Development of Renewable Biofuels Technology by Transcriptomic Analysis and Metabolic Engineering of Diatoms

Abstract

There is enormous interest in developing renewable sources of liquid fuels because of depletion of fossil fuel reserves, dependence on foreign sources, and increasing atmospheric CO2 levels. Algae produce neutral lipids that are readily converted into liquid fuels such as biodiesel or JP-8 equivalent, and are attractive sources because they are far more productive than plants (yielding 10 -100’s of time more lipid per land area), and can be grown on non-cultivatable land with non-potable (brackish or salt) water sources. Unicellular algae known as diatoms were the most thoroughly characterized species in the National Renewable Energy Laboratory’s Aquatic Species Program, whose goal was to develop microalgae as renewable fuel sources. Lipid accumulation in microalgae is generally induced by nutrient limitation, which involves a change in environmental conditions. Intrinsic variability in cellular response to environmental changes prevents a high degree of control over the process. Nutrient limitation also inhibits biomass accumulation; therefore a tradeoff between high biomass and lipid production occurs. The goal of this project was to develop metabolic engineering approaches for diatoms to enable induction of lipid accumulation by controllable manipulation of intracellular processes rather than from external environmental conditions, and to manipulate carbon partitioning within the cell betweenmore » lipid and carbohydrate synthesis to enable both abundant biomass and lipid accumulation. There were two specific objectives for this project; Objective 1:To perform comparative transcriptomic analysis in T. pseudonana and C. cryptica of lipid accumulation resulting from silicon and nitrogen limitation, to identify common and key regulatory steps involved in controlling lipid accumulation and carbon partitioning; and Objective 2: To metabolically engineer the cell to alter carbon partitioning to either trigger lipid induction without the need for nutrient limitation, or to enable lipid accumulation along with high biomass accumulation.The significance of this project is that it will enable greater control over lipid production in diatoms by manipulable intracellular processes rather than from variable environmental conditions, and it will possibly enable lipid accumulation under normal growth conditions. Current economics dictate the use of open outdoor raceway pond systems for commercial-scale microalgal growth for biofuels production (although advanced design enclosed bioreactors are under consideration, they are currently not cost effective). Outdoor systems are subject to large variability in environmental conditions. In microalgae, lipid accumulation generally occurs under nutrient limiting conditions, which prevents high biomass accumulation. Potentially, one could carefully adjust the level of a particular nutrient so that it would become limiting after sufficient biomass accumulated; however, given the variability inherent in microalgal cellular metabolism under different light, temperature, and nutrient regimes, this will be a relatively uncontrolled and poorly reproducible approach. A better strategy would be to provide ample nutrients, but trigger lipid accumulation “artificially” by manipulating intracellular processes through metabolic engineering. In addition, identifying the key regulatory steps involved in controlling carbon partitioning in the cell coupled with metabolic engineering should enable greater partitioning of carbon into lipids during non-limiting nutrient growth conditions. The approaches outlined in this proposal are aimed at achieving these goals, and are expected to have a substantial impact on the development of renewable biofuels technology. Development of the approaches described in this proposal will provide a rich interdisciplinary educational experience for high school and undergraduate students to foster their development in a scientific career.« less