmgRHmgS

HmgSAtoB

Dueber. 2009. Nat. Biotech. 27:753.

Component optimization and debugging

yields another 50 fold

0.0001

0.001

0.01

0.1

1

10

100

1000

Construct

Iso

pre

no

id (

mg

/L)

PMK MPD MK idi ispA HMGS atoB tHMGR Amorphadiene

Mevalonate pathway (TOP)

H

H

Mevalonate pathway

(BOTTOM)

OPP

Synthase

FPP Mevalonate

OH

OHCH3

HO2CH3C SCoA

O

Acetyl-

CoA

Fermentation optimization pushes yields

beyond 25 g/L!

0.0001

0.001

0.01

0.1

1

10

100

1000

Construct

Iso

pre

no

id (

mg

/L)

Fermentation and final microbe

optimization done by Amyris.

H

H

O

HO

Identify final enzyme in pathway (P450/AMO)

and transplant into E. coli

PMK MPDMK idi ispAHMGSatoB tHMGR Amorphadiene Artemisinic

Acid

ADS

Mevalonate pathway

(TOP)

H

H

Mevalonate pathway

(BOTTOM)

p450

OPP

Synthase Hydroxyase

Unit

FPPMevalonate CPR

OH

OHCH3

HO2C

H3C SCoA

O

Acetyl-

CoA

A. annua

H

H

H

H

O

HO

p450CPR

Amorphadiene Artemisinic Acid

Proposed artemisinin

biosynthetic pathwayCytochrome P450

monooxygenase

Alcohol

dehydrogenase

Aldehyde

dehydrogenase

CH2OH

OHC

HOOC

CH2OH

OHC

HOOC

FPP

Amorphadiene

Artemisinic acid

Artemisinin

An experimental path

for identifying the P450

Amorph-OH

Gene 1

Gene 2

Gene 3

Gene 4

Amorph

Amorph

Amorph

Amorph

Yeast

Amorph-OH

E. coli

Amorph

Construction of a yeast that produces

amorphadiene

Amorph

Amorph

Amorph

Amorph

The mevalonate pathway in yeast

produces ergosterol

G6P

FDP

G3PDHAP

PEP

PYR

AcCoA

OAA

MAL

CIT

IPP DMAPPMEV

Mevalonate pathway

FPP

ErgosterolParadise. 2008. Biotech. Bioeng. 100:371

Production of amorphadiene is very low

when the gene is introduced into yeast

G6P

FDP

G3P

PEP

PYR

AcCoA

CIT

IPP DMAPPMEV

Mevalonate pathway

FPP

Ergosterol

Amorphadiene 0

20

40

60

80

Construct

Am

orp

ha

die

ne

(m

g/L

)

A

L

Paradise. 2008. Biotech. Bioeng. 100:371

Overexpression of the HMG-CoA reductase

gene improves production

G6P

FDP

G3P

PEP

PYR

AcCoA

CIT

IPP DMAPPMEV

Mevalonate pathway

FPP

Ergosterol

0

20

40

60

80

Construct

Am

orp

ha

die

ne

(m

g/L

)

Amorphadiene

A

L

Paradise. 2008. Biotech. Bioeng. 100:371

Expression of a transcription factor

enhances expression of several

mevalonate pathway genes

G6P

FDP

G3P

PEP

PYR

AcCoA

CIT

IPP DMAPPMEV

Mevalonate pathway

FPP

Ergosterol

0

20

40

60

80

Construct

Am

orp

ha

die

ne

(m

g/L

)

Amorphadiene

A

L

Paradise. 2008. Biotech. Bioeng. 100:371

Turning down ergosterol production

enhances amorphadiene production

G6P

FDP

G3P

PEP

PYR

AcCoA

CIT

IPP DMAPPMEV

Mevalonate pathway

FPP

Ergosterol

0

20

40

60

80

Construct

Am

orp

ha

die

ne

(m

g/L

)

XAmorphadiene

A

L

Paradise. 2008. Biotech. Bioeng. 100:371

Expression of FPP synthase further

enhances amorphadiene production

G6P

FDP

G3P

PEP

PYR

AcCoA

A

L

CIT

IPP DMAPPMEV

Mevalonate pathway

FPP

Ergosterol

0

20

40

60

80

Construct

Am

orp

ha

die

ne

(m

g/L

)

XAmorphadiene

Paradise. 2008. Biotech. Bioeng. 100:371

Finding the remaining genes in the

biosynthetic pathway

Gene 1

Gene 2

Gene 3

Gene 4

Lettuce, chicory, and sunflower produce

isoprenoids like artemisininGermacrene A

(Chicory, sunflower and lettuce)Amorphadiene

(Artemisia annua)

H

H

O

O

O

OH

HO

OH

OH

O

H

H

O

O

O

H

H

OO

O

OO

H

lettucenin Aniveusin A Artemisinin

OHO

O

P450’s involved

Alignment of known

terpene hydroxylases

Ro. 2006. Nature 440:940-943

CH2OH

OHC

HOOC

FPP

Amorphadiene

Artemisinic acid

P450 candidate produces

artemisinic acid

121

24893

18879105 216136

16217314555 67

201 233

121

93 248

79 188105 136 216

16217314555 67 201 233

Re

lati

ve

Io

n A

bu

nd

an

ce

Peak 1

Yeast product

Peak 2

Artemisinic acid

m/z

Ro. 2006. Nature 440:940-943

HO

O

H

H

H

HO

O

H

H

H

Amorphadiene

Artemisinic

Acid

H

H

H

H

O

HO

HO

O

H

H

H

HO

O

H

H

H

H

H

O

HO

HO

O

H

H

H

H

H

O

HO

Artemisinic acid is

transported out of the cell

Ro. 2006. Nature 440:940-943

N-terminal parts for functionally

expressing P450s in E. coli

Chang. 2007. Nat. Chem. Biol. 3:274-277

FPP

CadS CadH,O2

CPR, NADPHH H

OH

cadinene 8-hydroxycadinene

The P450 is functional in E. coli

Chang. 2007. Nat. Chem. Biol. 3:274-277

Public-Private Partnerships

• Royalty-free, exclusive licenses for

production of artemisinin for the

Developed World

• Must be produced at cost

Patents

Patents

Licenses Licenses

Licenses

Acknowledgements

Funding

Department of Energy

National Science Foundation

Office of Naval Research

University of California Discovery Grant

Bill & Melinda Gates Foundation (OneWorld Health)

Keasling lab

Jennifer Anthony

Michelle Chang

Howard Chou

Jeffrey Dietrich

John Dueber

Connie Kang

Lance Kizer

Jim Kirby

Taek Soon Lee

Vincent Martin

Karyn Newman

Farnaz Nowroozi

Mario Ouellet

Eric Paradise

Chris Petzold

Brian Pfleger

Doug Pitera

Dae-Kyun Ro

Christina Smolke

Sydnor Withers

Gabriel Wu

Yasuo Yoshikuni

Amyris#

Jack Newman

Chris Paddon

Kinkead Reiling

Rika Regentin

Neil Renninger

#Jay Keasling has a financial

interest in Amyris & LS9.

Joint Genome Institute