Team:Reading/Project

Project Description

Contents

Biological photovoltaics (BPVs) have the potential to provide a more sustainable alternative to traditional photovoltaics. However, current implementations have not been able to provide sufficient electron output to be viable alternatives to solar panels. Our project aims to increase output by redirecting electron flow in our chosen cyanobacteria, Synechocystis sp. PCC 6803 (henceforth Synechocystis).

1. Project Overview
2. The Problem
3. Why Synechocystis?
4. Project Aims
5. Methods
6. References

The Problem

Solar photovoltaics are one of the main sources of renewable energy and are likely to become more widely used in the future . However, they are far from perfect. Although cyanobacteria have been predicted to have photosynthesis efficiency of around 11%¹ (maximum recorded current produced is 11pA/cell²), compared to 10-32%³ for traditional photovoltaics, BPVs could have several benefits. As they are made from living organisms, BPVs are capable of self-repair. Furthermore, they consume carbon dioxide during photosynthesis and are cheaper to produce, so have been proposed for use in lower income countries where less energy is required per person². Though BPVs are worth pursing as an alternative to traditional photovoltaics, their potential electrical output is yet to be realised. Our project seeks to improve upon current levels and so make BPVs more viable.

Why Synechocystis?

Photolithoautotrophic microorganisms derive their energy from sunlight, their electrons from inorganic sources, and their carbon from inorganic carbon sources. This is particularly useful as sunlight, water and carbon dioxide can provide these three vital resources respectively. Algae and cyanobacteria are both candidates for use in BPVs, but the latter provide several advantages: they are easier to manipulate genetically, the grow more quickly and have an increasing number of tools available⁴. Synechocystis was the first cyanobacteria to have its genome completely sequenced⁵, has been used extensively in mathematical modelling of cyanobacteria⁴, and seems to be the most commonly used in species BPVs.

Project Aims

To improve output in Synechocystis we have targeted key points in the photosynthetic electron transport chain (PETC) to increase the number electrons available, as this has been used successfully in the past⁶, and are attempting to improve the transfer of these electrons to the anode. The electrically insulating outer membrane (OM) appears to be the major limiter in preventing higher electron output, so methods to improve the release of electrons are very important. In general, electrons in BPVs may be donated to the anode through direct contact with the OM, or at a distance by using a soluble mediator or through other methods such as bacterial nanowires⁷. Although it is unclear exactly which of these mechanisms are used by Synechocystis², it seems direct contact is the most likely⁸. As direct contact, mediators and nanowires could all potentially be used, our project will target all 3 types of transfer.

The diagram below shows a simplified view of the photosynthetic and respiratory electron transport chains in Synechocystis. The cytoplasmic membrane at the top only contains the respiratory electron transport chain (RETC), while the thylakoid membrane below contains both the respiratory and photosynthetic transport chains. A significant feature of photosynthesis in cyanobacteria is that these two transport chains share several components. Furthermore, soluble mediators (shown in turquoise) can diffuse through the membrane to shuffle electrons between proteins, so the two chains are highly interlinked. The aspects of the PETC that we are targeting are shown in red; for more detail on these, see the methods section.

Methods

Targeting the PETC

Increasing the number of electrons available in the plastoquinone pool is one way of increasing electron output in Synechocystis. This can be done by removing the exit points of the PETC, namely the terminal oxidases and FNR. The former has been shown to increase electron output in the dark phase of diurnal growth cycle by removing the 3 major terminal oxidases⁶, while knocking out FNR seems to prove fatal to the cell as FNR is crucial for cell growth and survival. The removal of FNR ceases the movement of electrons from photosystem I to NADP⁺ that is vital for the energy transfer in the cell. The Flv proteins are terminal oxidases used by Synechocystis and other cyanobacteria that remove electron by reducing oxygen without producing reactive oxygen species (ROS), unlike other terminal oxidases⁹. They are therefore very important in controlling the redox state of the photosystems. 4 Flv proteins are present in Synechocystis; these form Flv1:3 and Flv2:4 heterodimers that attach to PSII and PSI respectively. They appear to provide an electron sink during sunlight¹⁰, with Flv2:4 providing photoprotection¹¹ and Flv1:3 allowing survival under fluctuating light¹². We will create knockouts of both dimers to try and increase the number of electrons available for release; this should increase electron output in light phase of growth cycle, possibly complementing the triple major terminal oxidase mutant.

Increasing pili

Bacterial nanowires are modified pili that are capable of transferring electrons out of the cell. They have only become known about fairly recently, with the first discovery in Geobacter sulfereducans in 2005¹³. Since then they appear to have been identified in Shewanella oneidensis strain MR-1 and Synechocystis sp PCC 6803¹⁴, the strain of cyanobacteria we are using.

Since 2006 there has been a considerable amount of work on nanowires, but mainly focusing on Geobacter spp. and S. oneidensis. To our knowledge, no further published work has confirmed or elaborated on the possibility of nanowires in Synechocystis. As such, despite the large role they play in some other species and that the method of electron transfer out of Synechocystis is essentially unknown², we cannot reasonably target nanowires as the core of our project. However, increasing the production of large pili, one of two types that can be seen on our strain¹⁵, could increase aggregation in biofilms (a benefit in itself as it could increase the number of bacteria growing directly on the anode), but it is possible that they will also increase the number of nanowires if they exist. Furthermore, a similar alteration that affects the number of pili has caused Synechocystis to no longer be transformable due to entanglement of the pili¹⁶, a possible added biosafety benefit. Conversely, hyperpiliation could cause our bacteria to become more transformable, a serious biosafety issue we will be taking into consideration.

To cause hyperpiliation, we will be inserting another copy of the PilA1 gene (one of the main pili components) along with a strong upstream promoter, Ptrc¹⁰, to initiate high-level expression¹⁷, and deleting the PilT1 gene, an ATPase that is involved in pilus retraction¹⁸.

Producing endogenous mediators

Mediators are redox molecules that can accept and donate electrons. They can therefore carry charge and, if soluble, can transport electrons from the cell to the anode. Mediators have been added to many BPVs to increase output, but this is no longer common practice². Mediators are often toxic so can potentially damage the cell; it is also thought to be less sustainable if mediator has to constantly be added to the BPV.

Endogenous mediators are those produced by the cell itself. They therefore do not need to be added, making it a more sustainable solution. Flavins are an example of an endogenous mediator. Previous iGEM teams have produced soluble mediators for expression in cyanobacteria; we hope to improve upon these parts.

Biosafety

Safety considerations have been embedded in synthetic biology since the beginnings of the field. As such there are several important, detailed reviews of biosafety^19–21, but almost all of these focus on Escherichia coli. They also focus on the use of plasmids that are maintained extra-chromosomally, rather than chromosomal modifications. Although E. coli and Synechocystis are both gram negatives, they pose significantly different risks, and very different technologies are used for modifying them. We will address the major differences and draw attention to the safety implications that follow from these.

During our project we will be considering the consequences of each of our modifications, and the potential safety problems and solutions faced in applying our technology. We will assume that the technology will make it to market, so will be used in areas where they could feature, such as on the roofs of houses.

References

1. Brenner, M. P. Engineering Microorganisms for Energy Production. (U.S. Department of Energy, 2006).
2. Bradley, R. W., Bombelli, P., Rowden, S. J. L. & Howe, C. J. Biological photovoltaics: intra- and extra-cellular electron transport by cyanobacteria. Biochem. Soc. Trans. 40, 1302–1307 (2012).
3. Crabtree, G. W. & Lewis, N. S. Solar energy conversion. Phys. Today 60, 37–42 (2007).
4. Berla, B. M. et al. Synthetic biology of cyanobacteria: unique challenges and opportunities. Front. Microbiol. 4, (2013).
5. Kaneko, T. et al. Sequence Analysis of the Genome of the Unicellular Cyanobacterium Synechocystis sp. Strain PCC6803. II. Sequence Determination of the Entire Genome and Assignment of Potential Protein-coding Regions. DNA Res. 3, 109–136 (1996).
6. Bradley, R. W., Bombelli, P., Lea-Smith, D. J. & Howe, C. J. Terminal oxidase mutants of the cyanobacterium Synechocystis sp. PCC 6803 show increased electrogenic activity in biological photo-voltaic systems. Phys. Chem. Chem. Phys. PCCP 15, 13611–13618 (2013).
7. Malvankar, N. S. & Lovley, D. R. Microbial Nanowires: A New Paradigm for Biological Electron Transfer and Bioelectronics. ChemSusChem 5, 1039–1046 (2012).
8. Cereda, A. et al. A Bioelectrochemical Approach to Characterize Extracellular Electron Transfer by Synechocystis sp. PCC6803. PLoS ONE 9, (2014).
9. Vermaas, W. F. Photosynthesis and Respiration in Cyanobacteria. (2001). at
10. Allahverdiyeva, Y. et al. Interplay between Flavodiiron Proteins and Photorespiration in Synechocystis sp. PCC 6803. J. Biol. Chem. 286, 24007–24014 (2011).
11. Zhang, P., Allahverdiyeva, Y., Eisenhut, M. & Aro, E.-M. Flavodiiron Proteins in Oxygenic Photosynthetic Organisms: Photoprotection of Photosystem II by Flv2 and Flv4 in Synechocystis sp. PCC 6803. PLoS ONE 4, e5331 (2009).
12. Allahverdiyeva, Y. et al. Flavodiiron proteins Flv1 and Flv3 enable cyanobacterial growth and photosynthesis under fluctuating light. Proc. Natl. Acad. Sci. 110, 4111–4116 (2013).
13. Reguera, G. et al. Extracellular electron transfer via microbial nanowires. Nature 435, 1098–1101 (2005).
14. Gorby, Y. A. et al. Electrically conductive bacterial nanowires produced by Shewanella oneidensis strain MR-1 and other microorganisms. Proc. Natl. Acad. Sci. 103, 11358–11363 (2006).
15. Yoshihara, S. & Ikeuchi, M. Phototactic motility in the unicellular cyanobacterium Synechocystis sp. PCC 6803. Photochem. Photobiol. Sci. 3, 512–518 (2004).
16. Nakasugi, K., Svenson, C. J. & Neilan, B. A. The competence gene, comF, from Synechocystis sp. strain PCC 6803 is involved in natural transformation, phototactic motility and piliation. Microbiology 152, 3623–3631 (2006).
17. Huang, H.-H., Camsund, D., Lindblad, P. & Heidorn, T. Design and characterization of molecular tools for a Synthetic Biology approach towards developing cyanobacterial biotechnology. Nucleic Acids Res. gkq164 (2010). doi:10.1093/nar/gkq164
18. Okamoto, S. & Ohmori, M. The Cyanobacterial PilT Protein Responsible for Cell Motility and Transformation Hydrolyzes ATP. Plant Cell Physiol. 43, 1127–1136 (2002).
19. Wright, O., Stan, G.-B. & Ellis, T. Building-in biosafety for synthetic biology. Microbiology 159, 1221–1235 (2013).
20. Schmidt, M. & de Lorenzo, V. Synthetic constructs in/for the environment: Managing the interplay between natural and engineered Biology. FEBS Lett. 586, 2199–2206 (2012).
21. Moe-Behrens, G. H. G., Davis, R. & Haynes, K. A. Preparing synthetic biology for the world. Microbiotechnology Ecotoxicol. Bioremediation 4, 5 (2013).

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