Genetic and biotechnological development of the pennate marine diatom Phaeodactylum tricornutum for high-value bioproducts and carbon bio-mitigation.
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Conflicting demands of food over fuel and the mushrooming need for feed and fibre are serious challenges for humankind at a time when we also need to address climate change challenges. Global demands for land based commodities will surpass increases in supply. Such divergence may bring civil unrest, hunger and malnutrition. Environmental issues associated with agriculture's environmental footprint pose challenges on a global scale including scarcity of fresh water, degradation of ecological biodiversity and the release of toxic wastes into the environment. One possible environmentally safe and sustainable solution would be the selection of alternative green energy sources that can be cultivated in marine waters or waste waters, and still offer us feed and fuel. Microalgae are miniature biofactories that can convert sunlight and CO2 into valuable biomass and into lipids that can be utilized as feedstock for biofuel, aquaculture feed and high value fatty acids. Marine microalgae such as diatoms represent potential targets for industrial production of useful fatty acids. In general, the fatty acids of marine microalgae predominate in saturated and monounsaturated fatty acids, with a high level of omega-3 polyunsaturated fatty acids (PUFA). Particularly promising candidate microalgae are those that can utilize seawater, can grow at high CO2 concentrations and can be used to produce higher-value products. The ease of growing microalgae has brought these photosynthetic machineries into the focus of intense biofuel market. We have developed strains of marine diatom Phaeodactylum tricornutum that differ significantly in lipid profile and fatty acids. Diatoms are marine microalgae belonging to the chromista which are photosynthetic sinks for capturing ambient carbon dioxide. Their carbon dioxide fixation rate is maintained through CO2 levels ranging from atmospheric concentrations to as high as 10% carbon dioxide. At present, the diatoms contribution to global photosynthetic CO2 fixation is comparable to that of the total rainforest contribution which make them ideal for capturing CO2 produced by fossil-fuel combustion. Encased in nanoporous silica shells, diatoms are a group of autotrophic organisms that arose by the evolutionary phenomenon of secondary endosymbiosis. Thus, their genome is composed of a diverse repertoire of both prokaryotic and eukaryotic genes. Diatoms also display an unmatched diversity in their intricate nanofabricated siliceous cell walls which can offer new directions for development of biological membranes that can aid in the separation of CO2 from flue gas exhaust from thermal power stations. The omega-3 fatty acid rich biomass of diatoms can provide a valuable feedstock for aquaculture. The CO2 biosequestration potential of the marine diatom Phaeodactylum tricornutum is demonstrated under high CO2 concentrations. P. tricornutum showed fastest growth rate of 0.502 day-1 at 4% v/v CO2 and showed maximum CO2 removal rate of 0.379 gC-1L-1 at 2% v/v CO2. P. tricornutum also accumulated high amounts of omega-3 polyunsaturated fatty acids (PUFA) with eicosapentaenoic acid as the major omega-3. The yield of microalgae is the paramount factor for the control of overall economics. The various factors that determine yield include growth rate, cell density and lipid content of the microalgae. Biomass yield and associated lipids which can be utilized for biofuels is under the control of complex set of environmental and genetic factors. For achieving best yield selection of potential genotypes is a prerequisite. There exists an immense genetic pool of algae; however no large-scale industrial production is possible using the wild-type strain. For genetic enhancement of lipid content, a wild strain of P. tricornutum was chemically mutagenised with ethylmethane sulfonate in whole-genome mutagenesis screens where new strains were identified that over-accumulated lipids and displayed altered fatty acid profiles. Two approaches were used for the selection of novel gain-of-function mutants from large populations of chemically mutagenised diverse populations of P. tricornutum. The two approaches were: (1) use of an antibiotic cerulenin for the isolation of novel P. tricornutum mutants with altered lipid profiles; and (2) use of Fluorescence-activated Cell Sorting (FACS) to isolate individual mutant cells (from which clonal populations can be established) with the highest levels of lipids compared to the progenitor strain. Through the development of these strategies a range of novel P. tricornutum mutants with altered lipid properties were isolated. The novel P. tricornutum strains showed 2-2.5 fold increases in total dry weight lipids over the wild type progenitor. Two of the mutant strains, designated as Ptcer12 and Ptcer13, accumulated very long chain fatty acids namely erucic (C22:1) and nervonic acid (C24:1) which have important industrial applications as biolubricants, cosmetics, pharmaceuticals and in dietary therapy. The novel P. tricornutum mutants screened and isolated using FACS accumulated 44-50% dry weight total lipids and higher biomass productivity. To facilitate targeted genome engineering of P. tricornutum we also used artificial microRNAs as a tool for gene knockdown experiments in P. tricornutum. P. tricornutum transformants were generated that expressed the mature artificial microRNAs which elicited down-regulation of an endogenous PHYTOENE SYNTHASE (PSY) gene and concomitant knockdown of carotenoid levels.
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