Harnessing Yarrowia lipolytica’s potential as a lipid and alkane production platform

Engineering cellular phenotype can enable the in vivo synthesis of renewable fuels, industrial precursors, and pharmaceuticals. Achieving economic viability requires the use of a cellular platform that generates high titers independent of fermentation condition, through either native or imported biosynthetic metabolism. While lacking fully developed genetic tools, the oleaginous yeast Yarrowia lipolytica has the native capacity to produce large titers of lipids and citric acid cycle intermediates. However, unlocking this biosynthetic capacity requires complete rewiring of native metabolism. To this end, this work focuses on the development and engineering of the yeast Y. lipolytica to rewire native metabolism and enable the production of lipids, alkanes, and itaconic acid. Precise control of gene expression is a requisite to enable metabolic and pathway engineering applications for any host organism. However, Y. lipolytica lacks promoter elements strong enough to manipulate intracellular metabolism. Thus, we utilized a hybrid promoter engineering approach to produce libraries of high-expressing, tunable promoters, seven-fold stronger than promoters previously characterized in Y. lipolytica 1,2. We successfully applied this approach to Saccharomyces cerevisiae, expanding transcriptional capacity of the strongest constitutive to highlight our hybrid approach as a generalizable method to increase expression capacity in eukaryotic organisms 3. We utilized our novel Y. lipolytica hybrid promoters to drive intracellular metabolism towards lipid production and to overexpress heterologous enzymes that enable alkane and itaconic acid production. Specifically, we implemented a global rewiring of Y. lipolytica’s native metabolism to increase lipogenesis more than sixty fold to 25.3g/L (the highest lipid production ever reported) and generated cells nearly 90% lipid content. We further expressed a lipoxygenase enzyme to catalyze the novel microbial production of the short-chain n-alkane, pentane. Finally, we exploited Y. lipolytica’s capacity to accumulate citric acid cycle intermediates by expressing a heterologous cis-aconitic acid decarboxylase enzyme to produce itaconic acid. Increasing substrate availability through media optimization and genomic engineering increased pentane and itaconic acid production threefold and eightfold, respectively 4. Collectively, these studies have facilitated the utilization of Y. lipolytica as an industrially relevant microbial platform, and represent a generic approach towards enabling biosynthetic control in microbial hosts will ill-defined gene expression technology.