Algal oils by design: a new biotech platform for high-value lipids.

Industrial biotechnology (IB) is the harnessing of biological processes and materials for the production of industrially useful products on a large scale. If offers novel solutions to product synthesis that avoid fossil fuels, but instead use plants, bacteria, fungi and algae as renewable sources. This bio-economy is currently worth trillions globally and new advances could bring major benefits to UK industry sectors including chemicals, renewable energy, materials, and health, and could create thousands of jobs in the coming years. A major area of IB is the intensive cultivation of specific microorganisms in industrial scale fermentors, where the microbes have been selected or engineered to produce high levels of a desired commercial product such as an antibiotic, a complex metabolite or a therapeutic protein. These fermentation processes generally use either bacterial or fungal species as the 'cell factory'. However, a very large and diverse group of microorganisms - the algae - represent a rich biological resource that has yet to be exploited in the biotech industry. These photosynthetic microorganisms not only offer a huge treasure trove of natural compounds that have potential application in drug discovery (e.g. as nutraceuticals and food additives, as natural colorants or stabilizers in food and cosmetics) but also offer the possibility of cultivation using sunlight and CO2 to drive biological production, rather than the expensive sugars needed for bacterial or fungal growth.

In this proposal we aim to develop algae as cell platforms for IB applications, and in doing so bring a new, disruptive technology to the fore. We will build on recent advances in our knowledge of the genetics, biochemistry and physiology of algae, together with developments in genetic engineering and high-throughput cell screening technologies. As a focus for this four year project we will develop the algal platform initially for the production of high value oils (=lipids) such as the edible omega-3 and omega-6 long-chain fatty acids that are an essential component of animal diets, and contribute, inter alia, to infant brain development and cognitive function in adults.

The multidisciplinary project has four integrated programmes of work and builds on the world-leading expertise of the five participating groups. We will combine our biological knowledge on the genomes, transcriptomes and lipid metabolomes of two chosen algal species with advanced metabolic modeling tools to build a detailed, robust and predictive model of lipid metabolism in the algal cell. Such as model will allow us to evaluate in silico the effects of manipulating the lipid biology (for example by adding new enzyme pathways). In a second programme, we will use our expertise in algal genetic engineering to develop synthetic biology tools and a design pipeline that will greatly accelerate the process of creating genetic engineered strains. This is critical since, even with a sophisticated model, a large number of different engineering parameters need to be evaluated in order to achieve a fully optimized strain. Such strain selection will also be facilitated by integrating the genetic engineering with a high-throughput cell evaluation technology developed by one of the groups. This system involves encapsulation of single cells in oil microdroplets and ultra-fast analysis and sorting using microfluidic technology coupled to analytical equipment. This will enable the rapid identification and recovery within large populations, of rare engineered or mutated cells that have the desired phenotype. Finally, the technologies will be brought together and tested in the final programme to confirm that we can design and create 'bespoke' strains producing high levels of a desired oil.

There is considerable interest worldwide in the exploitation of microalgae for the production of a wide range of molecules, including biofuels, bulk chemicals and high value products. These photosynthetic organisms offer many advantages in an industrial context - they do not compete with food crops for arable land, growth rates and productivity is very high for many species, and they are amenable to cultivation in fermenters or closed photobioreactors, where conditions can be controlled. However, to exploit them fully as platforms for industrial biotechnology, it is essential that they can be manipulated easily and robustly, as is possible for other commonly used hosts, such as E. coli, yeast and certain mammalian cell cultures. In this proposal we aim to establish the workflow and molecular tools for two marine microalgae: Phaeodactylum tricornutum (Pt) and Nannochloropsis gaditana (Ng), both of which have sequenced genomes, and accumulate large amounts of lipids. Pt is a marine diatom that is readily transformable, and for which a number of molecular tools are being developed. Ng is a eustigmatophyte alga, for which transformation by homologous recombination has been reported; this will provide the opportunity for knock-outs and knock-ins of genes of interest.

We will use genome and transcriptome data to develop metabolic network models of lipid metabolism, and then employ the models to inform strategies for which lipid biosynthetic enzyme(s) should be manipulated to optimize production of particular lipid molecules, focusing initially on the omega-3 fatty acid DHA. The metabolic engineering itself will be facilitated by development of synthetic biology approaches, including multiplex DNA assembly, registry of DNA parts, and development of synthetic circuits for regulation of transgene expression. We will also design and fabricate microfluidic devices for high-throughput selection of desired strains of algae.

There is considerable interest in the UK and indeed worldwide in developing an algal industrial biotechnology (IB) sector, both at the level of policy makers and research funders, but also industry. However, because exploitation is at such an early stage, there needs to be some form of proven platform technology before industry would feel confident about moving into the area.

In this application we aim to provide the basis of this knowledge. We will develop tools to enable much more rapid generation of constructs for metabolic engineering of industrially important marine microalgae. We will also develop mathematical models to define lipid metabolic pathways, thus working towards the goal of predictive design principles to engineer the algae to make any desired lipid molecule. Our focus in this grant will be on the important omega-3 fatty acid, DHA, but the principles we establish, together with the generic tools, will provide the means to do the same for many different lipid products. Finally, in an innovative cross-disciplinary collaboration, we will use a microdroplets platform to study the algae at the single cell level, and to sort populations with high throughput. Our links with industrial partners provide the means to take any promising strains we identify through to the next stage of technology readiness, with the prospect of commercialization within 5-10 years.

As the technological advances with microalgae proceeds, economic sustainability will be enhanced, and algal IB will have genuine societal impact. The following sectors will benefit: Food, Feed, Nutraceuticals, but also as the range of products that algae can be engineered to make expands, there will be impacts on the Speciality / platform chemicals, Energy, Fertiliser, and Cosmeceuticals, sectors.

Government and Public Bodies will also benefit, since the outputs of this project can have an impact on several pressing current issues: building the knowledge economy will lead to job creation, and contribute to sustainable economic growth, improve security of food and materials, and contribute to enhanced nutrition.

There will also be benefit to the general public, since the research may contribute to creation of jobs, as algal IB starts to become a reality. Adoption of research outcomes may result in nutraceuticals from algae (eg omega fatty acids, antioxidants and vitamins) becoming more affordable and widely available, leading to improved nutrition and public health. The outreach activities aim to increase awareness of the public on issues of energy, food and material security, and how the development of IB and a knowledge economy can benefit the UK. This will encourage pupils to adopt a career in science and technology. Outreach activities will also increase awareness of resource efficiency in the public, particularly energy awareness, and thus may contribute to lowering energy consumption and household bills.The benefit of these interactions on informed policy choices should start during the lifetime of the grant, and through introductions to algal lobbying bodies such as the European Algae Biomass Association, may have a lasting effect on decision makers in the public domain.

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.