What s for lunch? Microorganisms for biorefineries

Transcription

1 What s for lunch? Microorganisms for biorefineries Alfredo Martinez alfredo@ibt.unam.mx Biotechnology Institute National University of Mexico 3 rd SMIBIO Cuernavaca, Morelos 4/Nov/7

2 Where we are: Morelos Campus Cuernavaca: The Eternal Spring City What s for lunch?

3 Where we are: Morelos Campus Cuernavaca: The Eternal Spring City What s for lunch? Ácido Láctico: Yogurt Ác. Succínico Ác. Pirúvico: Piruvato de Creatinina Etanol: Mezcal

4 Biorefineries: Biofuels and Chemicals from Lignocellulose Artificial CO cycle CO The Sun Xylose Glucose Cellobiose Mannose Galactose Chemicals BioFuels Agricultural Residues Bagasses and Stovers Sugars Hydrolysis Fermentation Purpose: Design microorganism and process to transform ALL the SUGARS contained into lignocellulose (cellulose: glucose & hemicellulose: pentoses, hexoses, disaccharides) to biofuels or chemicals with homofermentative strains Carreón Rodríguez et al., 9 4

5 BIO-REFINERÍA Sol + CO Biomasa Combustibles: Sólidos, Gaseosos y Líquidos Productos de Fermentación Bio-Refinería Bio-Combustibles Bio-Plásticos Bio-Polímeros Bio-Resinas Bio-Químicos BIODEGRADABLE AMIGABLE CON EL MA RENOVABLE ~ año! vs Petróleo Costos Bio-Plásticos Son 3 R Bio-Etanol Martínez 9 5

6 Fermentation Products Escherichia coli E. coli uses a Wide range of sugars HEXOSES: Glc, Fru, Gal, Man PENTOSES Xyl, Ara, Rib, Xylu And Galacturonate D-Lactate D-Lactato Orencio et al., 8 Acetyl-P acka Acetato Acetate ldha pta NADH Glucose Xylose PEP Pyruvate pflb AcetylCoA ppc NADH Formate adhe Oxalacetate Cell Mass Citrate Acetaldehyde adhe CO Ethanol H frdabcd NADH Fumarate Succinate and make a mix of fermentation products Homo Fermentative 6

7 D-Lactogenic E. coli strain to use pentose-hexose mixtures MG655: ΔpflB, ΔadhE, ΔfrdA, ΔxylFGH, Evolved (ALE) Strain JU5 GLUCOSE XYLOSE PTS Escherichia coli MG655 ΔpflB, ΔadhE, ΔfrdA, ΔxylFGH Adaptive Evolution ΔxylFGH gatc S84L Pdh anaerobic expression ΔmidarpA XYLOSE GLUCOSE-6P FRUCTOSE-6P PEP Pyruvate NADH + H XylE Δreg 7.3 kb Ace Tolerance Xylose D-Lactate X FRUCTOSE-,6 BP DHA-P NADH + H X Ethanol NADH + H Formate Acetaldehyde adhe G3P PEP PYRUVATE X pflb Acetyl-CoA adhe X Acetyl-P ppc pta Oxalacetate ldha NADH + H ack Acetate XYLULOSE XYLULOSE-5P Pentoses Pathway X frda FADH + H FAD + D-Lactate Succinate Utrilla et al. 9 Fernández et al. Utrilla et al. Martínez et al. Martínez et al. 3 USP:

8 Specific Xylose Consumption Rate (gxyl / gdcw. h) Volumetric D-Lactate Productivity (glac / L.h) JU5 Evaluation on Xylose CL3 MG655 CL3 JU JU JU5 JU5 Utrilla et al. JIM&B 9 Utrilla et al. Met Eng. CL3 (MG655 pflb adhe frda) JU (MG655 pflb adhe frda xylfgh::km) JU5 (MG655 pflb adhe frda xylfgh::km Evolved) 8

9 JU5 and JU gatc xyle mutants J U 5 J U g a tc S 8 4 L T c R J U J U r e g 7.3 J U 5 x y le J U g a t C J U 5 g a tc S 8 4 L J U x y le J U g a t C x y le J U 5 g a tc S 8 4 L x y le Utrilla et al. Met Eng G r o w th r a te (h - ) gatc S84L Point Mutation Serine Leucine Position 84 of GatC Protein 9

10 A c e ta te (g /L ) C e ll m a s s (g /L ) X y lo s e (g /L ) G lu c o s e (g /L ) A ra b in o s e (g /L ) X y lo s e (g /L ) G lu c o s e (g /L ) A ra b in o s e (g /L ) D -L A (g /L ) A c e ta te (g /L ) D -L A (g /L ) A) Simulated hydrolysate B) SC-Bagasse hydrolysate A ) B ) T im e (h ) Y D -L A (g D -L A /g S u g a r s ) = Q D -L A (g D -L A /L h ) = Utrilla et. al. Bioresource Technol T im e (h ) Y D -L A (g D -L A /g S u g a r s ) =.. 3 Q D -L A (g D -L A /L h ) = Strain JU5

11 Stover from White Corn Sequential: Thermochemical Hydrolysis, Enzymatic Saccharification and Fermentation, without detoxification Moss-Acosta,

12 A c e ta te (g /L ) C e ll m a s s (g /L ) G lu c o s e (g /L ) X y lo s e (g /L ) A ra b in o s e (g /L ) G lu c o s e (g /L ) X y lo s e (g /L ) A ra b in o s e (g /L ) D -L A (g /L ) A c e ta te (g /L ) D -L A (g /L ) A) Simulated hydrolysate B) Corn Stover hydrolysate 5 A ) B ) T im e (h ) Y D -L A (g D -L A /g S u g a r s ) =.9 5. Q D -L A (g D -L A /L h ) =.3. 5 AV3: JU5 ΔpoxB, ΔackA-pta, ΔmgsA Simultaneous sugar consumption T im e (h ) Y D -L A (g D -L A /g S u g a r s ) = Q D -L A (g D -L A /L h ) =.. 5 Y >!!! Utrilla et al. Bioresource Technol. 6

13 PLA: PLLA sc-pla

14 L - Lactato Ópticamente Puro CH OH O OH OH OH O O O HO CH 3 O O O CH 3 OH OH OH OH O O O CH 3 n O Glucosa Láctico Dímero PLLA 4

15 nd Generation Bio-Plastics: Small Scale BioRef. Artificial CO cycle The Sun CO kg of Sugar Yield kg of Lactic Acid > USD / kg; PLLA: > 4 USA dol/kg Lactate Lignocellulose - Biomass Agricultural Residues Sugar Cane Bagasse Xylose, Cellobiose Glucose, etc. Cellulose, Hemicellulose Hydrolysis Fermentation Purpose: Design microorganism and process to transform Lignocellulose (cellulose & hemicellulose: pentoses, hexoses, disaccharides) to optically pure lactates (D&L): Biopolymer Precursors 5

16 Lactic acid (D and L) production with Metabolic Engineered E. coli strains ldha from E. coli was chromosomally substituted by ltce from B. subtilis No Plasmids D-Lactic D-Lactato acid L-Lactic acid Acetyl-P acka Acetato Acetate ldha ltce Bs pta NADH Glucose Xylose PEP Piruvate pflb AcetylCoA ppc NADH Formate adhe Oxalacetate Cell Mass Citrate Acetaldehyde adhe CO H Ethanol PLA Demand > Ton frdabcd NADH Fumarate Succinate Utrill Carreri et al., 9; Leal Reyes ; Martinez et al. WO PCT 676 A 6

17 Ethanologenic E. coli strain to use pentose-hexose mixtures MG655: ΔpflB, ΔadhE, ΔfrdA, ΔxylFGH, Δldh, PpflB::pdc-adh Zm Strain MS4 GLUCOSE XYLOSE PTS JU5: PpflB::pdc Zm -adhb Zm Pyruvate Ethanol GLUCOSE-6P FRUCTOSE-6P PEP Pyruvate NADH + H XylE Fernández-Sandoval et al. App. Microb. Biotechnol. J. Chem Technol Biotechnol. 7 X FRUCTOSE-,6 BP DHA-P NADH + H X Ethanol NADH + H ETHANOL Formate Acetaldehyde adhe G3P PEP PYRUVATE X pflb Acetyl-CoA adhe X XYLOSE Acetyl-P ppc pta Oxalacetate X ldha NADH + H ack Acetate XYLULOSE XYLULOSE Pentoses Pathway X frda FADH + H FAD + Lactate Succinate Utrilla et al. 9 Fernández et al. Utrilla et al. Martínez et al. Martínez et al. 3 USP:

18 Stover from White Corn: Sequential: Thermochemical Hydrolysis, Enzymatic Saccharification and Fermentation Total Sugar (g/l) Total Sugar (g/l) Ethanol (g/l) Acetate (g/l) Ethanol (g/l) Acetate (g/l) kg of Sugar Yields.5 kg of EtOH ~.5 USD / L 8 6 % H SO 4 3 Non-aerated Cultures with Ethanologenic E. coli MS4, 3.7 g/l,. L, 37 C, ph 7, rpm. No salts were added. No detox. 4 % H SO 4 3 Vargas-Tah et al., Bioresource Technol Comment Small Scale Biorefinery: Ethanol Time (h) All sugars are fermented to ethanol by ethanologenic E. coli MS4 in 36 h 8

19 Lactic acid, Ethanol Scale-up k L a 7. h - Corn stover hydrolysates. 4 L Yield: ~ 95% Productivity. (.5) g/l/h Fernández-Sandoval et al. J. Chem Technol Biotechnol. 7 Sierra-Ibarra 7, To be submitted 9

20 Glucosa, xilosa (g/l) Succínico, Pirúvico (g/l) Succinic acid: Strain JU5 ΔldhA frda Ptrc pck Δppc kg of Sugar Yields -.3 kg SA > 3 USD / kg.65 D-Lactate D-Lactato ldha NADH Glucose Xylose PEP AcetylCoA ppc..5 L-Lactate Acetyl-P acka Acetato Acetate CO pta Pyruvate pflb NADH CO Ethanol Formate adhe Oxalacetate Cell Mass Citrate GLUCOSA + XILOSA Acetaldehyde adhe CO H Tiempo (h) Ethanol frdabcd 6 8 NADH 4 Glc Xil Suc Pir Fumarate Succinate >. -.3

21 g kg of Sugar Yields.98 kg of Pyruvate > USD / kg.65 D-Lactate D-Lactato Pyruvic acid: Strain JU5 ΔldhA ldha NADH Glucose Xylose PEP AcetylCoA ppc..5 L-Lactate Acetyl-P acka Acetato Acetate CO pta Pyruvate pflb.98 NADH 8 7 CO Consumo Cell Mass glucosa/ producción pyr (78) Ethanol Formate adhe.5 Oxalacetate Citrate GLUCOSA + XILOSA Acetaldehyde adhe CO H Ethanol frdabcd NADH Fumarate Succinate > tiempo (h) Glucosa Pyr

22 Biomasa Biorefinery Plataforma Bioquímica Residuos Calor y Electricidad Gas Plataforma Termoquímica Azúcares Combustibles, Productos y Materiales Químicos Gases Escherichia coli as Microbial Cell Factory for the (Small Scale) Biorefinery Concept Etanol Butanol Propanol Metano Bio-Diesel Bio-Gasolina Isopentenol Bio-Queroseno Bio-Hidrógeno Bio-Electricidad,3 Propanodiol PHB Plásticos Polímeros Solventes Fenólicos Resinas (furfural) Ac. grasos Ac. orgánicos Pigmentos Detergentes

23 Who is Escherichia coli? What does E. coli do for humans? E. coli: Bacteria Approximately 33% of the therapeutic proteins for human use are currently produced with E. coli in industrial fermenters. Human growth hormones; interferons; interleukins; erythropoietin; among others L-fenilalanine, PHB, and Propanediol, among others Easy to manipulate & cultivate Sanchez-Garcia et al. MCF 6;5:33 3

24 Macromolecular composition of N. oleoabundans cells from batch and fed-batch cultures Culture mode Proteins (gprot/gdcw*) Carbohydrates (gcarb/gdcw*) Lipids (glip/gdcw*) S M S M S M Batch C/N = 7 4. (±.4) 43.7 (±.9) 3.7 (±.3) 3.9 (±3.5) 4.8 (±.3) 4. (±.9) Batch C/N = (±.7) 4.4 (±.) 3. (±.4) 33.3 (±.5) 3.3 (±.) 5.7 (±.7) Fed-Batch 4.7 (±.4).6 (±.9) 7.5 (±.) 54. (±.) 7.5 (±.9) 33.7 (±.6) S: at the start of the culture M: at the time of maximum cell mass produced

25 Combustibles Fósiles Necesidad de E. Renovables Biocombustibles Combustibles Fósiles Y Biocombustibles Actuales Fósiles Petróleo Gasolina Diesel Turbosina ra Generación Almidón Sacarosa Bio-Etanol Aceites de Plantas Bio-Diesel Islas y Martínez Lignocelulosa Bio-Etanol Bio-Butanol Oleaginosas no comestibles Biodiesel Bioturbosina da Generación CO Algas y Cianobacterias H Fotobiológico Bio-Diesel Bio-Petróleo Bio-Turbosina 3ra Generación ++++ Biorefinería Bioplásticos Biosolventes Proteínas Alimento Ácidos grasos Aceite comestible Etc. Biocombustibles Biorrefinerías Mediano y Largo Plazo 5

26 Gracias Preguntas CONACyT UNAM PAPIIT DGAPA 6