Building Solar-Powered, Carbon-Fixing Protoalgae

Algae are single-celled organisms that use sunlight to power their metabolism and convert atmospheric carbon dioxide into useful carbohydrates. Like plants, they have a sophisticated photosynthetic machinery that has evolved to efficiently capture light energy and convert it into a type of biological electricity. This electricity flows through circuits that are embedded in proteins - biological molecules built from linear chains of amino acids that adopt complex 3D structures informed by their amino acid sequence. These proteins also contain within them other types of biologically derived molecules that impart specific functions necessary for capturing light and forming electrical circuits. Since we now have a good, working understanding of the photosynthetic machinery at the atomic level, it is possible to consider the key steps in this process and incorporate them into entirely new, manmade proteins or redesigned natural proteins; thus, the photosynthetic process could be recapitulated in much simpler, scaled-down systems.

We have therefore selected both manmade and natural proteins to act as functional building blocks in an artificial photosynthetic pathway that will allow us not only to operate a simple photosynthetic process, but also to encapsulated it within an elementary cell-like assembly called a protocell. These entities are designed to facilitate the input of functional biological and artificial components in a very simple compartment, much like a living bacterial cell. Currently, since there is limited or no information storage capacity in these protocells, they cannot evolve or actively reproduce and they act as a safe platform for us to test our understanding of complex biological molecules and systems, while allowing us to explore aspects of the origin of life on Earth. Protocells can also exhibit unusual influences on biological molecules like proteins, increasing the rates of enzymes - proteins capable of performing chemical reactions - and providing a useful facsimile of the environment in which proteins and enzymes evolved. We therefore intend not only to build functional, manmade photosynthetic assemblies, but also to co-encapsulate these in a protocell with a natural enzyme capable of transforming carbon dioxide into a useful chemical building block. This will provide a primitive but versatile, solar-powered 'protoalgae' into which a more sophisticated metabolism can be constructed. Beyond the academic study of biological systems and molecules that can be achieved through this work, there is also great potential to deliver programmable cell-like entities that can perform truly useful chemistry unachievable in large-scale industrial process. Therefore, we strongly believe this work to be of interest both for the fundamental exploration of biological engineering and for the construction of protocells for addressing industrial issues.

Within the burgeoning field of synthetic biology, there is a central goal to tractably engineer biological or biologically-inspired parts and devices, and incorporate them into discrete, protocellular platforms. To enable these systems to do work and function in a useful manner, it is crucial to engineer into them energy capture and storage mechanisms, therefore maintaining a non-equilibrium state that is so vital to their living, natural counterparts. Here we propose to integrate engineered, protein-based components with functional nanomaterials to construct protein-based assemblies capable of harvesting light to power an elementary, protocell-encapsulated CO2-fixing metabolism.

We will achieve this using a combination of de novo protein design, natural protein engineering and bioconjugate chemistry, providing protein and nanoparticle modules for further integration into solar-powered NADP+ reducing assemblies. These, in turn, will be encapsulated with natural formate dehydrogenases within the context of a versatile, membrane-free protocell, thus creating a "protoalgal" platform that uses solar energy to reduce CO2 to formate. This will pave the way for the expansion of metabolic processes within the protocell, with the ultimate aim of producing high value organic molecules from simple 1-carbon substrates.

Elements of natural oxygenic photosynthesis will be imprinted within the individual modules of the protein:nanoparticle hybrid assemblies: natural NAD(P)+ reductases will be photosensitized - either with simple Ruthenium compounds or de novo designed maquettes incorporating photoactive tetrapyrroles - and light-harvesting quantum dot antennae will be bioconjugated using simple carbodiimide chemistry. De novo designed water splitting maquettes will be added to the chimeric assemblies, enabling the use of water as the electron source, or 'fuel' in the artificial photosynthetic process.

Scientific discovery is integral to the international competitiveness of the UK. Through the assembly of the first CO2-fixing protocells, this project will deliver an unprecedented advance in the Synthetic Biology BBSRC Strategic Priority Area, while delivering vital information that will further our fundamental understanding of protocell assembly and protein design and engineering. These advances will contribute significantly to the UK's position as a world leader in these areas.

We anticipate that this project will deliver significant impact upon the commercial sector. The creation of solar-powered NAD(P)H regeneration assemblies will be of particular use for industrial-scale biotransformations with enzymes such as the Cytochromes P450; while the construction of robot-like, programmable protocells that perform useful chemical tasks will be ripe for commercial exploitation. To fully maximize impact on the commercial sector we plan to undertake training in science business and innovation, establish close ties with the University Research and Development Office and establish and maintain contacts with industry.

Synthetic biology and bionanotechnology have been the focus of significant public concern and since our work is directly related to both these fields, we plan to allay such concern by regularly engaging and educating the public through University public outreach schemes and the media. JLRA will attend courses in communication skills and media training, continue participating in public outreach schemes run by the University and maintain accessible websites displaying information about our current research. Press offices of the BBSRC, Royal Society and the University of Bristol will be contacted when high profile research papers are accepted.

We anticipate that this fundamental research will significantly impact upon the third sector. We will maximize impact on policy-makers, funding bodies and academic institutions by providing clear evidence of the value of synthetic biology research and raising its profile within the UK. This research will be actively promoted through the scientific community and within the University of Bristol itself, with the aim of establishing links and new collaborations with other departments and disciplines. Training and expertise in this field will be offered to those involved in the project (PDRAs, PhD students, etc.), providing them with the skills to succeed in a future career in academia or industry.