1. 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

2. 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

3. 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

4. The proposed solution:
PSII
PSI e-
H2O O2 H+
macroscopic
membrane
hydrogenase H2
Brh
funding
31/03/2009
the photanol process

5. What is needed..
Use the auto-regenerative capacity of living organisms
A solution for solar fuel with as few conversions as possible (0.334 = 0.01!)
fuels
For any large-scale process, only H2O is a realistic electron donor
31/03/2009
the photanol process

6. Some current biofuel technologies (1)
Waste, chemicals, land. Not discuss economical/political. Algae better
From: Esper, Badura and Rögner (2006) Trends in Plant Science 11:
31/03/2009
the photanol process

7. Some current biofuel technologies (2)
1 Grow crops on land
2 Grow algae in ponds
Harvest organic matter
Harvest cells
Transport to bioreactor
& fractionate
Transport to separator
extraction
&
modification
Waste, chemicals, land. Not discuss economical/political. Algae better
fermentation
biofuel +
Waste
Biodiesel (e.g. fatty acid methyl esters)
Mostly ethanol
31/03/2009
the photanol process

8. 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”
31/03/2009
the photanol process

9. Unity of life & the broken circle
(plants, bacteria) E
CO2 + H2O
Cells + O2
CO2
(animals, fungi, bacteria)
Billions of years storage, released in 150 years
 energy
Earth’ surface
fossil fuels
31/03/2009
the photanol process

10. The 2 modes of life H2O reducing power + ATP + O2 Organic C
1 Light-dependent life (plants, bacteria)
((Chloro)Phototrophy)
H2O
reducing power + ATP + O2
Some high school biology
Reducing power + CO2 + ATP
Organic C
Cells
31/03/2009
the photanol process

11. Chloro-Phototrophy; optimized during billions of generations
The light reactions
of photosynthesis:
Light + dark reaction, focus latter
31/03/2009
the photanol process

12. Chloro-Phototrophy; optimized during billions of generations
NADPH + ATP O2
Light reaction
Dark reaction
Dark reaction
CO2
Light + dark reaction, focus latter
1/3 GAP
Glyceraldehyde-3-P
31/03/2009
the photanol process

13. GAP Phototrophy Cells CO2 H2O O2 hn Light reaction PS II PS I
NADP
NADPH
H2O O2
ATP hn
Dark reaction
Cells
GAP
CO2
ADP
Again, note GAP
31/03/2009
the photanol process

14. The 2 modes of life Organic C + O2 Organic C + O2 ATP + CO2 + H2O
2 Organic matter-dependent life
(Chemotrophy)
a) respiration
(animals, fungi, bacteria)
Organic C + O2
Organic C + O2
ATP + CO2 + H2O
Organic C + ATP
Cells
b) fermentation
(fungi, bacteria)
Organic C
Cells
+ FERMENTATION PRODUCTS
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

15. Chemotrophy: optimized for billions of generations
(Ethanol, propanol, butanol, propanediol, glycerol, acetone, lactate, acetate, )
Organic matter
Fermentation products
Pyruvate
Glyceraldehyde-3-P (GAP)
F-1,6-BP
NAD(P)H, ATP
NAD(P)H, (ATP)
The other mode but note GAP
31/03/2009
the photanol process

16. GAP Photofermentation CO2 Cells H2O O2 hn Fermentation products
Light reaction
PS II
PS I
NADP
NADPH
H2O O2
ATP hn
Dark reaction
CO2
GAP
Cells
ADP
Fermentation
Fermentation
products
Fusion of the 2 modes
CO2 + H2O  fuel + O2!!
31/03/2009
the photanol process

17. Fermentation pathways
31/03/2009
the photanol process

18. Some successful pathway insertions
Bermejo et al (Acetone production in E. coli (Clostridium acetobutylicum pathway) )
Deng and Coleman (EtOH production in Synechococcus sp. (pdc and adh from Zymomonas mobilis) )
Takahama et al (Ethylene in Synechococcus sp. (efe from P. syringiae) )
Fu (EtOH production in Synechocystis sp. PCC 6803)
Pirkov et al (Ethylene production in S. cerevisiae (efe from P. syringiae) )
Shen and Liao (1-Butanol and 1-Propanol in E. coli)
Tang et al (Propanediol in E. coli (genes from Clostridium butyricum))
31/03/2009
the photanol process

19. Synechocystis sp. PCC 6803 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
31/03/2009
the photanol process

20. Constructing a photofermentative strain
Host: phototrophic
Synechocystis PCC6803
Donor: chemotrophic
bacterial species
HOM1
HOM2
wt genome
plasmid
goi
GAP
EtOH genes
PCR
recombination
Standard gene technology
expression
31/03/2009
the photanol process

21. (in)complete segregation
Cloning in the psbA2 locus of Synechocystis
M P N
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
Tested clones
M C- C+
2kbp
5kbp
Example of incomplete segregation
31/03/2009
the photanol process

22. Ethanol synthesis by genetic engineering in Cyanobacteria
From: Ming-De Deng and John R. Coleman (1999) Applied & Environm. Microbiol. 65:
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

23. ‘Photofermentation’: the best of both worlds
cells
CO2 + H2O fuel + O2!!
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
Fusion of the 2 modes
31/03/2009
the photanol process 23

24. Biological incompatibility: methanogenesis
Fdred H2
CO2
Formyl-MFR
Formyl-H4MPT
Methenyl-H4MPT H2
Enzymes involved are extremely oxygen-sensitive and have several very uncommon cofactors
H2F420
What you would like but. Incomplete scheme but shows Different Cofactors, different protein synthesis, highly oxygen sensitive
Methyl-H4MPT
Methyl-S-CoM
HS-CoB
CH4
31/03/2009
the photanol process

25. Regulation of fuel formation: The GAP branchpoint
A~CO2 B
GAP A Eg Ep D E
Fluxgrowth = [Eg].vmax. [PGA]
Km + [PGA]
cassette
Cloning is easy, but what is the effect on flux
Fluxproduct = [Ep].vmax. [PGA]
Km + [PGA]
31/03/2009
the photanol process

26. The Photanol Process: Genetic Process control
A~CO2 B D E A
cassette
Promoter
product
NH4 +
GAP
CO2
Brute force but elegant
Ammonia availability is often used as a control parameter to regulate biomass formation
(cells: “C4H7O2N”)
31/03/2009
the photanol process

27. Nitrogen sensing in Synechocystis
N-excess
a-oxoglutarate + NH4 +
glutamate +
proteins
NtcA
NtcA-aOG X sE -
PSigE
SigE
Pgap1
Gene cassette
gap1
N-depletion
Gene cassette +
Pgap1
gap1
a-oxoglutarate + NH4
glutamate
proteins
NtcA
NtcA-aOG
PSigE
SigE sE ~
31/03/2009
the photanol process

28. N-dependent fuel cassette expression
N-excess
N starvation
2OG + N
Glu
protein
Ntca
12 3-P-Glycerate
12 1,3-bPG
6 CO2
5 R1,5bP
10 GAP
2 GAP P P
Growth
Hexose-P
5 FbP
thl
crt
NH4 +
growth
etf
4hbd
2 phases, +/- N, growth/prod
ald
bdh
Butanol
31/03/2009
the photanol process
time

29. N-dependent fuel cassette expression
N-excess
N starvation
2OG + N
Glu
protein
Ntca + 2OG
Ntca~2OG
12 3-P-Glycerate
12 1,3-bPG se
6 CO2 +
5 R1,5bP
10 GAP
2 GAP P +
Growth
Hexose-P
5 FbP
thl
NH4 +
crt
4hbd
etf
product
ald
bdh
Butanol
growth
growth
31/03/2009
the photanol process
time

30. ‘Back-of-the-envelope’ calculation
1 acre = m2
1 year has 107 seconds of sunlight ( )
Sunlight intensity (PAR): 600 μE.m-2.s-1
 Einstein/acre/year
Complete conversion of light energy to ethanol:
12 photons per ethanol: 2 CO2 + 3 H2O  C2H6O + 3 O2
Maximal productivity:
moles ethanol/year/acre
~ 100 ton ethanol/year/acre
31/03/2009
the photanol process

31. 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

32. 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) expression
f] 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

33. Circadian regulation of gene expression
Dong G and Golden SS (2008) How a cyanobacterium tells time. Curr Opin Microbiol. 11:
Figure 2. An overview of the molecular mechanism of the circadian clock in S. elongatus. The central oscillator is composed of KaiA, KaiB, and KaiC. KaiA stimulates KaiC phosphorylation, and KaiB inactivates KaiA when KaiC reaches a certain phosphorylation state (see Figure 1 for details). In the input pathway, both LdpA and CikA sense the cellular redox state, which is regulated by light and cell metabolism. LdpA affects the stability of CikA and KaiA through an unknown mechanism. Through its PsR domain, CikA binds quinone molecules directly, which destabilizes CikA. CikA affects the phosphorylation states of KaiC, but where and how it works in the signal transduction pathway is unknown. Pex is a transcriptional repressor of KaiA, and its abundance is sensitive to light, but it is not clear whether the pathway that regulates pex senses light directly or does it through cellular redox. In the output pathway, SasA interacts physically with KaiC and autophosphorylates, and then transfers the phosphoryl group to RpaA, a response regulator with a DNA-binding domain. The target of RpaA has not been identified. LabA works upstream of RpaA and downstream of KaiC, but its exact function is not clear. A SasA-independent and RpaA-independent output pathway might exist. The output pathway controls DNA topology, which is proposed to regulate global gene expression. A transcription/translation rhythm could interact with and reinforce the post-translational rhythm of KaiC activities. A solid line indicates a direct effect whereas a dotted line indicates an indirect effect or an effect whose mechanism is unknown. Arrows indicate the direction of the information flow or a stimulation of activity or both. Blunt ends represent an inhibition of protein activity or abundance, whereas an end with a filled circle suggests a regulation of unspecified direction.
7 sigma factors of three different classes
31/03/2009
the photanol process

34. 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

35. Summary of the Photanol Process
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
xCO2 + yH2O
CxH2yOz + (x+0.5y-0.5z)O2
31/03/2009
the photanol process

36. Dreams
31/03/2009
the photanol process 36