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)