ENGINEERING AN OLEOGINOUS YEAST FOR THE PRODUCTION OF BIODIESEL

Abstract

There are economic and social interests in replacing the current energy dependence we have on petroleum-based oleochemicals. Yarrowia lipolytica, an oleaginous yeast, has the ability to metabolize unique carbon sources, particularly hydrocarbons and to accumulate large amounts of lipids which could be developed into a source of biodiesel. The ability of Y. lipolytica to accumulate triacylglycerols in lipid droplets and the complete sequencing of its genome make Y. lipolytica a viable organism to genetically engineer for the production of large quantities of biodiesel precursors. The purpose of this project is to genetically modify Y. lipolytica to further increase its production of triacylglycerols by knocking out genes that encode enzymes involved in the β-oxidation of fatty acids. This genetic modification will be accomplished by using homologous recombination to disrupt the genes POX3-5 and POT1. The 5’ and 3’ untranslated regions of POX3-5 and POT1 were amplified by polymerase chain reaction and cloned to allow a drug resistance gene to be introduced between them. Following cloning, these genes will be knocked out from the Y. lipolytica genome using drug resistance as a marker. The disruption of these genes is expected to increase the accumulation of triacylglycerols in Y. lipolytica lipid droplets versus the wild-type. Progress towards the goals of this project will be reported.