Metabolic Engineering of Algae for Hydrogen Production

Dr. Iftach Yacoby, PhD Laboratory for Renewable Energy Studies. Department of Molecular Biology and Ecology of Plants The George S. Wise Faculty of Life Sciences Tel Aviv University, Tel Aviv 69978, Israel
Iftachy@post.tau.ac.il

Energy status in the near future

Global energy demand is predicted to rise from 13 TW in 2000 to 46 TW in 2100 (till then we will use 1300 TW.) International Energy Agency suggest that in addition to the 540 TW supply of oil that is reported to remain, coal and natural gas could supply an additional 1000 TW (total of 1540 TW)
2

The potential of solar energy

Solar energy is by far the largest (178,000 TW year) and capable of supplying 13,500 times the total global energy demand (13 TW year in 2000)

Major car companies have already presented hydrogen fueled cars

BMW The BMW Hydrogen 7 DaimlerChrysler F-Cell Ford Motor Focus FCV General Motors multiple models of fuel cell vehicles Hyundai Tucson FCEV Mazda RX-8 Nissan X-TRAIL FCV, Toyota The Toyota Highlander FCHV and FCHV-BUS

History of H2 production studies has been plagued by lack of efficiency

Hydrogen production was first reported by Jackson and Ellms in 1896. The modern era of research: Gaffron and Robin, 1940s. 1999-breakthrough, Melis and co workers found that sulfur deprivation increases algal hydrogen production efficiency by a factor of 10,000. Yet, an additional factor of at least 5-10 fold is necessary to make photosynthetic H2 production economically viable.

Photosynthesis 3 billion years old natural pathway for H2 production

Sugars

NADPH

NADP+

2H+

H2

Anaerobiosis No CO2

Linear e- Flow

2H2O

O2 + 4H+ + 4e-

We show that FNR is located on the photosynthetic membrane with unknown mechanism. Thus, FNR is the first in line to accept electrons !

NADPH

NADP+ 2H+

H2

Anaerobiosis No CO2

2H2O

O2 + 4H+ + 4e-

Shown also by Iwai M, et al. Nature (2010) 464(7292):1210-1213.

Yacoby et al. Proc Natl Acad Sci USA ,2011, 108: 9396-9401.

We show that more than 85% of electrons are transferred to NADPH production instead of H2 production at any situation tested Fd titration

NADPH

NADP+ 2H+

H2

Anaerobiosis No CO2

2H2O

O2 + 4H+ + 4e-

High Fd conc led to aggregation as reported earlier Fourmond Vet al. (2007) J Am Chem Soc 129(29):9201-9209
Yacoby et al. Proc Natl Acad Sci USA,2011, 108: 9396-9401.

We show that more than 85% of electrons are transferred to NADPH production instead of instead of H2 production at any situation tested HydA titration

NADPH

NADP+ 2H+

H2

Anaerobiosis No CO2

2H2O

O2 + 4H+ + 4e-

Yacoby et al. Proc Natl Acad Sci USA,2011, 108: 9396-9401.

Therefore, there are two bottlenecks that limit photosynthetic H2 production from becoming a commercially-viable energy production option 1) The irreversible inhibition of Hydrogenase by oxygen.

2) The inefficient shuttling of electrons from PSI to the Hydrogenase enzyme. 2H

NADPH NADP+

H2

Anaerobiosis No CO2

2H2O

O2 + 4H+ + 4e-

Ferredoxin is the best candidate for improving the efficiency of H2 production Ferredoxin (Fd) is: 1) A sole electron supplier to all competing processes. 2) A sole mediator of all electron transfer from Photosystem-I to all outgoing processes. 3) A soluble protein that is not physically linked to membranes or other cellular NADPH components.
?
NADP+ 2H+ H2

Anaerobiosis No CO2

2H2O

O2 + 4H+ + 4e-

Rationale Shifting electrons towards H2 production for increased efficacy

We hypothesize that linkage between Ferredoxin (Fd) and Hydrogenase (HydA) will divert electrons to produce H2 instead of sugars

NADPH

NADP+

2H+

H2

Anaerobiosis No CO2

2H2O

O2 + 4H+ + 4e-

Fd and HydA naturally form a functional complex, that we can easily stabilize by adding a synthetic linker
Hydrogenase

Ferredoxin
Structure modeled by Chang et al. (2007) Biophys J

Selection of candidates from a diverse battery of fusion proteins based on chemical reaction efficacy step 1 design
Design of different fusions with different linker lengths and orientations

Selection of candidates from a diverse battery of fusion proteins based on chemical reaction efficacy step 2 successful expression/purification
Expression & purification of fusions protein are shown in the protein gel picture

20aa% Fd&HydA% Fd&HydA% Fd&HydA% HydA&Fd% 20aa% 15aa% 10aa% Fd&HydA% Fd&HydA% Fd&HydA%

20aa% 15aa% 10aa% HydA&Fd% HydA% 15aa% Fd&HydA% 10aa% HydA&Fd% HydA% Fd&HydA% Fd&HydA% 160%
HydA% HydA%

HydA&Fd% 20aa% 15aa% 10aa% Fd&HydA% Fd&HydA% Fd&HydA%

2600#
20aa% Fd&HydA%

110% 160% %%80% 110% %%60% %%80% 160% %%50% %%60% 110% %%50% %%40% %%80%

16 1 %%8

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15aa% Fd&HydA%

1400#

HydA&Fd% 10aa% Fd&HydA%

400#

HydA%

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Selection of candidates from a diverse battery of fusion proteins based on chemical reaction efficacy - step 3 fusions activity measurements
HydA&Fd% 20aa% 15aa% 10aa% Fd&HydA% Fd&HydA% Fd&HydA% HydA%

160% 110% %%80%

%%60% %%40%

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Crude cells nmol H2 ml-1 min-1 Spec act mol H2 mg-1 min-1

2600#

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Reaction setting The blue electron donor (reduced methyl viologen)

Ferredoxin is an expression and maturation booster, developed by us as a purification tag

A'
60KDa'

C'terminal'Fd'

N'terminal'Fd'

B'

4l' 2l' 1l' 0.5l' TEV' TEV' TEV' TEV' +' 20l'of'15TEV'' fusion'at'1.5'mg/ml'

C'

D'

Pure'HydA'
50KDa'

Yacoby et al. PLoS ONE, (2012) 7(4): e35886

TEV'parHal' 'digesHon' TEV'complete' digesHon'

No'TEV'

Lab setup to measure light dependent H2 production

Reaction serum vial

H2

Light source

Hila Toporik, TAU

in vitro photochemical optimization of linker length between Fd and HydA no relation to chemical activities
Linker"length"eect"with"Pure"PSI"
12"

mol"H2/mg"Chl"x"h"

10" 8" 6" 4" 2" 0" 5" 10" 15" 20" 25" Linker"length"(amino"acids)" 30"

H2

35"
19

Dr. Sergii Phochkailov, MIT

in-vitro H2 production in competition with simultaneous NADPH production. The fusion is superior to the native as the native HydA is severely inhibited.
90 80 70

HydA - NADP + HydA + NADP + Fd-15aa-HydA + NADP

(mol(H2) x h x mg(Chl) )

-1

H2 production rate, AH2

60 50 40 30 20 10 0

-1

HydA$ 2H+ + 2eNADPH

DCMU$

H2

Fd$

HydA$
2 1

NADP+

FNR$

Fd$ Fd$

(mol(NADPH) x h x mg(Chl) )

NADPH production rate, ANADPH

60 50 40 30 20 10 0 0 10 20 30 40

PSII$ PQ$ PQ$ Mn$

Cytb6f$

PSI$

-1

-1

PC$
Ascorbate + DCIP

Ferredoxin concentration, Cf (M)

Yacoby et al. Proc Natl Acad Sci USA,2011, 108: 9396-9401.

4 fold improvement of H2 production by the bioengineered Fd-HydA fusion

HydA competition 16% 84%

H2

Fd-15aa-HydA competition 39%

H2
61%

Performance of Native enzyme in competition with sugar production

Performance of Bioengineered enzyme in competition with sugar production

Yacoby et al. Proc Natl Acad Sci USA,2011, 108: 9396-9401.

Summary - Steps towards commercial H2-producing Algae

1) Overcome the oxygen sensitivity problem 2) Study the main competitive pathways 3) Design and bioengineer a bypass route (the fusion protein) 4) Express and purify fusion proteins 5) Select photosynthetic active fusions 6) Proof of concept - H2 production under photosynthetic competitive state

7) Transformation of fusion protein into alga in progress 8) Transformation of fusion protein into cyanobacteria in progress 9) Scale up H2 production process with the most successful engineered H2 producing alga/cyanobacteria

Acknowledgements
People Dr. Suguang Zhang and his Lab members (MIT) Prof. Itai Benhar, Tel Aviv University Prof. Ehud Gazit, Tel Aviv University Prof. Nathan Nelson and Dr. Yuval Mazor (Tel Aviv University) Drs. Paul King, Maria Ghirardi and their lab members (NREL) Funding agencies past and current: TAU $25,000 past EMBO $90,000 past Yang Trust fund $120,000 past MITei $150,000 past NREL $120,000 past TAU Seed Grant $600,000 current TOTAL 1,105,000$