2-ketoacids are one of four classes of compounds that undergo recursive carbon-chain elongation in nature, and are of particular interest due to their role as precursors to higher alcohols, which are valuable renewable fuel compounds. While expression of heterologous enzymes and protein engineering have been successfully employed in E. coli to increase the maximum number of iterative elongation cycles, it remains difficult to achieve specific production of longer-chain 2-ketoacids. Here, we demonstrate that mitochondrial compartmentalization in the yeast Saccharomyces cerevisiae can increase specificity of recursive pathways toward longer-chain products. We show that overexpression of the 2-ketoacid elongation enzymes in mitochondria of a four-carbon alcohol production strain boosts five-carbon alcohol production more than 13-fold. Total alcohol production also shifts from a predominately four-carbon alcohol mixture to one with more than 80% five-carbon alcohols. Reducing the loss of intermediates to competing pathways further increases specificity toward five-carbon alcohol production, in addition to achieving the highest titer reported for S. cerevisiae. This work establishes the ability of mitochondrial compartmentalization of recursive metabolic pathways to enhance specificity toward larger products while maintaining high titers, exemplifying the potential benefits of harnessing organelles to engineer recursive pathways for product specificity.

Mitochondrial compartmentalization enables high-specificity recursive alcohol production in Saccharomyces cerevisiae

Macromolecular Chemistry: The Second Century

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ACS Spring 2020 National Meeting & Expo

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.