engineering microbial metabolism for production of fuels

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Engineering Microbial Metabolism for Production of Fuels and Chemicals Jay Keasling Joint BioEnergy Institute University of California, Berkeley Lawrence Berkeley National Laboratory

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Page 1: Engineering Microbial Metabolism for Production of Fuels

Engineering Microbial Metabolism for

Production of Fuels and Chemicals

Jay Keasling

Joint BioEnergy Institute

University of California, Berkeley

Lawrence Berkeley National Laboratory

Page 2: Engineering Microbial Metabolism for Production of Fuels

Hydrocarbon fuels

Gasolines

• High octane

• Short-chain alkanes

• Many branches

• Some aromatics

Jet fuels

• Long-chain alkanes

• Few branches

• Some aromatics

Diesels

• Appropriate cetane numbers

• Long-chain alkanes

• Few branches

• Some aromatics

Page 3: Engineering Microbial Metabolism for Production of Fuels

Linear hydrocarbons from the fatty acid biosynthetic

pathways

PEP

Pyruvate

Acetyl-CoA

CIT

ICT

OGA

SUC

FUM

OAA

MAL

CO2

Glyoxylate

SUC-CoA

Acetaldehyde

Ethanol

Propionyl-CoA Propanol

Acetoacetyl-CoA Butyryl-CoA Butanol

Even chain fatty acid

Aliphatic hydrocarbons

Odd chain fatty acid

G3P

IPP DMAPP

GPP

FPP

GGPP

Isopentenol Isopentanol

Geraniol

Farnesol

Geranylgeraniol

Monoterpene

Sesquiterpene

Diterpene

DHAP

FBP

F6P

G6P 6PG X5P

E4P

S7P

Valeryl-CoA Isopentanol

Glucose

Xylose

Mannose M6P

Galactose G’1P G1P

Xylulose

R5P

Ribulose

Arabinose

Various esters

Alcohols

Aromatic hydrocarbons

Short, highly-branched

hydrocarbons

Page 4: Engineering Microbial Metabolism for Production of Fuels

Linear hydrocarbons make great diesels

and jet fuels

Gasolines

• High octane

• Short-chain alkanes

• Many branches

• Some aromatics

Jet fuels

• Long-chain alkanes

• Few branches

• Some aromatics

Diesels

• Appropriate cetane numbers

• Long-chain alkanes

• Few branches

• Some aromatics

Page 5: Engineering Microbial Metabolism for Production of Fuels

Linear hydrocarbons from the fatty acid biosynthetic

pathways

PEP

Pyruvate

Acetyl-CoA

CIT

ICT

OGA

SUC

FUM

OAA

MAL

CO2

Glyoxylate

SUC-CoA

Acetaldehyde

Ethanol

Propionyl-CoA Propanol

Acetoacetyl-CoA Butyryl-CoA Butanol

Even chain fatty acid

Aliphatic hydrocarbons

Odd chain fatty acid

G3P

IPP DMAPP

GPP

FPP

GGPP

Isopentenol Isopentanol

Geraniol

Farnesol

Geranylgeraniol

Monoterpene

Sesquiterpene

Diterpene

DHAP

FBP

F6P

G6P 6PG X5P

E4P

S7P

Valeryl-CoA Isopentanol

Glucose

Xylose

Mannose M6P

Galactose G’1P G1P

Xylulose

R5P

Ribulose

Arabinose

Various esters

Alcohols

Aromatic hydrocarbons

Short, highly-branched

hydrocarbons Fatty acids

Page 6: Engineering Microbial Metabolism for Production of Fuels

Wild type E. coli produces no free fatty acids

because of strict acyl-ACP inhibition PEP

PYR

AcCoA

CIT

Fatty-ACP Fatty

acid

ACC FAS

Lipid

FadD Fatty

acyl-CoA

FadA

Steen et al. 2010.

Nature 463:559

FadB

FadE

β-keto

acyl-CoA

WT

Page 7: Engineering Microbial Metabolism for Production of Fuels

Production of fatty acid ethyl esters

PEP

PYR

AcCoA

CIT

Fatty-ACP Fatty

acid

ACC FAS

Lipid

FadD Fatty

acyl-CoA

FadA

AtfA

TesA

Acetaldehyde Ethanol

Pdc

AdhB

Fatty acid ethyl

esters

Steen et al. 2010.

Nature 463:559

FadB

FadE

β-keto

acyl-CoA

Page 8: Engineering Microbial Metabolism for Production of Fuels

Methyl ketones produced from fatty acyl-CoA

by overexpressing FadEBM PEP

PYR

AcCoA Fatty-ACP Fatty

acid

ACC FAS

Lipid

FadD Fatty

acyl-CoA

FadA

TesA

β-keto

acyl-CoA

FadM FadB

FadE

CIT

Methyl ketones

Goh et al. 2012.

AEM 78:70

Page 9: Engineering Microbial Metabolism for Production of Fuels

Diesel-range methyl ketones in E. coli

Goh et al. 2012 AEM 78:70

Goh et al. 2014 Metab. Eng. 26:67

Re-engineering β-oxidation Titers exceeding 1.4 g/L (1% Glu, M9)

40% of maximum theoretical yield

0.12

0.62

10

120

8.6

36

390

570

1350 1400

0.1

1

10

100

1000

10000

Me

thy

l ke

ton

es

(m

g/L

)

TSB

M9-MOPS (1% Gluc)

12020

Goh et al. 2012

‘tesA, fadE + + + + + + + + + +

fadM + + + + + + + +

aco, fadB, fadA + + + + + + + +

fadR, fadD + + + + +

poxB + + +

1-plasmid, co-aco

+ + +

ackA-pta + +

EG

S8

95

EG

S1

710

EG

S1

895

EG

S8

95

EG

S5

60

EG

S1

32

0

EG

S1

37

0

EG

S18

90

EGS860

EGS084

Page 10: Engineering Microbial Metabolism for Production of Fuels

TesA-FadD combination wastes ATP

ATP is consumed

when free fatty acids

are

esterified to CoA Haushalter et al. 2015. Metab. Eng. 30:1-6

Acyl-ACP inhibits fatty

acid biosynthesis

Page 11: Engineering Microbial Metabolism for Production of Fuels

Type I FAS nearly doubles fatty alcohol titers

compared to native FAS

• Improvement in

production

(2.9 g/L versus

1.6 g/L)

• Changes in

distribution of

fatty acids

Page 12: Engineering Microbial Metabolism for Production of Fuels

AcCoA

ACC

FAS1 FAS2

Fatty-CoA

TesA DGAT

FAR

AtfA

Tef1 FAS1

Chromosomal DNA

Tef1 FAS2

Tef1 ACC

b-oxidation

Engineered yeast strain WR1

! "

#! "

$! ! "

$#! "

%! ! " O

nOH

! "# $%"&'( )%

wt

mg/L

wt

TesA

WR1

TesA

WR1

! ACSs

TesA ! "

#"

$! "

$#"

%! "

O

O

O

O

O

O

n

n

n

! " #$%

wt wt

DGAT

WR1

DGAT

WR1

! ²-oxidation

DGAT

Lipid Content (%)

! "

#! "

$! "

%&! "

nOH

! "# $%%"&' ( ) ( &*%

mg/L

wt wt

FAR

WR1

FAR

WR1

! ²-oxidation

FAR

! "

#"

$"

%"O

nOEt

! " ##$%

mg/L

wt wt

AtfA

WR1

! ²-oxidation

AtfA

X

Fatty acid-derived fuels in S. cerevisiae

Page 13: Engineering Microbial Metabolism for Production of Fuels

Unbalanced FAEE pathway caused strain instability

PEP

PYR

AcCoA

CIT

Fatty-ACP Fatty

acid

ACC FAS

Lipid

FadD Fatty

acyl-CoA

FadA

AtfA

TesA

Acetaldehyde Ethanol

Pdc

AdhB

Fatty acid ethyl

esters

Steen et al. 2010.

Nature 463:559

FadB

FadE

β-keto

acyl-CoA

Page 14: Engineering Microbial Metabolism for Production of Fuels

Can FadR regulate FAEE production?

Peth

pdc adhB

PYR

Fatty-ACP Fatty

acid

ACC FAS FadD Fatty

acyl-CoA

TesA

Acetaldehyde Ethanol Pdc AdhB Fatty

esters

AtfA

AcCoA

PatfA

atfA PfadD

fadD

PBAD

fadR

PlacUV5

tesA

Zhang et al. 2012 Nat. Biotechnol. 30:354

Page 15: Engineering Microbial Metabolism for Production of Fuels

FadR regulation improves FAEE production

PlacUV5

PAR

PFL1

PFL2

PFL3

0

200

400

600

800

1000

1200

1400

1600

PlacUV5PAR

PFL1PFL2

PFL3Promoters for

ethanol production

Promoters

for atfA

FAEE titer (mg/L)

• Four-fold improvement

in FAEE production

• Significantly improved

stability of host

Zhang et al. 2012 Nat. Biotechnol. 30:354

Page 16: Engineering Microbial Metabolism for Production of Fuels

Melting points of various biofuels

12-methyl tetradecanoic acid methyl ester

Melting point = -5.3°C

pentadecanoic acid methyl ester

Melting point = 18°C

Farnesane

Melting point = <-78°C

Bisabolane

Melting point = <-78°C

13-methyl tetradecanoic acid methyl ester

Melting point = 6.4°C

Page 17: Engineering Microbial Metabolism for Production of Fuels

A few branches are important!

Gasolines

• High octane

• Short-chain alkanes

• Many branches

• Some aromatics

Jet fuels

• Long-chain alkanes

• Few branches

• Some aromatics

Diesels

• Appropriate cetane numbers

• Long-chain alkanes

• Few branches

• Some aromatics

Page 18: Engineering Microbial Metabolism for Production of Fuels

Production of branched-chain fatty acids

AcCoA

Fatty Acyl-ACP

ACC

FAS (E. coli FabH)

Mal-CoA Ketoacyl-CoA

Branched

Fatty Acyl-ACP

FAS (B. subtilis FabH)

Alkanes Esters Ketones Alcohols

Leu, Val, Ile BCKA

Decarboxylase

Page 19: Engineering Microbial Metabolism for Production of Fuels

Production of branched-chain fatty acids

• Branched-chain amino acid biosynthetic pathway produces precursors to branched-chain fatty acids

• B. subtilis fabH2 initiates fatty acid synthesis with branched acyl-CoA precursors

Melting point = -5C (methyl ester)*

Melting point = 18C (methyl ester)*

All genes in blue

were overexpressed

Haushalter et al. 2014. Met. Eng. 26:111-118

Page 20: Engineering Microbial Metabolism for Production of Fuels

Anteiso-branched fatty acid production

• E. coli MG1655ΔfadE

expressing ‘tesA and fadR

• only straight-chain saturated

and unsaturated fatty acids

Straight-chain, even carbon

Straight-chain, odd carbon

Iso-branched

Anteiso-branched

!

!

• Branched acyl-CoA production,

coupled with B. subtilis fabH2

• 22% anteiso branched species

Haushalter et al. 2014. Met. Eng. 26:111-118

Page 21: Engineering Microbial Metabolism for Production of Fuels

A few branches are important!

Gasolines

• High octane

• Short-chain alkanes

• Many branches

• Some aromatics

Jet fuels

• Long-chain alkanes

• Few branches

• Some aromatics

Diesels

• Appropriate cetane numbers

• Long-chain alkanes

• Few branches

• Some aromatics

Page 22: Engineering Microbial Metabolism for Production of Fuels

Branched-chain hydrocarbons

from isoprenoids

PEP

Pyruvate

Acetyl-CoA

CIT

ICT

OGA

SUC

FUM

OAA

MAL

CO2

Glyoxylate

SUC-CoA

Acetaldehyde

Ethanol

Propionyl-CoA Propanol

Acetoacetyl-CoA Butyryl-CoA Butanol

Even chain fatty acid

Aliphatic hydrocarbons

Odd chain fatty acid

G3P

IPP DMAPP

GPP

FPP

GGPP

Isopentenol Isopentanol

Geraniol

Farnesol

Geranylgeraniol

Monoterpene

Sesquiterpene

Diterpene

DHAP

FBP

F6P

G6P 6PG X5P

E4P

S7P

Valeryl-CoA Isopentanol

Glucose

Xylose

Mannose M6P

Galactose G’1P G1P

Xylulose

R5P

Ribulose

Arabinose

Various esters

Alcohols

Aromatic hydrocarbons

Short, highly-branched

hydrocarbons

Isoprenoids

Page 23: Engineering Microbial Metabolism for Production of Fuels

Branched-chain hydrocarbons

from isoprenoid biosynthetic pathway FDP

G3P DHAP

PEP

PYR

AcCoA IPP DMAPP

OAA

MAL

CIT

MEV

DXP

FPP quinones

GPP

Sesquiterpenes

Monoterpenes

Hemiterpenes

Page 24: Engineering Microbial Metabolism for Production of Fuels

DMAPP Monoterpenes

FDP

G3P DHAP

PEP

PYR

AcCoA IPP

OAA

MAL

CIT

MEV

DXP

FPP quinones

GPP

Sesquiterpenes

IPP 3-methyl-3-butenol

phosphatase

DMAPP 3-methyl-2-butenol

3-methyl-butanolreductase

0

2

4

6

8

10

12

14

16

18

20

C5

Alc

oh

ols

(m

g/L/

OD

)

HAD-likePhosphataseLibrary

(-)control

0

100

200

300

400

500

Iso

pen

tan

ol(

mg/

L)

(-)control

OYELibrary

OHOPP

OPP OH

OHBiofuel

Water solubility

(g/L)

Research Octane

Numbera

Energy

Density

3-methyl-2-butenol

6 + 0.34 90

3-methyl-3-butenol

9 + 0.15 102

3-methyl-butanol

15 + 3.2 102

96% of gasoline

(116

MJ/gal)

Hemiterpenes

Branched-chain hydrocarbons

from isoprenoid biosynthetic pathway

Page 25: Engineering Microbial Metabolism for Production of Fuels

DMAPP

FDP

G3P DHAP

PEP

PYR

AcCoA IPP

OAA

MAL

CIT

MEV

DXP

FPP quinones

GPP

Sesquiterpenes

Hemiterpenes IPP 3-methyl-3-butenol

phosphatase

DMAPP 3-methyl-2-butenol

3-methyl-butanolreductase

0

2

4

6

8

10

12

14

16

18

20

C5

Alc

oh

ols

(m

g/L/

OD

)

HAD-likePhosphataseLibrary

(-)control

0

100

200

300

400

500

Iso

pen

tan

ol(

mg/

L)

(-)control

OYELibrary

OHOPP

OPP OH

OHBiofuel

Water solubility

(g/L)

Research Octane

Numbera

Energy

Density

3-methyl-2-butenol

6 + 0.34 90

3-methyl-3-butenol

9 + 0.15 102

3-methyl-butanol

15 + 3.2 102

96% of gasoline

(116

MJ/gal)

Monoterpenes Limonene

synthase

Pinene

synthase

Pd/C, H2

Chemical

catalyst

Branched-chain hydrocarbons

from isoprenoid biosynthetic pathway

Page 26: Engineering Microbial Metabolism for Production of Fuels

DMAPP

FDP

G3P DHAP

PEP

PYR

AcCoA IPP

OAA

MAL

CIT

MEV

DXP

FPP quinones

GPP

Hemiterpenes

Monoterpenes Limonene

synthase

Pinene

synthase

Pd/C, H2

Chemic

al

catalyst

IPP 3-methyl-3-butenol

phosphatase

DMAPP 3-methyl-2-butenol

3-methyl-butanolreductase

0

2

4

6

8

10

12

14

16

18

20

C5

Alc

oh

ols

(m

g/L/

OD

)

HAD-likePhosphataseLibrary

(-)control

0

100

200

300

400

500

Iso

pen

tan

ol(

mg/

L)

(-)control

OYELibrary

OHOPP

OPP OH

OHBiofuel

Water solubility

(g/L)

Research Octane

Numbera

Energy

Density

3-methyl-2-butenol

6 + 0.34 90

3-methyl-3-butenol

9 + 0.15 102

3-methyl-butanol

15 + 3.2 102

96% of gasoline

(116

MJ/gal)

Sesquiterpenes

Peralta-Yahya et al. 2011. Nature Comm. 2:483

McAndrews et al. 2011. Structure 19:1876

Branched-chain hydrocarbons

from isoprenoid biosynthetic pathway

Page 27: Engineering Microbial Metabolism for Production of Fuels

DMAPP Monoterpenes

Branched-chain hydrocarbons

from isoprenoid biosynthetic pathway

FDP

G3P DHAP

PEP

PYR

AcCoA IPP

OAA

MAL

CIT

MEV

DXP

FPP quinones

GPP

Sesquiterpenes

IPP 3-methyl-3-butenol

phosphatase

DMAPP 3-methyl-2-butenol

3-methyl-butanolreductase

0

2

4

6

8

10

12

14

16

18

20

C5

Alc

oh

ols

(m

g/L/

OD

)

HAD-likePhosphataseLibrary

(-)control

0

100

200

300

400

500

Iso

pen

tan

ol(

mg/

L)

(-)control

OYELibrary

OHOPP

OPP OH

OHBiofuel

Water solubility

(g/L)

Research Octane

Numbera

Energy

Density

3-methyl-2-butenol

6 + 0.34 90

3-methyl-3-butenol

9 + 0.15 102

3-methyl-butanol

15 + 3.2 102

96% of gasoline

(116

MJ/gal)

Hemiterpenes

Page 28: Engineering Microbial Metabolism for Production of Fuels

Branched-C5 alcohols

are promising gasoline replacements

• High energy content

– 96% of gasoline

• Low solubility in

water

• High blending

octane number

OH

OH

OH

3-methyl-2-butenol

3-methyl-3-butenol

3-methyl-butanol

Page 29: Engineering Microbial Metabolism for Production of Fuels

Constructing de novo synthetic pathways

for biofuel targets

OH IPP 3-methyl-3-butenol

phosphatase

OH DMAPP 3-methyl-2-butenol

OH 3-methyl-butanol

reductase

OPP

OPP

Page 30: Engineering Microbial Metabolism for Production of Fuels

Novel phosphatases for synthesizing isopentenols

Superfamily Haloacid dehalogenase-like

(HAD) Phosphatase

Nudix Hydrolase

# sequenced >3000 >4000

# in E. coli 29 13

Mechanism Covalent Enzyme-

Substrate Intermediate

Metal-assisted hydrolysis

0

2

4

6

8

10

12

14

16

18

20

C5 A

lco

ho

ls (

mg

/L/O

D)

HAD-like Phosphatase Library

(-) control

Nudix Hydrolase Library

0

2

4

6

8

10

12

14

16

18

20

C5 A

lco

ho

ls (

mg

/L/O

D)

(-) control

2X

Howard Chou

Page 31: Engineering Microbial Metabolism for Production of Fuels

Constructing de novo synthetic pathways

for biofuel targets

OH IPP 3-methyl-3-butenol

phosphatase

OH DMAPP 3-methyl-2-butenol

OH 3-methyl-butanol

reductase

OPP

OPP

Howard Chou

Page 32: Engineering Microbial Metabolism for Production of Fuels

Novel reductases for synthesizing isopentanol

0

50

100

150

200

250

300

350

400

450

Iso

pen

tan

ol (m

g/L

)

Cultures fed 1g/L 3-methyl-2-butenol for 24 hours.

(-) control OYE Library

Williams & Bruce. Microbiol. 148, 1607-14 (2002).c

Old Yellow Enzyme Family

Howard Chou

Page 33: Engineering Microbial Metabolism for Production of Fuels

A Hybrid Sensor for IPP

AraC binding sites GFP

IPP isomerase

linker

AraC DNA

binding domain

AraC binding sites GFP

X

Mev IPP

IPP IPP

Mev added to culture

Mev absent from culture

DMAPP

Chou et al 2013 Nat Commun 4:2595

Page 34: Engineering Microbial Metabolism for Production of Fuels

A Hybrid Sensor for IPP

AraC binding sites GFP

X

Mev IPP

IPP IPP

Mev added to culture

DMAPP

Chou et al 2013 Nat Commun 4:2595

Page 35: Engineering Microbial Metabolism for Production of Fuels

Using the Hybrid Sensor to Select for Mutations that

Increase [IPP]

AraC binding sites MutD5

AraC binding sites MutD5

X

Mev IPP

IPP IPP

High [IPP] in cells Low mutation rate

Low [IPP] in cells High mutation rate

DMAPP

AcCoA

x

x

x

x

x

x

Chou et al 2013 Nat Commun 4:2595

Page 36: Engineering Microbial Metabolism for Production of Fuels

Mutated Cells with Increased [IPP] Produce more

Lycopene

0

1000

2000

3000

4000

5000

6000

7000

8000

0 72 144 216 288 360 432

Lyco

pen

e (

p.p

.m.)

Time (hrs)

AraC IA32 IA44

Mev IPP DMAPP

AcCoA

GGPP Lycopene

E. coli chromosome

Non-synonymous

SNPs

All SNPs

Chou et al 2013 Nat Commun 4:2595

Page 37: Engineering Microbial Metabolism for Production of Fuels

DMAPP

Branched-chain hydrocarbons

from isoprenoid biosynthetic pathway

FDP

G3P DHAP

PEP

PYR

AcCoA IPP

OAA

MAL

CIT

MEV

DXP

FPP quinones

GPP

Sesquiterpenes

Hemiterpenes IPP 3-methyl-3-butenol

phosphatase

DMAPP 3-methyl-2-butenol

3-methyl-butanolreductase

0

2

4

6

8

10

12

14

16

18

20

C5

Alc

oh

ols

(m

g/L/

OD

)

HAD-likePhosphataseLibrary

(-)control

0

100

200

300

400

500

Iso

pen

tan

ol(

mg/

L)

(-)control

OYELibrary

OHOPP

OPP OH

OHBiofuel

Water solubility

(g/L)

Research Octane

Numbera

Energy

Density

3-methyl-2-butenol

6 + 0.34 90

3-methyl-3-butenol

9 + 0.15 102

3-methyl-butanol

15 + 3.2 102

96% of gasoline

(116

MJ/gal)

Monoterpenes Limonene

synthase

Pinene

synthase

Pd/C, H2

Chemical

catalyst

Page 38: Engineering Microbial Metabolism for Production of Fuels

Engineering pinene production

IPP

DMAPP GPP Pinene

GPP Synthase Pinene Synthase

Sarria et al 2014 ACS Syn Bio In press

Page 39: Engineering Microbial Metabolism for Production of Fuels

GPP and pinene synthases from Grand Fir produce the

highest pinene titers

Sarria et al 2014 ACS Syn Bio In press

Page 40: Engineering Microbial Metabolism for Production of Fuels

Pinene synthase competes for GPP

IPP

DMAPP GPP Pinene

GPP Synthase Pinene Synthase

FPP Growth

Sarria et al 2014 ACS Syn Bio In press

Page 41: Engineering Microbial Metabolism for Production of Fuels

Linking enzymes to improve GPP access

IPP

DMAPP

GPP

Pinene

GPP Synthase Pinene Synthase

Sarria et al 2014 ACS Syn Bio In press

Page 42: Engineering Microbial Metabolism for Production of Fuels

GPPS at the N-terminus with a sufficient linker length is

best

Sarria et al 2014 ACS Syn Bio In press

Page 43: Engineering Microbial Metabolism for Production of Fuels

DMAPP

Branched-chain hydrocarbons

from isoprenoid biosynthetic pathway

FDP

G3P DHAP

PEP

PYR

AcCoA IPP

OAA

MAL

CIT

MEV

DXP

FPP quinones

GPP

Hemiterpenes

Monoterpenes Limonene

synthase

Pinene

synthase

Pd/C, H2

Chemic

al

catalyst

IPP 3-methyl-3-butenol

phosphatase

DMAPP 3-methyl-2-butenol

3-methyl-butanolreductase

0

2

4

6

8

10

12

14

16

18

20

C5

Alc

oh

ols

(m

g/L/

OD

)

HAD-likePhosphataseLibrary

(-)control

0

100

200

300

400

500

Iso

pen

tan

ol(

mg/

L)

(-)control

OYELibrary

OHOPP

OPP OH

OHBiofuel

Water solubility

(g/L)

Research Octane

Numbera

Energy

Density

3-methyl-2-butenol

6 + 0.34 90

3-methyl-3-butenol

9 + 0.15 102

3-methyl-butanol

15 + 3.2 102

96% of gasoline

(116

MJ/gal)

Sesquiterpenes

Peralta-Yahya et al. 2011. Nature Comm. 2:483

McAndrews et al. 2011. Structure 19:1876

Page 44: Engineering Microbial Metabolism for Production of Fuels

IPP DMAPP

FPP

GPP

Branched-chain hydrocarbons from the isoprenoid

biosynthetic pathway

FDP

G3P DHAP

PEP

PYR

AcCoA IPP

OAA

MAL

CIT

MEV

DXP

FPP quinones

Page 45: Engineering Microbial Metabolism for Production of Fuels

IPP DMAPP

FPP

GPP

Branched-chain hydrocarbons from the isoprenoid

biosynthetic pathway

FDP

G3P DHAP

PEP

PYR

AcCoA IPP

OAA

MAL

CIT

MEV

DXP

FPP quinones

Page 46: Engineering Microbial Metabolism for Production of Fuels

Bisabolane as a renewable diesel

No 2 Diesel Bisabolane

Cetane No. 41.6 41.9

Freeze pt. N/a <-81C

Cloud pt. -21C <-78C

Density 864.6 g/L 819 g/L

Bisabolene Bisabolane

Peralta-Yahya et al. 2010 Nature Comm. 2:483

Page 47: Engineering Microbial Metabolism for Production of Fuels

Finding the best bisabolene synthase

Peralta-Yahya et al. 2010 Nature Comm. 2:483

Page 48: Engineering Microbial Metabolism for Production of Fuels

Finding the best bisabolene synthase

McAndrew et al. 2011 Structure 19:1876

Page 49: Engineering Microbial Metabolism for Production of Fuels

Bisabolene (C15) Synthase Aristolochene (C15) Synthase

Squalene Hopene (C30) Cyclase Trichodiene (C15) Synthase

Structural comparison

Page 50: Engineering Microbial Metabolism for Production of Fuels

Pathway balance is essential to prevent

accumulation of toxic intermediates

FDP

G3P DHAP

PEP

PYR

AcCoA

OAA

MAL

CIT

IPP DMAPP

DXP

FPP

MEV

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)

Cell

Gro

wth

(O

D 6

00 )

Page 51: Engineering Microbial Metabolism for Production of Fuels

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

PMK MK PMD Idi IspA FPP ADS Mev Terpene

IPTG Arabinose

Page 52: Engineering Microbial Metabolism for Production of Fuels

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?

PMK MK PMD Idi IspA FPP ADS Mev Terpene

Page 53: Engineering Microbial Metabolism for Production of Fuels

Identification of FPP-responsive promoters

• No terpene synthase to

consume FPP

• Examine differences in gene

expression in the presence and

absence of a terpene synthase

PMK MK PMD Idi IspA Mev FPP

PMK MK PMD Idi IspA FPP ADS Mev Amorp

Dahl et al. 2013 Nat. Biotechnol. 31:1039.

Page 54: Engineering Microbial Metabolism for Production of Fuels

Expression of about 40 promoters significantly changed

in response to FPP

Without sesquiterpene

synthase

With sesquiterpene

synthase

FPP

Dahl et al. 2013 Nat. Biotechnol. 31:1039.

Page 55: Engineering Microbial Metabolism for Production of Fuels

Two FPP-responsive promoters

HMGS atoB tHMGR PMK MK PMD Idi IspA FPP ADS

IPTG

Plac Plac Plac

HMGS atoB tHMGR PMK MK PMD Idi IspA FPP ADS

PrstA PgadE PgadE

Inducible system

Dynamically-controlled system

Dahl et al. 2013 Nat. Biotechnol. 31:1039.

Page 56: Engineering Microbial Metabolism for Production of Fuels

0

300

600

900

1200

1500

1800

pADS prstA-ADS pADS prstA-ADS

lacUV5-MevT-MBIS pgadE-MevT-MBIS

Am

orp

ha

die

ne

Pro

du

cti

on

(m

g/L

)

-IPTG

+IPTG

FPP-responsive promoters

improve terpene production

HMGS atoB tHMGR PMK MK PMD Idi IspA

IPTG

Plac Plac

HMGS atoB tHMGR PMK MK PMD Idi IspA FPP

PgadE PgadE

FPP ADS

PrstA

FPP ADS

PrstA

ADS

IPTG

Plac

ADS

IPTG

Plac

High product titers with

inducer-free control

Dahl et al. 2013 Nat. Biotechnol. 31:1039.

Page 57: Engineering Microbial Metabolism for Production of Fuels

Fuel toxicity

a-pinene limonene

E. coli M9 Minimal Media (vol/vol)

No

rmal

ize

d C

ell

De

nsi

ty

Page 58: Engineering Microbial Metabolism for Production of Fuels

Bioprospecting for solvent resistance pumps (SRPs)

Many different pumps in many

microbes

How do we find the right ones?

Dunlop et al. 2011. Mol. Sys. Biol. 7:487.

Page 59: Engineering Microbial Metabolism for Production of Fuels

Bioprospecting for solvent resistance pumps (SRPs)

• Search genomes of all

sequenced gram-negative

bacteria for efflux pumps

• Final library contained

pumps distributed across

the homology profile

• Used SLIC-based strategy

to create efflux pump strain

library

Dunlop et al. 2011. Mol. Sys. Biol. 7:487.

Page 60: Engineering Microbial Metabolism for Production of Fuels

Pumps screened by competition

For this we designed custom microarrays which contain

probes for all known efflux pumps from sequenced bacteria.

Expression

plasmids and

individual

strains

Pool

strains in

equal

proportion

Pool is grown in M9 medium (control)

OR M9 medium with biofuel (test)

Arrays used

to identify

pumps

remaining in

the pool at

every

dilution

Plasmid isolated

from pools at

every dilution and

identify winner

Dunlop et al. 2011. Mol. Sys. Biol. 7:487.

Page 61: Engineering Microbial Metabolism for Production of Fuels

Biofuel competitions

Dunlop et al. 2011. Mol. Sys. Biol. 7:487.

Page 62: Engineering Microbial Metabolism for Production of Fuels

Engineering pinene tolerance into E. coli

Dunlop et al. 2011. Mol. Sys. Biol. 7:487.

a-pinene

Page 63: Engineering Microbial Metabolism for Production of Fuels

Microorganisms engineered

for consolidated bioprocessing

Problem: Enzymes

add significant

cost to the biofuel

Solution: Engineer fuel-producing

microbes to secrete enzymes

Biofuel

Pretreatment Enzymes

Page 64: Engineering Microbial Metabolism for Production of Fuels

E. coli growth on Beechwood Xlyan

Beechwood Xylan Xylodextrins

Xylose/

Arabinose

Endoxylanase β-xylosidase

M9 minimal medium with

0.2% ionic liquid-

extracted switchgrass

xyn10B OsmY xsa

Endoxylanase β-xylosidase

Beechwood xylan

Xylose

Bokinsky et al. 2011. Proc. Natl. Acad. Sci. USA. 108:19949.

Page 65: Engineering Microbial Metabolism for Production of Fuels

Endocellulase: OsmY-Cel Betaglucosidase: Cel3A

E. coli growth on phosphoric

acid-swollen cellulose

cel OsmY cel3A

Endocellulase β-glucosidase

Bokinsky et al. 2011. Proc. Natl. Acad. Sci. USA. 108:19949.

Page 66: Engineering Microbial Metabolism for Production of Fuels

Switchgrass Eucalyptus

E. coli growth on

ionic liquid-treated plant biomass

Bokinsky et al. 2011. Proc. Natl. Acad. Sci. USA. 108:19949.

Page 67: Engineering Microbial Metabolism for Production of Fuels

Growth on IL-treated garden waste

Bokinsky et al. 2011.

Proc. Natl. Acad. Sci. USA. 108:19949.

Page 68: Engineering Microbial Metabolism for Production of Fuels

Fatty acid ethyl esters (biodiesel)

Butanol (gasoline substitute)

Pinene (jet fuel precursor)

Jet fuel, diesel fuel, and gasoline

from switchgrass

Bokinsky et al. 2011. Proc. Natl. Acad. Sci. USA. 108:19949.

Page 69: Engineering Microbial Metabolism for Production of Fuels

Polyketide-based fuels

PEP

Pyruvate

Acetyl-CoA

CIT

ICT

OGA

SUC

FUM

OAA

MAL

CO2

Glyoxylate

SUC-CoA

Acetaldehyde

Ethanol

Propionyl-CoA Propanol

Acetoacetyl-CoA Butyryl-CoA Butanol

Even chain fatty acid

Aliphatic hydrocarbons

Odd chain fatty acid

G3P

IPP DMAPP

GPP

FPP

GGPP

Isopentenol Isopentanol

Geraniol

Farnesol

Geranylgeraniol

Monoterpene

Sesquiterpene

Diterpene

DHAP

FBP

F6P

G6P 6PG X5P

E4P

S7P

Valeryl-CoA Isopentanol

Glucose

Xylose

Mannose M6P

Galactose G’1P G1P

Xylulose

R5P

Ribulose

Arabinose

Various esters

Alcohols

Aromatic hydrocarbons

Short, highly-branched

hydrocarbons

Isoprenoids

Page 70: Engineering Microbial Metabolism for Production of Fuels

Erythromycin Nystatin

Lipomycin

Polyketides

Page 71: Engineering Microbial Metabolism for Production of Fuels

Erythromycin

AT ACP KS AT ACP

KR

DH

ER

TE KS AT ACP

KR

KS AT ACP

KRx

AT ACP

KR

KS AT

KR

KS AT ACP

KR

KS ACP

Donadio 1991 Science

Erythromycin synthase

DEBS1 DEBS2

Module 1 Module 2 Module 3 Module 4

DEBS3

Module 5 Module 6

Page 72: Engineering Microbial Metabolism for Production of Fuels

DEBS1 DEBS2

Module 1 Module 2 Module 3 Module 4

DEBS3

Module 5 Module 6

AT ACP KS AT ACP

KR

DH

ER

TE KS AT ACP

KR

KS AT ACP

KRx

AT ACP

KR

KS AT

KR

KS AT ACP

KR

KS ACP

Polyketide synthases (PKSs)

are very large, multi-activity proteins

Protein 1 Protein 2 Protein 3

Page 73: Engineering Microbial Metabolism for Production of Fuels

DEBS1 DEBS2

Module 1 Module 2 Module 3 Module 4

DEBS3

Module 5 Module 6

AT ACP KS AT ACP

KR

DH

ER

TE KS AT ACP

KR

KS AT ACP

KRx

AT ACP

KR

KS AT

KR

KS AT ACP

KR

KS ACP

Each extension module adds 2 carbons (plus pendant

groups) to the polyketide backbone

Extension

Module

Loadin

g

Module

Page 74: Engineering Microbial Metabolism for Production of Fuels

DEBS1 DEBS2

Module 1 Module 2 Module 3 Module 4

DEBS3

Module 5 Module 6

AT ACP KS AT ACP

KR

DH

ER

TE KS AT ACP

KR

KS AT ACP

KRx

AT ACP

KR

KS AT

KR

KS AT ACP

KR

KS ACP

Polyketide synthases (PKSs)

are very large, multi-activity proteins

Extension

Module

Loadin

g

Module

a

Termination

Module

Page 75: Engineering Microbial Metabolism for Production of Fuels

DEBS1 DEBS2

Module 1 Module 2 Module 3 Module 4

DEBS3

Module 5 Module 6

AT ACP KS AT ACP

KR

DH

ER

TE KS AT ACP

KR

KS AT ACP

KRx

AT ACP

KR

KS AT

KR

KS AT ACP

KR

KS ACP

Each domain in a PKS has an individual enzyme activity

Domains

Page 76: Engineering Microbial Metabolism for Production of Fuels

Basic PKS

AT KS AT ACP ACP TE

Load Extension Termination

SH SH

Page 77: Engineering Microbial Metabolism for Production of Fuels

Basic PKS

AT KS AT ACP ACP TE

Load Extension Termination

SH SH

HSCoA SCoA

O

R1

Page 78: Engineering Microbial Metabolism for Production of Fuels

Basic PKS

AT KS AT ACP ACP TE

Load Extension Termination

SHS

O

R1

Page 79: Engineering Microbial Metabolism for Production of Fuels

Basic PKS

AT KS AT ACP ACP TE

Load Extension Termination

SH

HSCoA

S

O

R1 SCoA

O

OH

O

R2

Page 80: Engineering Microbial Metabolism for Production of Fuels

Basic PKS

AT KS AT ACP ACP TE

Load Extension Termination

S

O

R1

S

O

OH

O

R2

Page 81: Engineering Microbial Metabolism for Production of Fuels

Basic PKS

AT KS AT ACP ACP TE

Load Extension Termination

CO2

S

O

R1

S

O

R2

Page 82: Engineering Microbial Metabolism for Production of Fuels

Basic PKS

AT KS AT ACP ACP TE

Load Extension Termination

SH S

O

R1

O

R2

Page 83: Engineering Microbial Metabolism for Production of Fuels

Basic PKS

AT KS AT ACP ACP TE

Load Extension Termination

SH S

O

R1

O

R2

OH

O

R1

O

R2

Page 84: Engineering Microbial Metabolism for Production of Fuels

Basic PKS

AT KS AT ACP ACP TE

Load Extension Termination

SH SH

OH

O

R1

O

R2

Page 85: Engineering Microbial Metabolism for Production of Fuels

Basic PKS

AT KS AT ACP ACP TE

Load Extension Termination

SH

KR

S

O

R1

O

R2

Page 86: Engineering Microbial Metabolism for Production of Fuels

Basic PKS

AT KS AT ACP ACP TE

Load Extension Termination

SH

KR

S

O

R1

HO

R2

Page 87: Engineering Microbial Metabolism for Production of Fuels

Basic PKS

AT KS AT ACP ACP TE

Load Extension Termination

SH

KR

SH

OH

O

R1

HO

R2

Page 88: Engineering Microbial Metabolism for Production of Fuels

Basic PKS

AT KS AT ACP ACP TE

Load Extension Termination

SH S

O

R1

O

R2

KR DH

Page 89: Engineering Microbial Metabolism for Production of Fuels

Basic PKS

AT KS AT ACP ACP TE

Load Extension Termination

SH

KR DH

S

O

R1

HO

R2

Page 90: Engineering Microbial Metabolism for Production of Fuels

Basic PKS

AT KS AT ACP ACP TE

Load Extension Termination

SH

KR DH

S

O

R1

R2

Page 91: Engineering Microbial Metabolism for Production of Fuels

Basic PKS

AT KS AT ACP ACP TE

Load Extension Termination

SH SH

KR DH

OH

O

R1

R2

Page 92: Engineering Microbial Metabolism for Production of Fuels

Basic PKS

AT KS AT ACP ACP TE

Load Extension Termination

SH

KR ER

DH

S

O

R1

O

R2

Page 93: Engineering Microbial Metabolism for Production of Fuels

Basic PKS

AT KS AT ACP ACP TE

Load Extension Termination

SH

KR ER

DH

S

O

R1

HO

R2

Page 94: Engineering Microbial Metabolism for Production of Fuels

Basic PKS

AT KS AT ACP ACP TE

Load Extension Termination

SH

KR ER

DH

S

O

R1

R2

Page 95: Engineering Microbial Metabolism for Production of Fuels

Basic PKS

AT KS AT ACP ACP TE

Load Extension Termination

SH

KR ER

DH

S

O

R1

R2

Page 96: Engineering Microbial Metabolism for Production of Fuels

Basic PKS

AT KS AT ACP ACP TE

Load Extension Termination

SH

KR ER

DH

SH

OH

O

R1

R2

Page 97: Engineering Microbial Metabolism for Production of Fuels

Polyketide synthase structure

Whicher et al. 2014. Nature. 510(7506): 560–564

KS

AT

KR

ACP

Page 98: Engineering Microbial Metabolism for Production of Fuels

ACP moves from active site to active site

like an arm on a robot

Whicher et al. 2014. Nature. 510(7506): 560–564

Page 99: Engineering Microbial Metabolism for Production of Fuels

6-dEB

PKSs function like

molecular assembly lines

Page 100: Engineering Microbial Metabolism for Production of Fuels

Lots of naturally occurring Load, Extension, and

Termination modules

AT KS AT ACP ACP TE

Load Extension Termination

S1 mAT ACPKSQ

mmAT(

S)

ACPKSQS2

ACPCoLS3 ERR

O

E1mAT ACPKS

OH

E2 mAT

ACPKS

KR(S)

OH

E3 mAT

ACPKS

KR(R)

E4mAT

ACPKS

KR

DH

E5mAT

ACPKS

KR

DH ER

O

E6 mmAT(

S)ACPKS

OH

E7ACPKS

KR(S)mmAT(

S)

OH

E8ACPKS

KR(R)mmAT(

S)

E9

OH

ACPKS

KR(S)mmAT(

R)

E10

OH

ACPKS

KR(R)mmAT(

R)

E11mmAT

ACPKS

KR

DH

E12ACPKS

KR

DH ER

mmAT(

S)

E13ACPKS

KR

DH ER

mmAT(

R)

E14

O

ACPKS

cMTmmAT

E15

O

ACPKS eAT (S)

E16ACPKS

KR

DH ER

eAT (S)

E17

O

ACPKS pAT (S)

TE

R O

O

TE

OH

O

Red-TE

H

O

Red-TEOH

ST TE

Page 101: Engineering Microbial Metabolism for Production of Fuels

Polyesters

• Used for

– Cushioning, clothing, bottles, films, liquid crystal

displays, holograms, filters, dielectric films, insulation for

wires

– Finishes on wood products, paints, etc.

Product Type 2002 (M tons/yr) 2008 (M tons/yr)

Textile-PET 20 39

Resin, bottle PET 9 16

Film-PET 1.2 1.5

Special polyester 1 2.5

Total 31.2 49

Page 102: Engineering Microbial Metabolism for Production of Fuels

Hydroxyacids are needed for polyesters

HO O

O

O

O

O

O

O

O

O

O

O

O

OH

O

3-hydroxy acids

Page 103: Engineering Microbial Metabolism for Production of Fuels

Erythromycin

AT ACP KS AT ACP

KR

DH

ER

TE KS AT ACP

KR

KS AT ACP

KRx

AT ACP

KR

KS AT

KR

KS AT ACP

KR

KS ACP

Erythromycin synthase

DEBS1 DEBS2

Module 1 Module 2 Module 3 Module 4

DEBS3

Module 5 Module 6

Page 104: Engineering Microbial Metabolism for Production of Fuels

Erythromycin

AT ACP KS AT ACP

KR

DH

ER

TE KS AT ACP

KR

KS AT ACP

KRx

AT ACP

KR

KS AT

KR

KS AT ACP

KR

KS ACP

Erythromycin synthase

DEBS1 DEBS2

Module 1 Module 2 Module 3 Module 4

DEBS3

Module 5 Module 6

Cortes et al. 1995. Science

Page 105: Engineering Microbial Metabolism for Production of Fuels

AT ACP KS AT ACP

KR

TE KS AT ACP

KR

Protein Engineering

DEBS1

Module 1 Module 2

Cortes et al. 1995. Science

Page 106: Engineering Microbial Metabolism for Production of Fuels

AT ACP KS AT ACP

KR

TE KS AT ACP

KR

Protein Engineering

DEBS1

Module 1 Module 2

Page 107: Engineering Microbial Metabolism for Production of Fuels

AT ACP KS AT ACP

KR

TE

Production of 3-hydroxyacids

DEBS1

Module 1

Satoshi Yuzawa, Clara Eng

Page 108: Engineering Microbial Metabolism for Production of Fuels

Potential polymers that can be produced with various 3-

hydroxyacids

HO O

O

O

O

O

O

O

O

O

O

O

O

OH

O

Interesting polyesters with novel properties

Page 109: Engineering Microbial Metabolism for Production of Fuels

LipPks1 1 LipPks2, LipPks3, LipPks4, LipNrps

b-Lipomycin

AT ACP KS AT ACP

KR

Lipomycin PKS

Yuzawa et al. 2013. Biochemistry 52:3791-3793.

Page 110: Engineering Microbial Metabolism for Production of Fuels

Lipomycin load has a broad substrate range

Substrate kcat (min-

1)

Km

(uM)

kcat/Km

(M-1/s-1)

isobutyryl-CoA 0.053 2.9 304.1

propionyl-CoA 0.056 13.4 70.3

n-butyryl-CoA 0.036 26.4 22.7

2-methylbutyryl-CoA 0.126 8.8 237.0

isovaleryl-CoA 0.290 128.1 38.0

pivaloyl-CoA 0.002 8.8 4.1

Yuzawa et al. 2013. Biochemistry 52:3791-3793.

Page 111: Engineering Microbial Metabolism for Production of Fuels

Constructing a 3-hydroxyacid synthase

AT ACP KS AT ACP

KR

TE

Thioesterase (TE) from DEBS PKS

Load and extension modules

from Lipomycin PKS

Load Extension

Yuzawa et al. 2013. Biochemistry 52:3791-3793.

Page 112: Engineering Microbial Metabolism for Production of Fuels

AT ACP KS AT ACP

KR

TE

Production of 3-hydroxy-2-methyl acids

with Lip PKS

+

Load

Extension

Product

Native substrate

Yuzawa et al. 2013. Biochemistry 52:3791-3793.

Page 113: Engineering Microbial Metabolism for Production of Fuels

Potential polymers that can be produced with various 3-

hydroxyacids

HO O

O

O

O

O

O

O

O

O

O

O

O

OH

O

Interesting polyesters with novel properties

Page 114: Engineering Microbial Metabolism for Production of Fuels

The acyltransferase (AT) determines the acyl-CoA

loaded onto the ACP

AT KS AT ACP ACP TE

Load Extension Termination

SH

HSCoA

S

O

R1 SCoA

O

OH

O

R2

Page 115: Engineering Microbial Metabolism for Production of Fuels

The acyltransferase (AT) determines the acyl-CoA

loaded onto the ACP

AT KS AT ACP ACP TE

Load Extension Termination

SH

HSCoA

S

O

R1 SCoA

O

OH

O

Specifies that a methylmalonyl group

will be loaded onto the ACP.

Page 116: Engineering Microbial Metabolism for Production of Fuels

Native DEBS1

Module 1 Module 2

AT ACP KS AT ACP

KR

KS AT ACP

KR

Acyl transferase (AT) specifies the acyl group that

gets loaded onto the ACP

Module 1 Module 2

AT ACP KS AT ACP

KR

KS AT ACP

KR

Modified DEBS1

TE TE

Oliynyk et al 1996 Chem. Biol. 3:883

Page 117: Engineering Microbial Metabolism for Production of Fuels

The acyltransferase (AT) determines the acyl-CoA

loaded onto the ACP

AT KS mAT ACP ACP TE

Load Extension Termination

SH

HSCoA

S

O

R1

Specifies that a malonyl group will be

loaded onto the ACP.

SCoA

O

OH

O

Page 118: Engineering Microbial Metabolism for Production of Fuels

AT ACP KS mAT ACP

KR

TE

Production of 3-hydroxyacids with Lip PKS

+

Load

Extension

Product

Native substrate

Yuzawa et al 2015 unpublished

Page 119: Engineering Microbial Metabolism for Production of Fuels

Ketones

• Solvents

– Methyl ethyl ketone (MEK)

– Ethyl ethyl ketone (EEK)

• Flavors and fragrances

– Long-chain alkyl groups

– Stereochemistry matters

• Fuels

– Diesels (C14-C16)

O

O

Page 120: Engineering Microbial Metabolism for Production of Fuels

- NADPH

Δ AT ACP KS AT ACP

KR

TE

In vitro production of ethyl ketones

LipPK1 + DEBS TE

Yuzawa et al 2015 unpublished

Page 121: Engineering Microbial Metabolism for Production of Fuels

Δ AT ACP KS AT ACP

KR

TE

In vivo production of ethyl ketones

LipPK1 + DEBS TE

X

Knocking out activity of ketoreductase (KR)

disables reduction of ketone to alcohol

Yuzawa et al 2015 unpublished

Page 122: Engineering Microbial Metabolism for Production of Fuels

Δ AT ACP KS mAT ACP

KR

TE

In vivo production of methyl ketones

X

Exchanging the native methylmalonyl-

CoA—specific acyltransferase (AT) for a

malonyl-CoA—specific AT enables

production of methyl ketones

Yuzawa et al 2015 unpublished

Page 123: Engineering Microbial Metabolism for Production of Fuels

Acknowledgements

• US Department of Energy for funding

• All of the members of JBEI

#Jay Keasling has a financial interest in Amyris, LS9, and Lygos.

4

Artemisinic Acid

O

O

O

O

O

H

HH

Glucose Artemisinin

A Novel Semi-synthetic Route

•US Department of Energy for

funding

•All of the members of JBEI

•US Department of Energy for

funding

•All of the members of JBEI

•US Department of Energy for

funding

•All of the members of JBEI

Bill & Melinda Gates Foundation

Department of Energy

National Science Foundation

Funding:

Page 124: Engineering Microbial Metabolism for Production of Fuels

Thanks to:

Eric Steen

Connie Kang

Greg Bokinsky

Robert Haushalter

Leonard Katz

Fuzhong Zhang

James Carothers

Andrew Hagen

Sean Poust

Ee-Been Goh

Harry Beller

Edward Baidoo

Chris Petzold

Tanveer Batth

Dan Groff

Sam Deutsch

Ted Chavkin

Simon Brunner

Satoshi Yuzawa

Clara Eng

Tristan de Rond

Clem Fortman

George Wang

Weerawat Runguphan

Funding:

US Department of Energy

National Science Foundation

LS9

University of California Discovery Grant Program

Institutes and Centers:

Joint BioEnergy Institute (JBEI)

Synthetic Biology Engineering Research Center (Synberc)

Disclosure:

Jay Keasling has a financial interest in Amyris & Lygos

Page 125: Engineering Microbial Metabolism for Production of Fuels

Thanks to …

DOE, NSF for funding

The JBEI Team

Disclosure: Jay Keasling has a financial interest in Amyris & Lygos.