Photo-Synthetic Biology for Fuels - ARPA-E | … Biology for Fuels James C. Liao Chemical and...

Photo-Synthetic Biology for Fuels

James C. Liao

Chemical and Biomolecular

Engineering

University of California, Los Angeles

The Current Biofuel Cycle

CO2

CO2

EthanolBiodiesel

The Direct Solar Fuel Cycle

CO2

CO2

Higher Alcohols

Biofuels

Ethanol Long chain alcohols/alkanes

Energy content Low High

Hygroscopicity High LowVehicle

retrofitting? Yes No

Vapor pressure High Low

Production yield High Zero/Low

Higher alcohols

Iso-propanol

n-propanol

n-butanol

Iso-butanol

2-methyl-1-butanol

3-methyl-1-butanol

yes

no

yes

no

no

no

Native producers?

Pathways for alcohol synthesis

n-Butanol

Pyruvate

Acetyl-CoA Acetaldehyde

EthanolAcetoacetyl-CoA

3-Hydroxybutyryl-CoA

Crotonyl-CoA ButyraldehydeButyryl-CoA

PDH

ADH

CoA-dependent pathway

NADH

NADH NADH

Alternative Pathways to Make Alcohols

n-Butanol

Pyruvate

Acetyl-CoA Acetaldehyde

EthanolAcetoacetyl-CoA

3-Hydroxybutyryl-CoA

Crotonyl-CoA ButyraldehydeButyryl-CoA

PDH PDC

ADH

CoA-dependent pathway

?Non-CoA pathway

Atsumi

et al. Nature 2008

NADH

NADH NADH

Generalization of keto acid decarboxylase chemistry

Pyruvate

Acetylaldehyde

Ethanol

PDC

ADH

R-aldehyde

R-OH

KDC

ADH

2-keto acid

A simple keto acidLonger chain keto acids

Ehrlich, F. Ber. Dtsch. Chem. Ges.1907

Opening the active site

Zymomonas mobilis PDC Lactococcus lactis KdcA

Production of Higher Alcohols in E.coli (from 2-keto acids)

Pro

duct

ion

(mM

)

KDC

ADH

Long Chain2-keto acids

R-aldehyde

2-ketobutyrate2-ketoisovalerate2-ketovalerate2-keto-3-methyl-valerate

2-keto-4-methyl-pentanoate

phenylpyruvate

1-pr

opan

ol

isob

utan

ol

1-bu

tano

l

2-M

-1-b

utan

ol

3-M

-1-b

utan

ol

2-ph

enyl

etha

nol

30 ํ C 40hr

Atsumi et al. Nature 2008

Further generalization of alcohol‐ producing chemistry

Atsumi et al. Nature 2008

Alternative Pathways to make Alcohols

Pyruvate

Acetyl-CoA Acetaldehyde

Ethanol

PDH PDC

ADH

?NADH

NADH NADH

Atsumi et al. Nature 2008

Long-chain Keto acids

Long-chain alcohols

Pyruvate

Non-polymeric Chain Elongation

XnXn+1

A B C D E F

Aceto-Hydroxy Acid Synthase (AHAS) Chain Elongation

CO2AHAS

(Ec) ilvCNADPH

(Ec) ilvD H2O

R

R

R

R

Pyruvate

CO2(Bs) alsS

(Ec) ilvCNADPH

2-keto-isovalerate

(Ec) ilvD H2O

Pyruvate

2-ketobutyrate

CO2

ilvGM

ilvC NADPH

2-keto-3-methyl valerate

ilvDH2O

Pyruvate

Novel Pathways for C4, C5 Alcohol Synthesis

Atsumi et al. Nature 2008

Isobutanol Synthesis

Pyruvate

acetolactate

Pyruvate

CO2

ilvIH (also ilvMG, ilvNB)

ilvC

2,3 dihydoxy-isovalerate

NADPH

2-keto-isovalerate

ilvDH2OilvE

valineglutamate2-KG

Ethanol

Iso-butanol

46x2/180=0.51

CO2 NADH

74/180 =0.41 g/g

0.3 Adjustedenergyyield

0.37 Adjustedenergy yield

CO2 NADH

Improvement of isobutanol production

2.2 g/LGlucose

PyruvateIlvIH

IlvC

IlvD

2-keto-isovalerate

Isobutanol

KDC

ADH30 ํ C

M9 + 3.6% glc

Atsumi et al. Nature 2008

Effects of Gene Deletions

adhldhfrdfnrptapflB

knoc

kout

gene

s

0

2000

4000

6000

8000

10000

12000

14000

16000

0

4

8

12

16

isob

utan

ol (g

/L)

30 ํ C 64hr

Pyruvate

Acetyl-CoA

LactateLdhA

AdhEEthanol

PtaAcetate

PflB

Glucose

PEPTCA cycle

Succinate

Citrate

PDHc

E.coli metabolism

Isobutanol

FrdABCD

Fnr

High yield production of isobutanol: 86% of Theoretical

21.9 g/LGlucose

Pyruvate

AlsS

IlvC

IlvD

2-keto-isovalerate

Isobutanol

KDC

ADH

Clostridium (1-butanol)

19.6 g/L

83% of theor.

Atsumi et al. Nature 2008

86% of theoretical yield

LeuABCD chain elongation

R

RR

R(Ec) leuCD

(Ec) leuB NADH

CO2Spontaneous

R

(Mj) cimA*(Ec) LeuA*

R2-ketoisovalerate

OH

O

O

2-Keto-4-methylvalerate

(Ec) LeuA

2-ketovalerate

OH

O

O

2-ketohexanoate (C6)

OH

O

O

(Ec) LeuA*

Novel Pathways for C5 alcohol synthesis

Atsumi et al. Nature 2008Connor and Liao, 2008 AEM

Novel Pathway for C3 alcohol synthesis

Atsumi et al. Nature 2008Shen and Liao 2008, Metabolic Engineering

Novel Pathway for C4 alcohol synthesis

Atsumi et al. Nature 2008Shen and Liao 2008, Metabolic Engineering

Novel Pathways for C5 alcohol synthesis

Atsumi et al. Nature 2008Cann and Liao, 2008 AMB

Pyruvate

2‐ketobutyrate 

2‐keto‐3‐methylvalerate (C6)

2‐ketovalerate  2‐ketoisovalerate 

2‐keto‐4‐methylvalerate

1‐butanol(C4)

1‐propanol(C3)

2‐methyl‐1‐butanol (C5)

3‐methyl‐1‐butanol(C5)

Isobutanol(C4)

(B)

(B)

(A)

(A)

(B)

(A) Leucine biosynthesis (leuABCD)

Isoleucine biosynthesis (ilvGM(ST)‐ilvCD)

Synthetic Network for Higher Alcohols

LeuABCD chain elongation

R

RR

R(Ec) leuCD

(Ec) leuB NADH

CO2Spontaneous

R

(Mj) cimA*(Ec) LeuA*

R2-ketoisovalerate

OH

O

O

2-Keto-4-methylvalerate

(Ec) LeuA

2-ketovalerate

OH

O

O

2-ketohexanoate (C6)

OH

O

O

(Ec) LeuA*

Engineering LeuA to produce non-natural alcohols

2-keto-5-methylheptanoate (C8)

2-keto-5-methylheptanoate (C8)

2-keto-6-methyloctanoate (C9)

(Ec) LeuA*(Ec) LeuA*

2-keto-4-methylhexanoate (C7)

(Ec) LeuA*

COOH

O

COOH

O

COOH

O

COOH

O

COOH

O

COOH

O

2-keto-3- methyl

valerate (C6)

2-keto-4-methylhexanoate (C7)

Synthetic pathways for non-natural metabolites

Pyruvate

2‐ketobutyrate 

2‐keto‐3‐methylvalerate (C6)

2‐keto‐4‐methylhexanoate (C7)

2‐keto‐5‐methylheptanoate (C8)

2‐ketovalerate 

2‐ketohexanoate 

2‐ketoheptanoate 

2‐ketoisovalerate 

2‐keto‐4‐methylvalerate

2‐keto‐5‐methylhexanoate

1‐butanol(C4)

1‐pentanol(C5)

1‐propanol(C3)

2‐methyl‐1‐butanol (C5)

3‐methyl‐1‐butanol(C5)

3‐methyl‐1‐

pentanol (C6)

4‐methyl‐1‐hexanol (C7)

2‐keto‐6‐methyloctanoate (C8)

5‐methyl‐1‐heptanol (C8)

1‐hexanol(C6)

4‐methyl‐1‐

pentanol

(C6)

Isobutanol(C4)

(A)

(B)

(A) (A)

(A)

(A)

(A)

(B)

(A)

(A)

Synthetic Network for Higher Alcohols

Zhang et al, 2008 PNAS

What’s new in this strategy?

Harnesses cell’s most

abundant  pathway for production of fuels

and  chemical stocks.

Allows direct synthesis of fuels from  cellulose and CO2

CO2 to isobutanol using Photosynthetic Bacteria

time (day)

isob

utan

ol(m

g/L)