Repurposing the Saccharomyces cerevisiae peroxisome for compartmentalizing multi-enzyme pathways

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Will DeLoache, Hanson Lee, John Dueber

UC Berkeley, United States

Engineered microorganisms promise to enable the renewable and environmentally friendly production of fuels, bulk chemicals, and therapeutics. To achieve commercial viability for these processes, the most difficult challenge is not necessarily how to build a pathway to produce a desired molecule; rather, it is to optimize that pathway for both yield (i.e. product generated per input consumed) and productivity (i.e. rate of product synthesis). Widely applicable strategies for pathway optimization must be developed in order to get products to market cheaper and faster. A chief consideration for developing such strategies is limiting crosstalk between high-flux engineered metabolic pathways and the cellular processes of the production host. Evolution has solved the problem of crosstalk for many cellular processes by segregating functions into membrane-bound organelles. The goal of this project is to repurpose one of these organelles — specifically the peroxisome of Saccharomyces cerevisiae — to compartmentalize heterologous metabolic pathways. As a proof-of-principle, we are working to demonstrate encapsulation of a short enzymatic pathway within the peroxisome and show that compartmentalization reduces the accumulation of off-pathway side products. Additionally, we are investigating methods to improve the cargo capacity of the peroxisome by both removing endogenous proteins and modulating the expression level of biogenesis genes. Ultimately, this work will contribute to the development of a synthetic organelle as a tool to limit metabolic crosstalk and improve the predictability and efficiency of engineered microorganisms.