Energy G eneration C arbon M anagem ent Methodology
Chen, T.; Barton, S.; Binyamin, G.; Gao, Z.; Zhang, Y.; Kim, H.-H.; Heller, A. J. Am. Chem. Soc. 2001, 123, 8630-8631. Miniature Biofuel Cells.
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Energy Generation
Carbon Management
Methodology
Energy Generation
Biofuel Cells
Feedstocks
EtOH, H2, Biodiesel, CH4
Reactions
O
OH
OH
OH
OH
HO
O
O
OH
OH
OH
HO
Os+3Os+2
Os+3Os+2
H2O O2
e
7µm carbon fiber
7µm carbon fiber
laccase(ox) laccase(red)
glucose oxidase
(ox)
glucose oxidase
(red)
400µmanode
cathode
Chen, T.; Barton, S.; Binyamin, G.; Gao, Z.; Zhang, Y.; Kim, H.-H.; Heller, A. J. Am. Chem. Soc. 2001, 123, 8630-8631.
Miniature Biofuel Cells
O
O CO2
H
O2H2O
marine sediment
seawater
e
Desulfuromonas acetoxydans
Desulfuromonas acetoxidans
anode
cathode
Microbial Fuel Cells
Bond, D. R.; Holmes, D. E.; Tender, L. M.; Lovley, D. R. Science 2002, 295, 483-485.Reimers, C. E.; Tender, L. M.; Fertig, S.; Wang, W. Environ. Sci. Technol. 2001,35, 192-195.http://math.usc.edu/~grosen/mudfuelcell/Park, D. H.; Zeikus, J. G. Appl. Environ. Microbiol. 2000, 66, 1292-1297.
CO2
O
OH
OH
OH
OH
HO
OOH
OHHO
O
OH
OH
OH
OH
starch
hemicellulose
cellulose
CH3CH2OH or H2
D-glucose
D-xylose
L-arabinose
HO
O
H
HOOHH
OHH
1
HO
O
H
HOOHH
HH
O
OH
H
H
O O
H
HOOHH
OHH
H
O O
H
HOOHH
OHH
H
H
O
amylose
4
1
1
4
4
1
O
H
HOOHH
OHH
1
HO
O
H
HOOHH
HH
O
OH
H
O
4
1
O
H
HOOHH
OHH
H
H
O O
H
HOOHH
OHH
H
H
O O
H
HOOHH
HH
H
O
4
1
1
4
4
1O
CH2
H
6
1,6-link(a branch point)
amylopectin
4
H
4
1,4-link
O
H
O
H
HO
HOHH
OH
O
H
H
HH
HO
H
O
OH
H
O
H
H
HH
HO
H
O
OH
O
H
O
H
HO
HOHH
OHH
O
OH OH1
4
6
4 6 6
1 4
1 4
cellobiose unit
cellulose chain
1
n
6
Cellulase(1) Improve catalytic activity(2) Eliminate feedback inhibition
Cellulose SynthaseTemperature-sensitive rsw1 rsw2 alleles produce noncrystalline, soluble cellulose in Arabidopsis.
Lane, D. R.; Wiedemieier, A.; Peng, L.; Hofte, H.; Vernhettes, S.; Desprez, T.; Hocart, C. H.; Birch, R. J.;Baskin, T. I.; Burn, J. E.; Arioli, T.; Betzner, A. S.; Williamson, R. E. Plant Physiol. 2001, 126, 278-288.Arioli, T.; Peng, L.; Betzner, A. s.; Burn, J.; Wittke, W.; Herth, W. Camilleri, C.; Hofte, H.; Plazinski, J.;Birch, R.; Cook, A.; Glover, J.; Redmond, J.; Williamson, R. E. Science 1998, 279, 717-720.
O
O
HOOH
OO O
OH
O O
HOOH
OO
O
HOOH
OO O
OH
O O
HOOH
OO
OH
OHO
O
CH3O
HO
HO
CH3O
O
O
O
OH
OH
1
1
heteroxylan
3
44
Approximately 80% of the backbone xylose residues are substituted by side chains.Ferulic acid is attached to arabinose side chains.Dimerization of the ferulic acids contributes to the cross-linking.
Saulnier, L.; Thibault, J.-F. J. Sci. Food Agric. 1999, 79, 396-402.Zaldivar, J.; Ingram, L. O. Biotechnol. Bioeng. 1999, 66, 203-210.
CH3CH2OH
Carbon source product
D-glucose D-xylose L-arabinose
Zymomonas mobilis
Saccharomyces cerevisiae
Escherichia coli
+
+
+ + +
+
+
_
_
_
_
_
H3C CO2H
O
CO2H3C H
O
NADH
CH3CH2OH
BioEthanol: Before Biotechnology
fermentablecarbon source
Pdc Adh
CH3CH2OH
carbon source product
D-glucose D-xylose L-arabinose
Zymomonas mobilis
Saccharomyces cerevisiae
Escherichia coli
+
+
+ + +
+
+
+
+
+
+
+
BioEthanol: After Biotechnology
H3C CO2H
O
CO2H3C H
O
NADH
CH3CH2OHfermentablecarbon source
Pdc Adh
Ingram, L. O.; Conway, T.; Flavio, A. U.S. Patent 5 000 000, 1991.Zhang, M.; Eddy, C.; Deanda, K.; Finkelstein, F.; Picataggio, S. Science 1995, 267, 240-243.Walfridsson, M.; Hallborn, J.; Penttila, M.; Keranen, S.; Hahn-Hagerdahl, B. Appl. Environ. Microbiol.
1995, 4184-4190.Ho, N.W.Y.; Chen, Z.; Brainard, A. P. Appl. Environ. Microbiol. 1998, 64, 1852-1859.
H2O
O2
H2O
O2 CO2
[CH2O]2 NADPH
H2
H2
Direct Biophotolysis
Photosystems Ferredoxin Hydrogenase
Indirect Biophotolysis
Photosystems Ferredoxin Hydrogenase
Bio Hydrogen
Benemann, J. Nature Biotechnol. 1996, 14, 1101-1103.
Light Reactions
H2
2H+
H2ase
CO2H
O
OH
O
CO2
Fdox
Fdred
carbonsource
ClostridiaCitrobacterBacillusEscherichia
Options: 1) cogeneration
2) organic waste streams
3) stationary phase metabolism
Bio Hydrogen
Dark Reaction
Van Ginkel, S.; Sung, S.; Lay, J.-J. Environ. Sci. Technol. 2001, 35, 4726-4730.Woodward, J.; Orr, M.; Cordray, K.; Greenbaum Nature 2000, 405, 1014-1015.Adams, M. W. W.; Stiefel, E. I. Science 1998, 282, 1842-1843.
O
O
OX
O
"R
R'
O
ROH
HOOH
OH
RO2C(CH2)7CH CH(CH2)7CH3
RO2CCH2CH2(CH2)12CH3
RO2CCH2CH2CH2CH2(CH2)12CH3
RO2C(CH2)7CH CH
CH2 CH
CH(CH2)4CH3
RO2C(CH2)7CH CH
CH2 CH
CH
CH2 CH
CHCH2CH3
caustic soda160 C
palmitate ester
stearate ester
oleate ester
linoleate ester
linolenate ester
glycerol
Bio Diesel
Murray, S. Wall Str. J. 2002 (May 9), CCXXIX (91), A4.
H2O
H+
H2O
H+
CH3-S-CoM + HS-CoB
CoM-S-S-CoB
CH3-S-CoM
CO2 + MF
2e-, 2H+
formyl-MF
formyl-MF + H4MPT formyl-H4MPT + MF
formyl-H4MPT methenyl-H4MPT+
methenyl-H4MPT+ + F420H2 methylene-H4MPT + F420
methylene-H4MPT + F420H2 methyl-H4MPT + F420
methyl-H4MPT + HSCoM
CH4 + CoM-S-S-CoB
2e-, 2H+
HSCoM + HSCoB
CH4
Ferry, J. FEMS Microbiol. Rev. 1999, 23, 13-38.
Pyrococus furiosus
1 atm CO90 °C
B2H6
1 atm CO40 °C
Clostridium thermoaceticum
Ni or 2CuO•Cr2O3
200-300 atm H2150-250 °CRCO2H RCH2OH
Reactions:
Reduction
Huber, C.; Skopan, H.; Feicht, R.; White, H.; Simon, H. Arch. Microbiol. 1995, 164, 110. Simon, S.; White, H.; Lebertz, H.; Thanos, I. Angew. Chem. Int. Ed. Engl. 1987, 26, 785.White, H.; Strobl, G.; Feicht, R.; Simon, H. Eur. J. Biochem. 1989, 184, 89. White, H.; Feicht, R.; Huber, C.; Lottspeich, F.; Simon, H. Biol. Chem. Hoppe-Seyler 1991, 372, 999.Johnson, M. K.; Rees, D.C.; Adams, M.W.W. Chem. Rev. 1996, 96, 2817. Kletzin, A.; Adams, M. W. W. FEMS Microbiol. Rev. 1996, 18, 5.Adams, M. W. W.; Kletzin, A. Adv. Prot. Chem. 1996, 48, 101.Fraisse, L.; Simon, H. Arch. Microbiol. 1988, 150, 381.Huber, C.; Caldeira, J.; Jongejan, J. A.; Simon, H. Arch. Microbiol. 1994, 162, 303.van den Ban, E. C. D.; Willemen, H. M.; Wassink, H.; Laane, C.; Haaker, H. Enz. Microb. Technol. 1999, 25, 251.
NADH NAD
H2O
CH3OHCH4 + O2
methane monooxygenase
Reactions:
Oxidation
Merkx, M.; Kopp, D. A.; Sazinsky, M. H.; Blazyk, J. L.; Muller, J.; Lippard, S. J.Ang. Chem. Int. Ed. 2001, 40, 2782-2807.
Carbon Management
CO2 Fixation
CO2 Immobilization
CH2OPO32-
C
C
O
OH
C
H
H
CH2OPO32-
OH
CH2OPO32-
C
C
OH
OH
CH
CH2OPO32-
OH
CO2
O2
CH2OPO32-
C
C
CO2-
O
CH
CH2OPO32-
OH
HO H2O
CH2OPO32-
C
C
O
O
CH
CH2OPO32-
OH
HO OH H2O
CH2OPO32-
C
CO2-
OHH
CH2OPO32-
CO2-
CO2-
CH
CH2OPO32-
OH
ribulose 1,5-bisphosphate enediol
2
3-phosphoglycerate
3-phosphoglycerate
phosphoglycolate
CO2 Fixation
ribulose 1,5-bisphosphate carboxylas/oxygenase
Can Rubisco be improved?Stemmer, W. P.; Subramanian, V.; Selifonov, S. WO 00/2808, 2000.
CO2 Fixation
CO2-
OPO32-
HCO3-
CO2-
O-O2C P
HO O-O-
O+
phosphoenolpyruvate oxaloacetate
+
phosphoenolpyruvate carboxylase
pyruvate carboxylase
NHHN
S
O
Enzyme
NHN
S
O
Enzyme
-O
O
ATP
CO2-
O
HCO3-
SCoA
O Mg2+
NHN
S
O
Enzyme
-O
O
NHHN
S
O
Enzyme
ADP
CO2-
O-O2C
PHO O-
O-
O+ + + +
+
pyruvate
+
oxaloacetate
Peters-Wendisch, P. G.; Schiel, B.; Wendisch, V. F.; Katsoulidis, E.; Mockel, B.; Sahm, H.; Eikmanns, B. J.J. Mol. Microbiol. Biotechnol. 2001, 3, 295-300.
Vemuri, G. N.; Eiteman, M. A.; Altman, E. Appl. Environ. Microbiol. 2002, 68, 1715-1727.
CO2
O
O
O
O
O
OH
HO OHOH
OHOH
OH
O
starch
lactic acidglucose
Lactobacillus
ring-openingpolymerization
low molecular wt.polylactide prepolymer
high molecular wt.polylactide polymer
lactidedimer
H O CH
CH3
n
C OH
O
H O CH
CH3
n
C OH
O
CO2 Immobilization
PLA
Gruber, P. R. In Carbon Management: Implications for R&D in the Chemical Sciences and Technology;National Academy Press: Washington, 2001, Chap 11, p 166-184.
Kharas, G. B.; Sanchez-Riera, F.; Severson, D. K. in Plastics from Microbes; Mobley, D. P., Ed.; Hanser:New York, 1994, Chap 4, p 93137.
CO2
NH
NH2O
O
OH
HO OHOH
OH
NH
O
N CH2
H
CO2
NH3
NH3
C
O
starch
L-lysineglucose
+
-
+
5 n
-aminocaprolactam caprolactam nylon 6
CO2 Immobilization
BioNylon 6
CO2
CO2
H
N CH2
H
O CH
CH3
C
O
OHOH
O
NH
O
lactic acid
n
5 n
caprolactamBioNylon 6
PLA
monomerrecovery
bio-degradation
bio-degradation
monomerrecovery
C
O
OH
x x
If 1 x 109 lb of Nylon 6 were made from glucose:
petroleum equivalent 9 x 106 barrels of petroleum
If asphalt was replaced with BioNylon 6:
20 x 106 MT/yr of asphalt1.3 kg petroleum/kg asphalt
petroleum equivalent 140 x 106 barrels of petroleum
Lesson:(a) large-volume commodity chemical(b) multiple commodity chemicals(c) expand individual commodity chemical volumes
HO OH
OH
CO2H
HO2C
OHCH2 CH2O
R
N CH2
H
N C
H
CH2 C
O O
C
O
C O
O
glucose
1,3-propanediolnCorterra (Shell)
Sorona (Dupont)
glucose
adipic acid
( )6
( )4
Nylon 66
glucose
n
Bakelite resinsNovolac resins
CO2H
HOO C O
OO C C
O Oglucose
Xydar
CH2 O( )3
Skraly, F. A.; Lytle, B. L.; Cameron, D. C. Appl. Environ. Microbiol. 1998, 64, 98-105.Niu, W.; Draths, K. M.; Frost, J. W. Biotechnol. Prog. 2002, 18, 201-211.Gibson, J. M.; Thomas, P. S.; Thomas, J. D.; Barker, J. L.; Chandran, S. S.; Harrup, M. K.;
Draths, K. M.; Frost, J. W. Ang. Chemie Int. Ed. 2001, 40, 1945-1948.Amaratunga, M.; Lobos, J. H.; Johnson, B. F.; Williams, E. D. U. S. Patent 6 030 819, 2000.
Methodology
Metabolic Engineering
Directed Evolution
HO OH
OH
H2O3PO OHOH
CO2CO2H
HO2CO
glycerolglycerolfacilitator ATP ADP glycerol
3-phosphate
oxaloacetateATP ADPcarbondioxide
C6
C5
C5
C3
C1
ADPATP
D-xylose
O
OHOH
OHOH
cytoplasmperiplasm
O
OH
OH
OH
OHH2O3PO
D-glucose
O
OH
OHHO
OH
OH
pyruvic acidPEP
O
HOOH
OHHO
L-arabinose
carbohydrate phospho- transferase
high-affinity permease
D-glucose 6-phosphate
ATP ADP
high-affinity permease
H2O3PO OHO
OH
OH
D-xylulose 5-phosphate
ATP
ADPXylA XylB
AraD
ATP ADP
AraA
AraB
pyruvic acid
Yield and Titer
D-glucose
O
OHHO OH
OHOH
O
OHHO OH
OHOH
O
OHHO OH
OHOH
O
OH
OH
OHOH
H2O3PO
D-glucose 6-phosphate
periplasm cytoplasm
carbohydratephosphotransferase system
facilitatedglucosediffusion
pts
glf
CO 2H
OPO 3H2
CO 2H
O
CO 2
glk
ADP
ATP
PEP pyruvate
Yield and Titer
Patnaik, R.; Liao, J. C. Appl. Environ. Microbiol. 1994, 60, 3903-3908Snoep, J. L.; Arfman, N.; Yomano, L. P.; Fliege, R. K.; Conway, T.;
Ingram, L. O. J. Bacteriol. 1994, 176, 2133.
Yield and Titer
Product Toxicity
Product Export
“A New Type of Transporter with a New Type of Cellular Function: L-Lysine Export from Corynebacterium glutamicum” Vrljic, M.; Sahm, H.; Eggeling, L. Molecular Microbiol. 1996, 22, 815.
“Unbalance of L-Lysine Flux in Corynebacterium glutamicum and Its Use for the Isolation of Excretion Defective Mutants” Vrljic, M.; Kronemeyer, W.; Sahm, H.; Eggeling, L. J. Bacteriol. 1995, 177, 4021.
mechanism-based
osmotolerance
0
10
20
30
40
50
60
0 12 18 24 30 36 42 480
10
20
30
40
50
glucose
O
OH
OHOH
HO OH OHHO OH
CO2HHO
quinate
QP1.1/pKD12.138
Glucose-Rich Culture of QP1.1/pKD12.138
QA
, DH
Q (
g/L
)
dry
ce
ll w
eig
ht
(g/L
)
time (h)
25 g/L 7 %
titer:yield:
OHOH
CO2HHO
O
3-dehydroquinate
46 g/L14 %
Catalytic Lifetime
Kolter, R. J. Bacteriol. 1999, 181, 697-699.Schellhorn, H. E.; Audia, J. P.; Wei, L. I. C.; Chang, L. J. Bacteriol. 1998, 180, 6283-6291.Rowe, D. C. D.; Summers, D. K. Appl. Environ. Microbiol. 1999, 65, 2710-2715.Yamada, M.; Talukder, A. A.; Nitta, T. J. Bacteriol. 1999, 181, 1838-1846.
Transcriptome and Proteome Analysis
mRNA cDNA hybridization radiolabeled cDNAw/ arrayed genomic ORFs
transcript quantitation
reverse
transcriptase
[-33P]-dCTP
Tao, H.; Gonzalez, R.; Martinez, A.; Rodriguez, M.; Ingram, L. O.; Preston, J. F.; Shanmugam, K. T.J. Bacteriol. 2001, 183, 2979-2988.
Gonzalez, R.; Tao, H.; Shanmugam, K. T.; York, S. W.; Ingram, L. O. Biotechnol. Prog. 2002, 18, 6-20.
Expression
Promoters
inducers
host range
mRNA stability
“all or none”
Transcription/Translation
codon usage
folding
proteolysis
Smolke, C. D.; Khlebnikov, A.; Keasling, J. D. Appl. Microbiol. Biotechnol. 2001, 57, 689-696.Jahng, D.; Wood, T. K. Appl. Environ. Microbiol. 1994, 60, 2473-2482.
xx x
x xx
xx
xxxx
xx x
fragmentwith DNAseI
xx x
x xx
xx
xxxx
xx x
reassemblefragments
xx x
x xx
xx
xxxx
xx x
xxx x x xxx
select bestrecombinants
repeat for multiple cycles
Stemmer, W. P. Proc. Natl. Acad. Sci. 1994, 91, 10747-10751Zhao, H.; Giver, L.; Shao, Z.; Affholter, J. A.; Arnold, F. H. Nature Biotechnol. 1998, 16, 258-261.
Gene Shuffling
Citrobacter freundii
Klebsiella pneumoniaeEnterobacter cloacae
Yersinia enterocolitica
Cephalosporinase Genes
8-fold increased resistance8-fold increased resistance8-fold increased resistance
8-fold increased resistance
Plate on moxalactam Select best mutants
Single gene shuffling
Multi-gene shuffling
Library of point mutants
Library of chimaeras
Plate on MoxalactamSelect best mutant:270-540-fold increased resistance
Family Shuffling
Crameri, A.; Raillard, S.-A.; Bermudez, E.; Stemmer, W. P. C. Nature 1998, 391, 288-291.
SF1
SF21
NTGNTGNTGNTGNTGNTGNTGNTGNTGNTGUVUVUVNSHNO2HNO2HNO2UVHNO2HNO2
GS1, GS2
GS
GS
NTG
Classicalstrainimprovement
(20 years106 assays)
Genomeschuffling
(1 year24,000 assays)
Strain
SF1
SF21
GS1
GS2
titre (rel. g l-1)
+_
+_+_
+_ 0.11.0
6.2
8.1
6.2 +_
2.4
1.2
1.2
Genome Shuffling
Zhang, Y.-X.; Perry, K.; Vinci, V. A.; Powell, K.; Stemmer, W. P. C.; del Cardayre, S. B. Nature 2002, 415, 644-646.
Energy Generation
Carbon Management
Methodology
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