Malaria - Amazon Simple Storage Service · Malaria • Economists have proposed that malaria decreases the GDP of affected countries by as much as 50%. • 1-3 million people die

Engineering microbes for production of low-cost, effective, anti-malarial drugs

Jay D. Keasling

MCB 113

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 infected

Source: Roll Back MalariaWorld Malaria Report 2005

• Most widely-used drugs to treat malaria

NCl

NHH3C

H3C

H3C

Chloroquine-based drugs

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

Source: Roll Back MalariaWorld Malaria Report 2005

Disease burden on the rise as resistance grows

Trends in Africa Particularly Troubling

Source: Roll Back Malaria

Ann

ual d

eath

s (in

mill

ions

)

Treatment:Quinine only

Heavy use ofDDT spraying

Resistance increasing

0.0

1.0

2.0

3.0

4.0

1900 1930 1950 1970 1990 2000

Africa

Americas

China & NE Asia

S. Asia & Middle East

ArtemisininArtemisia annua

O

O

O

OO

H

HH

Artemisinin is produced by trichomes in Artemisia annua leaves

O

O

O

OO

H

HH

Artemisinin... ...is produced by trichomes... ...found on Artemisia annua leaves...

A brief history of artemisinin

Active ingredient (artemisinin) isolated 1972

Zhou Hou Bei Ji Fang (Handbook of Prescriptions for Emergency Treatments)

Fevers (malaria)

340 A.D.

Recipes For 52 Kinds Of Diseases found in the Mawangdui Han Dynasty tomb

Hemorrhoids

168 B.C.

Isoprenoids

Essential oils

OHMenthol

C-10 Monoterpene

Carotenoids

LycopeneC-40 Tetraterpene

Chemotherapeutics

TaxolC-20 Diterpene

EleutherobinC-20 Diterpene

> 50,000 known molecules

Isoprenoid 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)2OGeranylgeranyl 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

Amorphadiene

Farnesyl pyrophosphate(FPP)

Geranyl pyrophosphate(GPP)

Dimethylallyl pyrophosphate(DMAPP)

Isopentenyl pyrophosphate(IPP)

ArtemisininO

O

O

OO

H

HH

Pathways for isoprenoid precursor biosynthesis

G6P

FDP

G3PDHAP

PEP

PYR

AcCoA

OAA

MAL

CIT

IPP DMAPP

GPP

FPP

MEV

Mevalonate pathway

DXP

DXP pathway

GGPP

Monoterpenes

Sesquiterpenes

Diterpenes

TCACycle

Gly

coly

sis

Artemisinin-based drugs• The current cost for an artemisinin-

based drug is approximately $2.50.–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

O

O

O

OO

H

HH

A chemical synthesis route to

artemisinin

H HOH

H OH

H

O

HI

H

OH

H

O

HI

H

OH

H

O

Si((SiMe)3)3

O

OH

H

O

H

OH

H

O

H

O

OH

H

O

H

SS

+

+ +

OH

H

O

H

SS

OMeH

H

O

HO

SS H

OMeH

H

O

O

SS H

OMeH

H

O

O

SS H

H3C

+

OMeH

H

O

O

O H

H3C O

O

O

OO

H3C

CH3

H

CH3

H

H

Artemisinin

OH

H

O

HI

OH

H

O

H

O

GoalReduce the cost of artemisinin-based anti-malarial drugs by an order of magnitude.

ApproachEngineer a microorganism to produce artemisinin from an inexpensive, renewable resource.

Designing a microbe to synthesize artemisinin Identify the chemistry

CH 2OHCH 2OH

OHCOHC

HOOCHOOC

CH 2OHCH 2OH

OHCOHC

HOOCHOOC

FPP5

6 7 811

12 13

4

Find and clone (or synthesize) the genes

Designing a microbe to synthesize artemisinin

Supply of intracellular precursors


Well characterized geneticcontrol system (chassis, parts, devices)


Microbial production of artemisinin

• Production not affected by weather conditions

• Consistent supply• Pure product can be made (free of other

contaminating terpenes)• Access to artemisinin can be controlled to

minimize production of mono-therapies.

Advantages

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 platform host and a host of metabolic engineering tools

Challenges

Designing a microbe to synthesize artemisinin Identify the chemistry

CH 2OHCH 2OH

OHCOHC

HOOCHOOC

CH 2OHCH 2OH

OHCOHC

HOOCHOOC

FPP5

6 7 811

12 13

4

CH 2OH

OHC

HOOC

CH 2OH

OHC

HOOC

FPP

Amorphadiene

Artemisinic acid

Artemisinin

Bertea (2005) Planta Med. 71:40-47

Proposed artemisininbiosynthetic pathway

Find and clone (or synthesize) the genes

Synthesis of artemisinin inmicrobes

CH 2OH

OHC

HOOC

CH 2OH

OHC

HOOC

FPP

Amorphadiene

Artemisinic acid

Artemisinin

Bertea (2005) Planta Med. 71:40-47

Proposed artemisinin biosynthetic pathway

Production of a model isoprenoid in E. coli

Tobacco as an early model

5-epi-aristolochene

IPP DMAPP

GPP

DXP pathway

FPP

Production of a model isoprenoid in E. coli

Very low production of isoprenoid resulted when using the native gene

5-epi-aristolochene

IPP DMAPP

GPP

DXP pathway

FPP

0.0001

0.001

0.01

0.1

1

10

100

1000

ConstructIs

opre

noid

(mg/

L)

Production of the artemisinin precursor (amorphadiene) in E. coli

amorphadiene

IPP DMAPP

GPP

DXP pathway

FPP

Gene synthesis

atcttgtgat catcccaaga caaaaccaga gaaaaagacc tgtctgtttt tttaagaagtctttatatta ttttttttgt cggagaatct tataagcatg gcttcaggag gatcaaagtcggcagctttc atgcttctga tgctgaatct tggtctctat ttcgtcatca ccatcatcgcttcttgggct gttaatcacg gcatcgagag aactcgcgag tctggtaact aacaaagataacaactgatt aagtaacaat taatccaacg ttagaaaatg tcatcatcaa tcttctttttgtggtatttt gcagcgtcga cactgtcact tccggcgaag atattcccga tatacttcccggtggggaac atggcgaccg gttttttcgt aatattcacg ttaatcgccg gcgtcgtcgg

atcttgtgat catcccaaga caaaaccaga gaaaaagacc tgtctgtttt tttaagactttatatta ttttttttgt cggagaatct tataagcatg gcttcaggag gatcaaaggcagctttc atgcttctga tgctgaatct tggtctctat ttcgtcatca ccatcatttcttgggct gttaatcacg gcatcgagag aactcgcgag tctggtaact aacaaagacaactgatt aagtaacaat taatccaacg ttagaaaatg tcatcatcaa tcttcttgtggtatttt gcagcgtcga cactgtcact tccggcgaag atattcccga tatacttggtggggaac atggcgaccg gttttttcgt aatattcacg ttaatcgccg gcgtcgt

• Remove rare codons• Insert restriction sites

Gene resynthesis improves amorphadiene production

142-fold improved production!

amorphadiene

IPP DMAPP

GPP

DXP pathway

FPP

0.0001

0.001

0.01

0.1

1

10

100

1000

Construct

Isop

reno

id (m

g/L)

DXP PathwayG6P

FDP

G3PDHAP

PEP

PYR

AcCoA

OAA

MAL

CIT

DXP

DXP pathway

IPP DMAPP

GPP

FPP AmorphadieneQuinones

Eliminating bottlenecks in the DXP pathway

G6P

FDP

G3PDHAP

PEP

PYR

AcCoA

OAA

MAL

CIT

IPP DMAPP

GPP

FPP

DXP

Amorphadiene

DXS

IdI

IspA

Engineering E. coli’s native pathway improves production

Three-fold improvement in production

P

P

R

A

CIT

IPP DMAPP

GPP

FPP

DXP

Amorphadiene

DXS

IdI

IspA

0.0001

0.001

0.01

0.1

1

10

100

1000

ConstructIs

opre

noid

(mg/

L)

Enhancing all of the steps in the DXP pathway

G6P

FDP

G3PDHAP

PEP

PYR

AcCoA

OAA

MAL

CIT

IPP DMAPP

GPP

FPP

DXP

Amorphadiene

IdI

IspA

pyridoxinethiamine

Pathways for isoprenoid precursor biosynthesis

G6P

FDP

G3PDHAP

PEP

PYR

AcCoA

OAA

MAL

CIT

IPP DMAPP

GPP

FPP

MEV

Mevalonate pathway

DXP

DXP pathway

AmorphadieneTCA

Cycle

Gly

coly

sis

Recruiting the mevalonate pathway from yeast

Yeast

G3P

PEP

PYR

cCoA

CIT

IPP DMAPP

GPP

MEVMevalonate pathway

DXP

DXP pathway

FPPCAycle

Amorphadiene

Acetyl-CoAP

HMGS tHMGRatoBMevTMevalonate

FPP

MBISP

PMK MPDMK idi

Mevalonate

ispA

Construction of synthetic mevalonate pathway operons

Constructing the mevalonate pathway

FDP

G3PDHAP

PEP

PYR

AcCoA

OAA

MAL

CIT

IPP DMAPP

FPP

DXP

Amorph

MEV

Exogenous MEV

The yeast mevalonate pathway improves yields ~90-fold

G3P

PEP

PYR

cCoA

CIT

IPP DMAPP

GPP


DXP

DXP pathway

FPPCAycle

Amorphadiene0.0001

0.001

0.01

0.1

1

10

100

1000

ConstructIs

opre

noid

(mg/

L)

Acetyl-CoAP


FPP

MBISP

PMK MPDMK idi

Mevalonate

ispA

Optimizing the bottom part of the mevalonate pathway

Optimizing the mevalonate pathway

FDP

G3PDHAP

PEP

PYR

AcCoA

OAA

MAL

CIT

IPP DMAPP

FPP

DXP

Amorph

MEV

Exogenous MEV


FDP

G3PDHAP

PEP

PYR

AcCoA

OAA

MAL

CIT

IPP DMAPP

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.


FDP

G3PDHAP

PEP

PYR

AcCoA

OAA

MAL

CIT

IPP DMAPP

FPP

DXP

Amorph

MEV

Exogenous MEV

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)


FDP

G3PDHAP

PEP

PYR

AcCoA

OAA

MAL

CIT

DXP

MEV

Exogenous MEV

IPP DMAPP

FPP Amorphadiene


FDP

G3PDHAP

PEP

PYR

AcCoA

OAA

MAL

CIT

DXP

MEV

Exogenous MEV

IPP DMAPP

FPP Amorphadiene

High expression of amorphadiene synthase relieves inhibition

FDP

G3PDHAP

PEP

PYR

AcCoA

OAA

MAL

CIT

IPP DMAPP

FPP

DXP

Amorph

MEV

Exogenous MEV

0

100

200

300

400

500

600

700

0 20 40 60 80 100

Am

orph

adie

ne (m

g/L)

Time (hours)

Mevalonate kinase limits amorphadiene production

MBISP

PMK MPDMK idi ispA

ispA

idi

MPD

PMK

MK


FDP

G3PDHAP

PEP

PYR

AcCoA

OAA

MAL

CIT

DXP

MEV

Exogenous MEV

IPP DMAPP

FPP Amorph

Pathway mutation to prevent accumulation of toxic intermediates?

Mini-summary: Two lessons

• Even native metabolic intermediates can be toxic at high concentrations.

• “Pulling” on a pathway is just as important as “pushing”.

Acetyl-CoAP


FPP

MBISP

PMK MPDMK idi

Mevalonate

ispA

Optimizing the top part of the mevalonate pathway

The top part of the mevalonate pathway

MevalonatetHMGR

Diffusesout of cell

AtoBAcetyl-CoA Acetoacetyl

-CoA HMG-CoAHMGS

HMGS tHMGRatoBMevT

Growth inhibition by MevT pathway

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)

Cel

l Gro

wth

(OD

600)

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)

Cel

l Gro

wth

(OD

600)

HMGS tHMGRatoB

HMGS tHMGR

tHMGR

HMGS

MevalonatetHMGRAtoB

Acetyl-CoA Acetoacetyl-CoA HMG-CoA

HMGS

HMGSatoB

Accumulation of the toxic intermediate Hmg-CoA

Ac-CoA AcAc-CoA HMG-CoA Mev

AtoB HmgS tHmgR

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)

Cel

l Gro

wth

(OD

600)

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)

Cel

l Gro

wth

(OD

600)

0

10

20

30

40

50

60

0 2 4 6 8Time (hours)

HM

G-C

oA (n

M/O

D 600

) D

0

10

20

30

40

50

60

0 2 4 6 8Time (hours)

HM

G-C

oA (n

M/O

D 600

) D

C159A mutation in HMGS knocks out activity

HMGS tHMGRatoB


AtoB tHmgR

E E S G N T D I E G I D T T N A C Y G G T A AE E S G N T D I E G I D T T N A C Y G G T A AE E S G N T D I E G I D T T N A C Y G G T A AE E S G N T D I E G I D T T N A C Y G G T A AE E S G N T D V E G I D T T N A C Y G G T A AQ D S G N T D I E G I D T T N A C Y G G T A A

Human fibroblastHuman adrenalChinese hamster ovaryRat liverChicken liverRat liver – mitochondrial

S. cerevisiae L F G E N T D V E G I D T L N A C Y G G T N A

E E S G N T D I E G I D T T N A C Y G G T A AE E S G N T D I E G I D T T N A C Y G G T A AE E S G N T D I E G I D T T N A C Y G G T A AE E S G N T D I E G I D T T N A C Y G G T A AE E S G N T D V E G I D T T N A C Y G G T A AQ D S G N T D I E G I D T T N A C Y G G T A A

Human fibroblastHuman adrenalChinese hamster ovaryRat liverChicken liverRat liver – mitochondrial

S. cerevisiae L F G E N T D V E G I D T L N A C Y G G T N A

Comparison of active pathway, inactive pathway, and vector only

HMGS tHMGRatoB

HMGS tHMGRatoB

HMG-CoA stress only

Protein expression stress only

Protein andHMG-CoA stress

Inactive pathway

Active pathway

Vector only

Functional genomics pipeline

0 5 10 15 20 25 Time [min]

AspPheGlu

Pro

Ile

Leu

Lys

Arg Val

His Met

Metabolomics

Proteomics

T0

T2T1

Gro

wth

(OD

)

Time

Transcriptomics

AnalysisBiomass production

Functional genomics pipeline

Denature and reduce cysteine bridges

Cation Exchange Cleanup

Analyze by LC-MS/MS

Digest

114 115 116 117

Digest Digest Digest

Pool

Proteins

Transcripts

Metabolites

15 20 25 30 35 40 Time [min]15 20 25 30 35 40 Time [min]

Methionine Sulfone (ISD)

Valine Betaine

Glutamine

Lysine

Arginine

Analysis

Biomass production

MevT up-regulates genes involved in fatty acid biosynthesis

Acetyl-CoA

Malonyl-CoA Malonyl-ACP

Acetyl-ACP

FabD

FabHAccD, AccB

Acetoacetyl-ACP

ACPSH, CO2

FabBAccC, AccA FabB

Proteomics

Transcriptomics

Saturated fatty acids

Unsaturated fatty acids

FabBFabA

UPDOWN

Stress regulons up-regulated in MevT strain

HMG-CoA

Fe-S ClusterRepair

Sideophores

Hydroperoxidases

NADPH Generation

Oxidative Stress

ChaperonesProteases

Inclusion BodyProteins

Protein Misfolding

Protectants

PSPPorins Porins

Membrane/Osmotic Stress

Proteomics

Transcriptomics

HMG-CoA and malonyl-CoAaccumulation

Active pathway

Inactive pathway

HMG-CoA Malonyl-CoAActive

pathway

Inactive pathway

0 5 10 15 20 25 Time [min]

AspPheGlu

Pro

Ile

Leu

Lys

Arg Val

His Met

0 5 10 15 20 25 Time [min]

AspPheGlu

Pro

Ile

Leu

Lys

Arg Val

His Met

Metabolomics

Saturatedfatty acids

Unsaturatedfatty acids

Presence of MevT enriches unsaturated fatty acids

Inactive

Inactive

Active

Active

Pathway

0 5 10 15 20 25 Time [min]

AspPheGlu

Pro

Ile

Leu

Lys

Arg Val

His Met

0 5 10 15 20 25 Time [min]

AspPheGlu

Pro

Ile

Leu

Lys

Arg Val

His Met

Metabolomics

MevT up-regulates fatty acid biosynthesis

Acetyl-CoA


Acetyl-ACP

FabD

FabHAccD, AccB

Acetoacetyl-ACP

ACPSH, CO2

FabBAccC, AccA FabB


Unsaturated fatty acids

FabBFabA

0 5 10 15 20 25 Time [min]

AspPheGlu

Pro

Ile

Leu

Lys

Arg Val

His Met

0 5 10 15 20 25 Time [min]

AspPheGlu

Pro

Ile

Leu

Lys

Arg Val

His Met

Metabolomics

Proteomics

Transcriptomics

UPDOWN

Three additional pieces of information

• Malonyl-CoA is very similar in structure to HMG-CoA

• HMG-CoA reductase (tHMGR) activity increases with induction, then decreases sharply.

• HMG-CoA accumulation in organisms that produce it natively is known to cause oxidative stress.

HMG-CoAMalonyl-CoA

AccD, AccB

AccC, AccA

A model for MevT effects on fatty acid biosynthesis

UPDOWN

Acetyl-CoA


Acetyl-ACP

FabD

FabH

Acetoacetyl-ACP

ACPSH, CO2

FabB FabB


Unsfat

FabBFabA

AccD, AccB

AccC, AccA


UPDOWN

Acetyl-CoA


Acetyl-ACP

FabD

FabH

Acetoacetyl-ACP

ACPSH, CO2

FabB FabB


Unsfat

FabBFabA

HMG-CoA

Mev

AtoB

HmgS

tHmgR

Acetoacetyl-CoA

AccD, AccB

AccC, AccA


UPDOWN

Acetyl-CoA


Acetyl-ACP

FabD

FabH

Acetoacetyl-ACP

ACPSH, CO2

FabB FabB


Unsfat

FabBFabA

HMG-CoA

Mev

AtoB

HmgS

tHmgR

Acetoacetyl-CoA

FabD


HMG-CoA

Mev

AtoB

HmgS

tHmgR

Acetoacetyl-CoA

UPDOWN

Acetyl-CoA


Acetyl-ACPFabH

AccD, AccB

Acetoacetyl-ACP

ACPSH, CO2

FabBAccC, AccA FabB


Unsfat

FabBFabA

Accumulation of the toxic intermediate HMG-CoA

mRNA


AtoB HmgS tHmgR

P

HMGS tHMGRatoB

Balancing the mevalonate pathway by tuning mRNA stability and

translation efficiency

mRNA

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

P1

HMGS tHMGRatoB

RNase Esite

RNase Esite

RBS RBS RBS

Combinatorial design of intergenicregions D

CA

B

EE E E

atoB hmgS hmgRE E

P1

HMGS tHMGRatoB

How do we choose?atoB hmgS hmgR

E E

atoB hmgS hmgRE E

atoB hmgS hmgREE




A fluorescent screen for improved mevalonate production

MEVMEV

Ac-CoA

MEV

Biomass

E. coliGFP-engineeredMEV auxotroph

E. coliMEV Producer

Mutant pathway

B.)B.)

Best producers make about 7X more mevalonate

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

Down-regulation of last two genes relieves toxicity, increases Mev production, and increases Ac-CoA concentration

RBS

RBSatoB hmgS hmgR

Mini-summary: Two more lessons

• Functional genomics is a useful tool for diagnosing problems in engineered cells.

• Pathway balancing is much harder than pathway construction!

Large-scale production of amorphadiene O2

G3P

PEP

PYR

cCoA

CIT

IPP DMAPP

GPP


DXP

DXP pathway

FPPCAycle

Amorphadiene

0

0.5

1

1.5

2

2.5

3

3.5

0 2 4 6 8 10 12 14

Time (Hours)

Am

orph

adie

ne

conc

entra

tion

(mg/

L)Amorphadiene is volatile

and is lost from bioreactors O2

Amorphadiene is lost from bioreactors O2

Condenser

Amorphadiene

Amorphadiene production in a two-phase fermentation O2

Oil

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)

A two-phase fermentation collects all of the amorphadiene

1 million fold improved production of amorphadiene

G3P

PEP

PYR

cCoA

CIT

IPP DMAPP

GPP


DXP

DXP pathway

FPP

Amorphadiene0.0001

0.001

0.01

0.1

1

10

100

1000

ConstructIs

opre

noid

(mg/

L)

Completing the pathway

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

Proposed artemisinin

biosynthetic pathway

Cytochrome P450 monooxygenase

Alcohol dehydrogenase

Aldehyde dehydrogenase

CH 2OH

OHC

HOOC

CH 2OH

OHC

HOOC

FPP

Amorphadiene

Artemisinic acid

Artemisinin

Cytochrome P450’s• Heme-containing oxygenases• Important for metabolizing

toxic compounds in the human body

• Often required as one of the initial steps in natural products synthesis

• Plant- and animal-derived versions require additional proteins for function– Cytochrome P450 reductase

partners• Often bound to the membrane

http://www.p450.kvl.dk/gallery/CYP3A4.jpg

Cloning the remaining genes in the Artemisinin biosynthetic pathway• Cytochrome P450’s are traditionally difficult to

express in E. coli

• A good host for cytochrome P450 expression and screening would aid in elucidating the remaining steps in the biosynthetic pathway–A Saccharomyces cerevisiae (yeast) strain

capable of producing amorphadiene would be very beneficial

An experimental path for identifying the P450

Amorph-OH

Gene 1

Gene 2

Gene 3

Gene 4

Amorph

Amorph

Amorph

Amorph

YeastAmorph-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 DMAPP

GPP


FPP

Ergosterol

Production of amorphadiene is very low when the gene is introduced

into yeast

G6P

FDP

G3P

PEP

PYR

AcCoA

AA

L

CIT

IPP DMAPP

GPP


FPP

ErgosterolAmorphadiene 0

20

40

60

80

Construct

Am

orph

adie

ne (m

g/L)

Overexpression of the HMG-CoA reductase gene improves production

G6P

FDP

G3P

PEP

PYR

AcCoA

AA

L

CIT

IPP DMAPP

GPP


FPP

Ergosterol0

20

40

60

80

Construct

Am

orph

adie

ne (m

g/L)

Amorphadiene

Expression of a transcription factor enhances expression of many of the

genes in the mevalonate pathway

G6P

FDP

G3P

PEP

PYR

AcCoA

AA

L

CIT

IPP DMAPP

GPP


FPP

Ergosterol0

20

40

60

80

Construct

Am

orph

adie

ne (m

g/L)

Amorphadiene

Turning down ergosterol production enhances amorphadiene production

G6P

FDP

G3P

PEP

PYR

AcCoA

AA

L

CIT

IPP DMAPP

GPP


FPP

Ergosterol0

20

40

60

80

Construct

Am

orph

adie

ne (m

g/L)

XAmorphadiene

Expression of FPP synthase further enhances amorphadiene production

G6P

FDP

G3P

PEP

PYR

AcCoA

AA

L

CIT

IPP DMAPP

GPP


FPP

Ergosterol0

20

40

60

80

Construct

Am

orph

adie

ne (m

g/L)

X

Amorphadiene production by yeast is 1/10th of that in E. coli … but good enough to screen for P450’s.

Amorphadiene

The genes encoding the P450 and redox partners must be present in

the same cell

Amorph-OH

Gene 3Amorph

Cytochrome P450 redoxpartners from A. annua

Finding the remaining genes in the biosynthetic pathwayGene 1

Gene 2

Gene 3

Gene 4

Lettuce, chicory, and sunflower produce isoprenoids like artemisinin

Germacrene A(Chicory, sunflower and lettuce)

Amorphadiene(Artemisia annua)

H

H

OO

OOH

HO

OH

OH

O

H

H

O

O

O

H

H

OO

O

OO

H

lettucenin Aniveusin A Artemisinin

OHOO

P450’s involved

Alignment of known terpene hydroxylases

71C171C271C3

71C471D12

71D1371D18

71D1671D20

71AV173A1

98A379F1

701A393C1

75A175B2

76B6706B1

84A188A3

90A190B1

50 changes

100100

90

74

79

91

86

100

90

66

92

100

90

9785

88

60

86100

100

71C171C271C3

71C471D12

71D1371D18

71D1671D20

71AV173A1

98A379F1

701A393C1

75A175B2

76B6706B1

84A188A3

90A190B1

50 changes

100100

90

74

79

91

86

100

90

66

92

100

90

9785

88

60

86100

100

P450 candidate is functional in yeast!

YeastCPR/CYP71AV1

YeastCPR

A. Annua

28.0

628

.06

IS

20 35Time (min)

1

2

*

*

Tota

l Ion

Col

lect

ion

P450 candidate produces artemisinic acid!

121

2489318879

105 21613616217314555 67 201 233

121

93 24879 188

105 136 21616217314555 67 201 233

Rel

ativ

e Io

n A

bund

ance

Peak 1Yeast product

Peak 2Artemisinic acid

m/z

YeastCPR/CYP71AV1

YeastCPR

A. Annua

28.0

628

.06

IS

20 35Time (min)

1

2

*

*

Tota

l Ion

Col

lect

ion

CH 2OH

OHC

HOOC

FPP

Amorphadiene

Artemisinic acid

Proposed artemisininbiosynthetic pathway

121

2489318879

105 21613616217314555 67 201 233

121

93 24879 188

105 136 21616217314555 67 201 233

Rel

ativ

e Io

n A

bund

ance

Peak 1Yeast product

Peak 2Artemisinic acid

m/z

HO

O

H

H

H

HO

O

H

H

H

Amorphadiene

ArtemisinicAcid

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

Expression of amorphadiene oxidase in yeast

• Amorphadiene oxidase catalyzes all three steps–amorphadiene alcohol aldehyde

artemisinic acid• The amorphadiene oxidase is functional in

vivo• Artemisinic acid is secreted from yeast and

adsorbs to the outside of the cells• Artemisinic acid can readily released from the

cells by a pH change

Production of artemisinic acid in E. coli

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

Production of hydroxylated terpenes in E. coli

FPP

CadS CadH,O2

CPR, NADPHH H

OH

cadinene 8-hydroxycadinene

Completing the biosynthetic pathway in E. coli

p450

Amorphadiene Artemisinic Acid

1 2 3

>25 g/LCurrent titer

in E. coli(lab scale)

> 1 g/L

P450/AMO Catalyzes 3 Separate Oxidations

Research, Development & Delivery

Keasling Laboratory

Amyris Biotechnologies

Institute forOneWorldHealth

Artemisinin costs

$1.00/gCurrent cost of API

$.10/gCost with new process

Conclusions• E. coli has been engineered to produce

amorphadiene at yields in excess of 0.5 g/L.• S. cerevisiae has been engineered to produce

amorphadiene at yields of approximately 0.1 g/L.• A cytochrome P450 and its redox partners can

oxidize amorphadiene to artemisinic acid.• S. cerevisiae expressing amorphadiene oxidase

produces artemisinic acid, which is secreted from the cells.

• E. coli can functionally express cytochrome P450’s that oxidize terpenes.

Acknowledgements

FundingNational Science FoundationOffice of Naval ResearchMaxygenDiversaUniversity of California Discovery GrantBill & Melinda Gates Foundations (OneWorld Health)

Keasling labJennifer AnthonyMichelle ChangConnie KangLance KizerJim KirbyVincent MartinKaryn NewmanFarnaz NowrooziMario Oulet

Eric ParadiseChris PetzoldBrian PflegerDoug PiteraDae-Kyun RoChristina SmolkeSydnor WithersYasuo Yoshikuni

Amyris Biotechnologies#

Jack NewmanChris PaddonKinkead ReilingRika RegentinNeil Renninger

#Jay Keasling has a financial interest in Amyris Biotechnologies.

Joint Genome Institute

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