Highlights d Deletion of pentose phosphate pathway genes causes isobutanol hypersensitivity d Deletion of GLN3 increases yeast tolerance specifically to branched-chain alcohols d Isobutanol production is greatly increased by deleting GLN3 in engineered strains d The nitrogen starvation response induced by isobutanol is evaded in gln3D strains Authors

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The mitochondria, often referred as the powerhouse of the cell, offers a unique physicochemical environment enriched with a distinct set of enzymes, metabolites, and cofactors ready to be exploited for metabolic engineering. In this review, we discuss how the mitochondrion has been engineered in the traditional sense of metabolic engineering or completely bypassed for chemical production. We then describe the more recent approach of harnessing the mitochondria to compartmentalize engineered metabolic pathways, including for the production of alcohols, terpenoids, sterols, organic acids, and other valuable products. We explain the different mechanisms by which mitochondrial compartmentalization benefits engineered metabolic pathways to boost chemical production. Finally, we discuss the key challenges that need to be overcome to expand the applicability of mitochondrial engineering and reach the full potential of this emerging field.

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