The World is faced with the considerable challenge of supplementing, and ultimately replacing, its fossil fuel-based economy with one based on clean energy technologies such as biofuels. Currently, commercially available biofuels (e.g. bioethanol and biodiesel) are derived from crop plants such as maize and soybean. However, there are major concerns regarding both the use of valuable agricultural land for production of biofuel crops, and the sustainability and energy balance of such technologies. A potential alternative source of biofuels is microalgae - aquatic photosynthetic organisms that do not require fertile land for cultivation; grow considerably faster than plants, and which can accumulate significant quantities of high-energy compounds such as oils. Furthermore, such aquatic cultivation could be coupled to waste streams such as CO2 output from industry and nutrient-rich effluent, thereby using this waste to promote algal growth. However, industrial-scale cultivation of microalgae for biofuels faces considerable challenges, not just in terms of technical feasibility, but also in terms of the economics and achieving a net positive energy balance. It is recognised that success will probably require the development of superior algal strains in which genetic engineering methods have been used to radically alter and tailor the cell metabolism's towards maximal biofuel productivity under industrial conditions. Currently, the molecular tools needed to create such strains are decided limited and algal metabolic engineering is still in its infancy. In this project, we will develop advanced tools for algae along the lines of the 'synthetic biology' technology now being used to design and create novel bacterial and yeast strains. A particularly, attractive feature of our approach is that we will exploit the ability to introduce new genes into two separate genetic compartments, the nucleus and the chloroplast, thereby allowing elaborate strategies for engineering that employ multiple new genes and create novel biosynthetic pathways within the chloroplast, but which can regulated from the nucleus. We will validate this new technology by creating a series of designer algae that produce two potentially useful fuel molecules - the short-chain hydrocarbon, isoprene and the alcohol, geraniol.

Technical Abstract:
The aim of this project is to establish tools for straightforward and predictable metabolic engineering in the model green alga Chlamydomonas reinhardtii, as a means to generate strains producing novel biofuel molecules. We will use synthetic biology principles to design a series of PhycoBrick parts that will enable rapid assembly of different genetic elements (coding region, plus regulatory elements such as promoters, enhancers, riboswitches, 3'UTRs, and targeting and epitope tags). In particular we will take advantage of two inducible systems we have discovered in Chlamydomonas, the METE promoter, which is repressed by vitamin B12, and riboswitches in the THI4 gene, which undergoes alternative splicing in the presence of thiamine pyrophosphate. These elements will thus allow tight yet reversible regulation of nuclear transgenes with natural metabolites. A selected subset of these PhycoBrick parts will be assembled into devices and tested for activity using a reporter gene (codon-optimized luciferase, targeted to the chloroplast and with an HA-tag) to provide quantitative output, to establish predictive behaviour of the PhycoBrick parts. Using these data, we will design devices for inducible nuclear expression of higher plant genes for either isoprene synthase (IPS) or geraniol synthase (GES) in Chlamydomonas. This should cause diversion of isoprenoid intermediates to produce isoprene or geraniol respectively. These are small volatile hydrocarbons that have the potential to be used directly as fuel molecules. Further refinement of the isoprene/geraniol-producing strains will be achieved by introduction of trans-operons into the chloroplast genome for genes encoding enzymes for synthesis of IPP (the isoprenoid precursor) and/or down regulation of competing pathways using artificial microRNAs. We will also explore the potential of PhycoBricks for metabolic engineering in other algal species for which transformation procedures have been established.

Potential Impact:
The topic of research in this application is relevant to a number of the major research challenges (so-called grand challenges) we face today: CO2 emissions and resulting climate change; dwindling reserves of fossil fuels, particularly those for liquid transport fuels, but also as feedstock for bulk and high-value chemical production; diminishing areas of arable land suitable for food crop production; and water management - both supplies of fresh water and waste-water treatment. Microalgae offer an enormous, as yet essentially untapped resource, which if exploited appropriately could lead to novel solutions to address ALL of the above. Many species have very fast rates of growth, and can accumulate high amounts of lipids, which can be used as fuel molecules. They can capture CO2 from flue-gas and scrub nutrients from effluent, and they do not require fertile land for cultivation. This has been recognized around the World by both governments and industry, leading to considerable investment in both research and development for algal biofuel production. Nevertheless, successful implentation of microalgal biotechnology will require much greater understanding of these organisms than we currently possess. In this application we will be developing tools that will enable much more rapid generation of constructs for metabolic engineering of the model green alga Chlamydomonas reinhardtii. The so-called PhycoBrick parts will establish a standard that can be used to permute the different DNA elements needed for this process into different devices. Using these tools, we will then explore the possibility of engineering Chlamydomonas to make two different fuel molecules, by introduction of one or more of these devices. We will make the Phycobrick parts openly available to the academic community. Both applicants have extensive connections with industry, from small start-up biotech companies, to large multinationals in the chemical and fuel sectors. We will engage with these industrial partners to explore the possibility of exploitation of the PhycoBricks parts, and also the strains that we generate. The likelihood is that scale-up and regulatory issues will require further R&D, but it is conceivable that commercial operations with these strains could occur within the next 5 years. Both applicants are very heavily involved in providing expert knowledge related to the use of algae for production of biofuels and other chemicals, and also to the impact of algae in the environment, such as waste water treatment and bioremediation on the one hand, and removal of algal contaminants on the other. We are frequently asked to give expert opinion by the media, and government agencies, and will continue to do so as part of this project. As well as commercial and academic sectors, the work we will do will have impact on our understanding of algal biology generally. There are over 300,000 different algal species, and marine species contribute up to half of all global CO2 fixation, so the study of these organisms has much wider implications than biotechnological exploitation. Our project will ensure that there are scientists with skills for studying algal biology, not just the PDRAs employed on the grant but also other members of our groups. We will carry out a number of public outreach activities, in which the PDRAs and students will also participate. The activities will build on our experience with mounting an exhibit at the Royal Society Summer Science Exhibition this year entitled 'Meet the Algae: Diversity, Biology and Energy'. As well as the stand, we are generating web-based information and resources to enable the general public to find out more about these beautiful organisms.