Next Generation Biofuels

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Next Generation Biofuels 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 E.coli Long Chain 2-keto acids KDC 2-keto acids) R-aldehyde ADH 2008 2-phenylethanol (from Nature 3-M-1-butanol et al. 2-M-1-butanol p henylpyruvate Atsumi Production of Higher Alcohols1-butanol in 2-keto-4-methyl-pentanoate 2-keto-3-methyl-valerate isobutanol 2-ketovalerate 1-propanol 2-ketoisovalerate Production (mM) Production 2-ketobutyrate ํC 40hr 30 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 E.coli metabolism Glucose 1600016 14000 Succinate FrdABCD 1200012 10000 TCA cycle PEP 80008 6000 Citrate 40004 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).
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