Mitochondrial Engineering: Harnessing the Powerhouse of the Cell for the Production of Fuels and Chemicals

Yeast metabolic engineering has mostly focused on the construction of metabolic pathways in the cell cytoplasm. However, there is huge potential in harnessing the diversity of environments, metabolites and enzymes that exist in the different organelles of eukaryotic cells, to benefit engineered metabolic pathways. Among these, the mitochondrion is of particular interest due to its diversity of enzymes and metabolic networks, wealth of cofactors, and dynamic physiology. To facilitate mitochondrial engineering, we developed new molecular tools that expedite the construction of metabolic pathways targeted to either the cytoplasm or the mitochondria. With these tools, we show that targeting whole biosynthetic pathways to the mitochondria of the yeast Saccharomyces cerevisiae improved the production of advanced biofuels by as much as 500%, compared to identical pathways targeted to the cytoplasm. The mechanisms of this enhancement include (1) the elimination of metabolic bottlenecks, (2) increased availability of intermediates of the engineered pathways, and (3) increased local concentrations of enzymes due to their confinement inside the smaller volume of mitochondria. Mitochondrial engineering also has the potential to circumvent cytoplasmic regulatory networks that undermine engineered pathways, and to alleviate the adverse effects of pathways that contain cytotoxic intermediate metabolites, by confining these metabolites to the mitochondria, away from their cytoplasmic or nuclear targets. Mitochondrial engineering thus offers multiple new strategies to enhance engineered metabolic pathways.