Photo-Synthetic Biology for Fuels

James C. Liao Chemical and Biomolecular Engineering University of California, Los Angeles The Current Biofuel Cycle

CO2

CO2

Ethanol Biodiesel The Direct Solar Fuel Cycle

CO2

CO2

Higher Alcohols Biofuels

Long chain Ethanol alcohols/alkanes Energy content Low High Hygroscopicity High Low Vehicle Yes No retrofitting?

Vapor pressure High Low

Production High Zero/Low yield Higher alcohols

Native producers? yes Iso-propanol

no n-propanol

yes n-butanol

no Iso-butanol

no 2-methyl-1-butanol

no 3-methyl-1-butanol Pathways for alcohol synthesis

CoA-dependent pathway Pyruvate

PDH NADH Acetyl-CoA Acetaldehyde NADH NADH ADH

Acetoacetyl-CoA Ethanol

3-Hydroxybutyryl-CoA

Crotonyl-CoA Butyryl-CoA Butyraldehyde n-Butanol Alternative Pathways to Make Alcohols

CoA-dependent pathway Pyruvate Non-CoA pathway PDH NADH PDC Acetyl-CoA Acetaldehyde NADH NADH ADH ?

Acetoacetyl-CoA Ethanol

3-Hydroxybutyryl-CoA

Crotonyl-CoA Butyryl-CoA Butyraldehyde n-Butanol

Atsumi et al. Nature 2008

Generalization of keto acid decarboxylase chemistry

Longer chain keto acids A simple keto acid

Pyruvate 2-keto acid PDC KDC Acetylaldehyde R-aldehyde ADH ADH Ethanol R-OH

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)

Long Chain 2-keto acids 2-M-1-butanol 1-butanol 3-M-1-butanol 2-phenylethanol 1-propanol isobutanol KDC

Production (mM) R-aldehyde

2 2 2 2 - 2 - - p -k k -ke ke k h e e e e t to t to to n o i o -3 -4 yl b s v - - p u ov al m m y ty a e e e ru r l rat th th a er y y v ADH te ate e l- l- a va p t e e le n ra ta t n e o 30 ํC 40hr a te

Atsumi et al. Nature 2008 Further generalization of alcohol‐ producing chemistry

Atsumi et al. Nature 2008 Alternative Pathways to make Alcohols

Pyruvate Pyruvate PDH NADH PDC ? Acetyl-CoA Acetaldehyde NADH NADH ADH Ethanol Long-chain Keto acids

Long-chain alcohols Atsumi et al. Nature 2008 Non-polymeric Chain Elongation

A B C D E F X Xn n+1 Aceto-Hydroxy Acid Synthase (AHAS) Chain Elongation

2-ketobutyrate Pyruvate

R Pyruvate Pyruvate ilvGM AHAS (Bs) alsS CO2 CO2 CO2

R NADPH NADPH (Ec) ilvC (Ec) ilvC ilvC NADPH

R ilvD (Ec) ilvD H2O (Ec) ilvD H2O H2O

R

2-keto-isovalerate 2-keto-3-methyl valerate Novel Pathways for C4, C5 Alcohol Synthesis

Atsumi et al. Nature 2008 Isobutanol Synthesis

46x2/180 Pyruvate Ethanol =0.51 CO2 NADH ilvIH Pyruvate (also ilvMG, ilvNB) 0.3 CO2 Adjusted acetolactate energy NADPH yield ilvC

2,3 dihydoxy-isovalerate ilvD ilvE H2O valine 2-keto-isovalerate Iso-butanol 2-KG glutamate 74/180 =0.41 g/g CO2 NADH 0.37 Adjusted energy yield Improvement of isobutanol production

2.2 g/L Glucose

Pyruvate IlvIH

IlvC IlvD 2-keto-isovalerate KDC

ADH Isobutanol 30 ํC M9 + 3.6% glc

Atsumi et al. Nature 2008 Effects of Gene Deletions

16000 E.coli metabolism Glucose 14000 16 12000 Succinate FrdABCD 1000012 8000 TCA cycle PEP 8 6000

40004 Citrate 2000 LdhA isobutanol (g/L) Lactate Pyruvate 00

s adh       

e

n ldh e        PDHc g frd t        u fnr Fnr PflB o    

k

c pta  

o

n pflB   k AdhE Ethanol 30 ํC 64hr Acetyl-CoA Isobutanol Acetate Pta High yield production of isobutanol: 86% of Theoretical

21.9 g/L

Glucose

Pyruvate AlsS IlvC IlvD Clostridium (1-butanol) 2-keto-isovalerate 19.6 g/L KDC 86% of 83% of theor. ADH theoretical yield Isobutanol

Atsumi et al. Nature 2008 LeuABCD chain elongation 2-ketoisovalerate 2-ketovalerate O R OH

O (Mj) cimA* (Ec) LeuA* (Ec) LeuA* (Ec) R LeuA

(Ec) leuCD R

(Ec) leuB NADH RR

O Spontaneous CO2 OH O OH O

R O 2-ketohexanoate (C6) 2-Keto-4-methylvalerate Novel Pathways for C5 alcohol synthesis

Connor and Liao, 2008 AEM Atsumi et al. Nature 2008 Novel Pathway for C3 alcohol synthesis

Shen and Liao 2008, Metabolic Engineering Atsumi et al. Nature 2008 Novel Pathway for C4 alcohol synthesis

Shen and Liao 2008, Metabolic Engineering Atsumi et al. Nature 2008 Novel Pathways for C5 alcohol synthesis

Cann and Liao, 2008 AMB Atsumi et al. Nature 2008 Synthetic Network for Higher Alcohols Pyruvate

2‐ketobutyrate (B) (A) 1‐propanol 2‐ketovalerate (B) 2‐ketoisovalerate (C3) (A) 1‐butanol Isobutanol (C4) (C4) 2‐keto‐3‐methyl 2‐keto‐4‐methyl valerate (C6) valerate 2‐methyl‐ 3‐methyl‐1‐butanol 1‐butanol (C5) (C5)

(A) Leucine biosynthesis (leuABCD)

(B) Isoleucine biosynthesis (ilvGM(ST)‐ilvCD) LeuABCD chain elongation 2-ketoisovalerate 2-ketovalerate O R OH

O (Mj) cimA* (Ec) LeuA* (Ec) LeuA* (Ec) R LeuA

(Ec) leuCD R

(Ec) leuB NADH RR

O Spontaneous CO2 OH O OH O

R O 2-ketohexanoate (C6) 2-Keto-4-methylvalerate Engineering LeuA to produce non-natural alcohols Synthetic pathways for non-natural metabolites

2-keto-3- 2-keto-4-methyl 2-keto-5-methyl methyl hexanoate (C7) heptanoate (C8) valerateO (C6) O COOH COOH COOH O (Ec) LeuA* (Ec) LeuA* (Ec) LeuA*

O COOH COOH COOH

O O 2-keto-4-methyl 2-keto-5-methyl 2-keto-6-methyl hexanoate (C7) heptanoate (C8) octanoate (C9) Synthetic Network for Higher Alcohols Pyruvate

2‐ketobutyrate (B) (A) 1‐propanol 2‐ketovalerate (B) 2‐ketoisovalerate (C3) 1‐butanol (A) (A) Isobutanol (C4) (C4) 2‐keto‐3‐methyl 2‐keto‐4‐methyl 2‐ketohexanoate valerate (C6) valerate 1‐pentanol (A) 2‐methyl‐ (A) (A) 3‐methyl‐1‐butanol (C5) 1‐butanol (C5) (C5) 2‐keto‐4‐methyl 2‐keto‐5‐methyl 2‐ketoheptanoate hexanoate (C7) hexanoate (A) 1‐hexanol 3‐methyl‐1‐ 4‐methyl‐1‐ (C6) pentanol (C6) 2‐keto‐5‐methyl pentanol heptanoate (C8) (C6) 4‐methyl‐ (A) 1‐hexanol (C7) 2‐keto‐6‐methyl octanoate (C8) 5‐methyl‐ 1‐heptanol (C8) 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 isobutanol (mg/L)

time (day)