Algal biofuels: novel approaches to strain improvement.

The development of biofuels as an alternative to fossil fuels is of world-wide importance. Currently there is considerable interest in replacing biofuels based on crop plants with more economic and ecologically acceptable sources of biomass. One promising alternative is microalgae since these photosynthetic organisms have very high growth rates, can be cultured without using fertile land and have the potential for coupling biomass production to CO2 capture from industrial flue gas, or to wastewater treatment. Whilst there are many engineering and downstream processing issues that need to be addressed before algal biofuels become a commercial reality, the major biological challenge is the development of suitable strains that have the necessary characteristics for mass culture. These include high lipid content, rapid growth, tolerance to high CO2, and ease of harvesting. The goal of this studentship is to develop novel genetic approaches to strain improvement. In particular, to produce strains with high lipid content during active growth. The project builds on the expertise of the academic supervisor in algal molecular biology, and the industrial partner in lipid biology and analysis to ensure a broad and effective student training and a successful project. The three key aspects of the project are: 1: A genetic screen to isolate high-lipid mutants and to identify regulatory genes involved in carbon partitioning. The production of storage lipids (triacylglycerides, TAGs) in algae is maximal under conditions of nutrient stress where growth is slow, and is the result of a shift in the partitioning of fixed carbon between carbohydrates and TAGs. Mutations that disrupt this partitioning control should allow the isolation of algae that accumulate high levels of TAGs during non-stress conditions where growth is maximal. Furthermore, molecular analysis of the mutants will provide valuable insight into the control mechanism. Mutant colonies that have elevated levels of storage lipids during active growth will be identified by mutagenising a cell population, and then screening the colonies for those with increased fluorescence using a lipophilic fluorescent dye. Two species will be used in this screen: Chlamydomonas reinhardtii will be used since genomic and molecular-genetic tools are already available for the mapping and characterisation of the affected genes, and Neochloris oleoabundans since this oleaginous alga is expected to yield mutants with very high TAG content, and may therefore be of commercial value. ii). Metabolic engineering of lipid biosynthesis. In algae, lipid biosynthesis takes place exclusively in the chloroplast. Since the genetically engineering of the Chlamydomonas chloroplast genome is well-established, it should be feasible to up-regulate TAG biosynthesis by introducing genes for biosynthetic enzymes likely to improve the flux of the pathway. The Purton lab has developed a series of chloroplast expression vectors and these will be used to insert various gene combinations into the chloroplast genome. The lipid profile of resulting strains will be examined to determine the effect of these genetic manipulations. iii) Novel algal hybrids via protoplast fusion. Cell fusion of protoplasts to create heterokaryons is a well-established technique that has been widely used to produce somatic hybrid lines of plants and yeasts. However, despite promising early research on algae, this technique has not been exploited to create novel algal hybrids. The project will develop protoplast fusion for algae and test whether we can create stable hybrids that combine desirable phenotypes from different algal species. Molecular markers will be used to assess the genetic state and stability of the heterokaryon. This somatic breeding approach is particularly attractive since it allows the creation of novel strains by crossing species boundaries and exploiting the diversity found amongst the microalgae, without using GM technology.