Engineering & construction of a Bio-photo-generator

Transcription

1 Engineering & construction of a Bio-photo-generator Roy I. Pinhassi 4, Gadi Schuster 1, Noam Adir 2 and Avner Rotchild 4 1. Faculty of Biology 2. Schulich Faculty of Chemistry 3. Department of Materials Engineering 4. Grand Technion Energy Program Free Powerpoint Templates

2 Lecture outline 1. The concept 2. Introduction: photosynthesis, photosystem II (PSII), D1 and psba. 3. Ways to reap electrons from PSII: a. Using chemical electron carriers as electron reapers b. Engineering of an alternative electron path in tobacco

3 The concept Using leaf extract to harvest sunlight, split water and inject e - to an external electric circuit through an electron carrier

4 Photosynthesis Conversion of CO 2 to organic compounds using energy from sunlight. Catalyzed by four multi-subunit membrane-protein complexes clustered in the Thylakoid: Photosystem I (PSI) Photosystem II (PSII) The cytochrome b 6 f complex F-ATPase I. McConnel et. al., Chem. & Bio. Review (2010)

5 Photosynthesis PSII commits the first stage in photosynthesis: Absorbs sunlight Splits H 2 O and provides e - flow to eventually reduce NADP + Creates H + gradient for ATP production Releases O 2

6 Chloroplasts in plant cells In plants photosynthesis take place in chloroplasts. Originate from endosymbiosis of cyanobacteria followed by massive gene transfer to host cell (~120 of ~3000 genes remain in ptdna). Have two membranes and their own ribosomes. A leaf cell contains ~100 chloroplasts, total of ~10,000 ptdna/cell. In most plants chloroplast are inherited maternally.

7 Y. Umena et al., Nature (2011) D1 protein Proteins D1 & D2 bind the redox active components involved in the electron transfer. D1 is encoded by psba gene. Cyanobacteria has three isoforms, while higher plants have only one found in the chloroplast DNA. D1 is a target of oxidative damage arising from water splitting. It is constantly degraded and resynthesized in PSII repair cycle.

8 O.D. 600nm Anticipated Energy Production Rate Hill reaction shows electron flow rate of: mol 8 e mg Chl S From 1000ml thylakoid suspension of 0.25mg (Chl/ml) we anticipate a current of: mol C C A 8 e mgChl 96, mg Chl S mole S 1.0 Hill Reaction LIGHT DARK Time (S)

9 How can we extract electrons from PSII? C. Herrero et Al., Coo. Chem. Review (2008)

10 How can we extract electrons from PSII? 1. Using chemical electron acceptors (quinones) to transfer electrons from WT thylakoid extract to anode. 2. Transforming plants to introduce binding sites for protein electron carriers.

11 1. Quinones as electron mediators Quinone is a class of organic compounds derived from aromatic compounds by conversion of CH= groups into C(=O) groups. Redox potential is controllable. PSII

12 1. Quinones as electron mediators DCBQ

13 1. Quinones as electron mediators

14 2. Engineering alternative electron path in PSII S. Larom et al.,pnas (2010)

15 2. Engineering alternative electron path in PSII Transgenic Cyanobacteria in which the thylakoid PSII protein D1 was genetically modified showed binding and reduction of cytochrome C. Moreover, a photocurrent was measured implying that electricity may indeed be transformed from PSII to external electric circuit. Applying such mutation to plants may greatly increase energetic efficiency

16 2. Engineering alternative electron path in PSII Photosynthesis is evolutionary conserved, yet differ between organisms: Compartmentalization (chloroplasts Vs. cytoplasm) Thylakoid structure (defined grana stacks Vs. loose layers) Light harvesting machinery (LHC Vs. phycobilisome) Gene location and protein production site (Nucleus Vs. nucleoid) Regulation of gene expression

17 2. Engineering alternative electron path in PSII

18 2. Engineering alternative electron path in PSII Vector cloning and propagation in E. Coli Preparation of DNA coated micro-projectiles Biolistic particle bombardment on plant target Reconstruction of transformed cells on selective media M. Pikko et. Al, Mol. Biotech. (1999) Maliga & Bock, Plant Physiol.(2011)

19 2. Engineering alternative electron path in PSII Selection of transformed cells is aided by introducing a selective marker, e.g. the gene aada aada confers resistance to spectinomycin enabling greening and shoot regeneration in culture. Maliga & Bock, Plant Physiol. (2011)

20 2. Engineering alternative electron path in PSII psba aada 5 F psba P psba T psba T psba P psba 3 F psba

21 2. Engineering alternative electron path in PSII Amplification of psba from tobacco DNA extract M 54.0 o cont o cont o cont o cont o cont o cont o cont.

22 Plastid transformation in tobacco TA-Cloning of psba to compotent cells (XL1-Blue)

23 Goals Transplastomic tobacco plants Exploring Quinones as e - carriers Constructing an efficient BioPhotoGenerator

24 Questions?