As core components of a sustainable bio-economy, microalgae have the potential to become a major source of biofuels and valuable chemicals with minimal environmental impacts. These photosynthetic microbes use solar energy, grow quickly, consume atmospheric carbon dioxide, and can be cultivated on non-arable land. However, there are presently considerable practical limitations in the photosynthetic production of biofuels from microalgae, resulting in low productivity and high costs. In order to engineer microalgae for efficient production of fuels and chemicals, it is necessary to understand their metabolism and how it is regulated at the whole-organism level. This project will study the unicellular green alga Chromochloris zofingiensis, which naturally accumulates high levels of oil as a carbon storage product, making it a promising biofuel feedstock. This alga also has a unique metabolic flexibility and can reversibly switch between growth modes with different levels of oil production. Taking advantage of C. zofingiensis genomic resources previously developed by the research team, this project will re-design this alga to produce high levels of oil from carbon dioxide. A comprehensive, large-scale integrative metabolic analysis will be conducted to understand how energy flow in the cell is regulated and redirected toward oil production. Guided by computational modeling approaches, genome-editing tools will be used to engineer this alga's metabolism to substantially increase oil productivity. As a result of this research, C. zofingiensis will be developed as a reference system for algal fuel production, expanding the range of platform organisms for the production of biofuels and bioproducts relevant for DOE-BER's Genomic Sciences program.