31/03/2009the photanol process The ‘Photanol’ Process: Cyanobacteria for simple solar fuel...
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Transcript of 31/03/2009the photanol process The ‘Photanol’ Process: Cyanobacteria for simple solar fuel...
31/03/2009 the photanol process
The ‘Photanol’ Process:Cyanobacteria for simple solar fuel
Kornel Golebski, Andreas Angermayr, Ginny Anemaet
Joost Teixeira de Mattos & Klaas J. Hellingwerf
Swammerdam Institute for Life Sciences &
Netherlands Institute for Systems Biology
University of Amsterdam
31/03/2009 the photanol process
A little bit of history
A Round-Table Discussion held during the 10th FEBS Meeting in Paris (July 25, 1975) considered the different approaches by which Biological Systems might be used to convert ambient solar energy into more useful energy forms.
31/03/2009 the photanol process
The problem:
“Man does not have much choice. Either we trust the physicist to make us a sun without blowing us up, or we let the bioenergeticists use our present one. Otherwise, we won’t last more than a hundred years or so. This is an exciting challenge for the bioenergetics of tomorrow.”
31/03/2009 the photanol process
The proposed solution:
PSII PSIe-
H2O
O2
H+
macrosco
picm
emb
rane
hydrogenase
H2
Brh
funding
31/03/2009 the photanol process
What is needed..
Antenna
ReactionCenter Catalytic
Site 2
CatalyticSite 1
e-
e-
R (H+, CO2)
P (H2, MeOH)
R (H2O, HA) P (O2, A, H+)
EET
e-Antenna
ReactionCenter Catalytic
Site 2
CatalyticSite 1
e-
e-
R (H+, CO2)
P (H2, MeOH)
R (H2O, HA) P (O2, A, H+)
EET
e-
For any large-scale process, only H2O is a realistic electron donor
fuels
• Use the auto-regenerative capacity of living organisms• A solution for solar fuel with as few conversions as possible (0.334 = 0.01!)
31/03/2009 the photanol process
Some current biofuel technologies (1)
From: Esper, Badura and Rögner (2006) Trends in Plant Science 11: 543-549.
31/03/2009 the photanol process
Some current biofuel technologies (2)
2 Grow algae in ponds
biofuel+
Waste
Harvest cells
extraction
&
modification
Transport to separator
1 Grow crops on land
Harvest organic matter
fermentation
Transport to bioreactor& fractionate
Mostly ethanol Biodiesel (e.g. fatty acid methyl esters)
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The photanol approach
• First generation: Starch from corn or sugar cane fermented into ethanol by yeasts or palm oil trans-esterified to biodiesel.• Second generation:More recalcitrant bio-polymers fermented to alcohol(s) or biodiesel produced by marine algae.• Third generation: “Photanol”
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CO2 + H2O Cells + O2
(plants, bacteria)
E
(animals, fungi, bacteria)
fossil fuels
CO2
Earth’ surface
Unity of life & the broken circle
energy
31/03/2009 the photanol process
1 Light-dependent life (plants, bacteria)
H2O reducing power + ATP + O2
Reducing power + CO2 + ATP
The 2 modes of life
Cells
((Chloro)Phototrophy)
Organic C
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Chloro-Phototrophy; optimized during billions of generations
The light reactionsof photosynthesis:
31/03/2009 the photanol process
Glyceraldehyde-3-P
CO2
H2O
NADPH + ATP
O2
Light reaction
Dark reaction
Chloro-Phototrophy; optimized during billions of generations
1/3 GAP
Dark reaction
31/03/2009 the photanol process
Light reaction
PS II
PS I
NADP
NADPH
H2O O2
ATP
h
Dark reaction
Cells
GAP
CO2
ADP
Phototrophy
31/03/2009 the photanol process
2 Organic matter-dependent life
Organic C + O2 ATP + CO2 + H2O
(animals, fungi, bacteria)
(Chemotrophy)
a) respiration
Organic C + ATP
Organic C + O2
Cells
Organic C Cells + FERMENTATION PRODUCTS
b) fermentation (fungi, bacteria)
The 2 modes of life
b): occurs when O2 is lacking or organic C is abundant; well-known as “overflow metabolism” in E. coli, LAB and yeast
31/03/2009 the photanol process
(Ethanol, propanol, butanol, propanediol, glycerol, acetone, lactate, acetate, ..........)
Organic matter
Fermentation products
Pyruvate
Glyceraldehyde-3-P (GAP)
F-1,6-BP
Chemotrophy: optimized for billions of generations
NAD(P)H, ATP
NAD(P)H, (ATP)
31/03/2009 the photanol process
Light reaction
PS II
PS I
NADP
NADPH
H2O O2
ATP
h
Dark reaction
CO2
GAP
Cells
ADP
Fermentation
Fermentationproducts
Photofermentation
CO2 + H2O fuel + O2!!
31/03/2009 the photanol process
Fermentation pathways
31/03/2009 the photanol process
Bermejo et al. 1998 (Acetone production in E. coli (Clostridium acetobutylicum pathway) )
Deng and Coleman. 1999 (EtOH production in Synechococcus sp. (pdc and adh from Zymomonas mobilis) )
Takahama et al. 2003 (Ethylene in Synechococcus sp. (efe from P. syringiae) )
Fu. 2008 (EtOH production in Synechocystis sp. PCC 6803)
Pirkov et al. 2008 (Ethylene production in S. cerevisiae (efe from P. syringiae) )
Shen and Liao. 2008 (1-Butanol and 1-Propanol in E. coli)
Tang et al. 2009 (Propanediol in E. coli (genes from Clostridium butyricum))
Some successful pathway insertions
31/03/2009 the photanol process
Unicellular prokaryote Genome sequenced Auto- and heterotrophic Effective photosynthesis Model organism for photosynthesis Defined (simple) growth media Naturally transformable Grows to high densities
Circadian rhythm doubling time ~8h 6 to 10 genomes per cell Low maintenance energy req.
EM photograph, scale bar 200nm
Synechocystis sp. PCC 6803
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Host: phototrophicSynechocystis PCC6803
EtOH genes
PCR
recombination
expression
GAP
Constructing a photofermentative strainDonor: chemotrophicbacterial species
HOM1 HOM2
HOM1 HOM2
wt genome
plasmid
pAAA170 9 1 bp
p s b up (H O M 1 )
p s b dn (H O M 2 )
f1 (+ ) o r iA m p R
e fe
P la c
T7 pro m o te r
T3 pro m o te r
P A m pR
R B S
K a nR
Eco R I (2530)
Sa c I (4 8 93)
X ba I (4 8 65)
X hoI (6 6 9)
Sa l I (14 59 )
Bam H I (4 0 4 9)
Pst I (254 0 )
Pst I (3129 )
Sma I (254 4 )
Sma I (4 0 45)
K pnI (6 58 )
K pnI (4 158 )
K pnI (4 59 0)
goi
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(in)complete segregation
Tested clonesM C- C+
2kbp
5kbp
Example of incomplete segregation
M P N 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
Colony PCR of pAAA2 transformants. M is marker. P is positive control. N is negative control. Transformants grown on 4ug/ml kanamycin. No correct insertion in transformants 4, 5, 7, 8, 10; not fully segregated transformants 1, 2, 3, 6, 9; full segregation in 11, 12, 13, 14, 15, 16, 17
Cloning in the psbA2 locus of Synechocystis
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Ethanol synthesis by geneticengineering in Cyanobacteria
From: Ming-De Deng and John R. Coleman (1999) Applied & Environm. Microbiol. 65: 523-528 FIG. 4. Cell growth and
ethanol synthesis in Synechococcus sp. strain PCC 7942 transformed with pCB4-LRpa. Cells were grown at 30°C in the presence of light in a 500-ml liquid batch culture aerated by forcing air through a Pasteur pipette. Samples were taken at intervals in order to monitor cell growth (OD730) and ethanol accumulation in the culture medium. The PDC and ADH activities in cell lysates on day 5 were 320 and 170 nmol · min 1 · mg of total protein 1, respectively.
31/03/2009 the photanol process
CO2 + H2O fuel + O2!!
‘Photofermentation’: the best of both worlds
cellscells
• Cells are auto-regenerative catalysts of the process• The fuel molecules can stably coexist with oxygen• Production is not limited by the storage capacity of the cells• It is possible to form the product in volatile form• Process can be run in a closed large-scale photobioreactor
31/03/2009 the photanol process
CO2H2
Formyl-MFR
Methenyl-H4MPT
Formyl-H4MPT
Methyl-H4MPT
Methyl-S-CoM
HS-CoBCH4
H2H2F420
Fdred
Biological incompatibility: methanogenesis
Enzymes involved are extremely oxygen-sensitive and have several very uncommon cofactors
31/03/2009 the photanol process
Regulation of fuel formation: The GAP branchpoint
A~CO2B
GAPA
DE
Fluxgrowth = [Eg].vmax. [PGA]
Km + [PGA]
Fluxproduct = [Ep].vmax. [PGA]
Km + [PGA]
cassette
EgEp
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A~CO2B
DE
A
E
cassette
Promoter
product
NH4
+
GAP
The Photanol Process: Genetic Process control
Ammonia availability is often used as a control parameter to regulate biomass formation(cells: “C4H7O2N”)
CO2
31/03/2009 the photanol process
N-excess-oxoglutarate + NH4
+ glutamate
proteinsNtcA+
NtcA-aOG
PSigE SigE X E
Gene cassette-
Pgap1 gap1
N-depletion
Gene cassette+
Pgap1 gap1
-oxoglutarate + NH4+ glutamate
proteinsNtcA+
NtcA-aOG
PSigE SigE E~
Nitrogen sensing in Synechocystis
31/03/2009 the photanol process
6 CO25 R1,5bP
12 3-P-Glycerate 12 1,3-bPG
Growth 5 FbPHexose-P
N-dependent fuel cassette expression
time
NH4
+
growth
N-excess2OG + N Glu protein
N starvationNtca
2 GAP
Pth
l
crt
4h
bd
etf
bd
h
ald
Butanol
P
10 GAP +
31/03/2009 the photanol process
N-excess
6 CO25 R1,5bP
12 3-P-Glycerate 12 1,3-bPG
Growth 5 FbPHexose-P
2OG + N GluN starvation
Ntca + 2OG
2 GAP
Ntca~2OG
+
+P
thl
crt
4h
bd
etf
bd
h
ald
Butanol
time
growth
e
protein
10 GAP +
product
NH4
+
growth
N-dependent fuel cassette expression
31/03/2009 the photanol process
‘Back-of-the-envelope’ calculation
• 1 acre = 4 . 103 m2
• 1 year has 107 seconds of sunlight (3600 . 12 . 235)• Sunlight intensity (PAR): 600 μE.m-2.s-1
24 . 106 Einstein/acre/year
• Complete conversion of light energy to ethanol:
• 12 photons per ethanol: 2 CO2 + 3 H2O C2H6O + 3 O2
Maximal productivity:
2 . 106 moles ethanol/year/acre
~ 100 ton ethanol/year/acre
31/03/2009 the photanol process
Large-scale culturing
Tubular system Raceway pond Flat panel system
• Extensive expertise is being generated with respect to the scale-up of culturing systems; systems can be used in ‘open’ and ‘closed’ form (e.g. Wijffels c.s.)• All systems have in common that the fuel-producing cells are exposed to oscillating light regimes, with typical frequencies ranging from minutes (depending on mixing regime) to 24 hrs.
31/03/2009 the photanol process
Some regulatory mechanisms in the photosynthesis of Synechocystis
a] State transitions of phycobilisomes b] Non-photochemical, IsiA and/or OCP-mediated quenching c] zeaxanthin cycle d] Regulation of expression ratio of PSI/PSII/Antennae e] Circadian regulation of gene (photosystem) expressionf] NDH (and FNR) mediated cyclic electron transfer around PSI g] Cyclic electron transfer around PSII h] PSI trimerization, PSII dimerization, IsiA and iron limitation i] Variation of antenna size (j] Chromatic adaptation)
a Systems Biology-based optimization is necessary
31/03/2009 the photanol process
Circadian regulation of gene expression
Dong G and Golden SS (2008) How a cyanobacterium tells time. Curr Opin Microbiol. 11: 541-546.
7 sigma factors of three different classes
31/03/2009 the photanol process
Cyanobacteria do it during the day
• Two interesting physiologies may occur at night:
1] oxidative catabolism (‘glycogen’ CO2)
2] anaerobic fermentation
(‘glycogen’ organic acids)
• Feasibility of supportive LED illumination during the night?
31/03/2009 the photanol process
Summary of the Photanol Process
xCO2 + yH2O
CxH2yOz + (x+0.5y-0.5z)O2
ATP, NADPH
cells
Clean fuel production
CO2 consuming
Cheap technology
Not competing with food stocks
Principle generally applicable: ethanol, butanol, etc
Yield per year per surface: up to 20x higher than plant crops
31/03/2009 the photanol process
Dreams