(188bb) Peroxisome Engineering for Improved Heterologous Biochemical Production

Numerous medically, industrially, and scientifically relevant compounds are being produced in ever-increasing quantities, and at decreasing costs, thanks to the continued advances of metabolic engineering. Many of these compounds are naturally produced by organisms that cannot reliably be grown at an industrial scale. By expressing the genes responsible for the formation of these interesting compounds in organisms that do function well as industrial hosts, high production rates can be achieved. Unfortunately, complications can arise when competing reactions in the new host detract from product formation. One solution to this problem is compartmentalization. By separating the newly engineered pathway from the bulk of the cell, many competing reactions can be avoided. Furthermore, localization of a reaction pathway to an isolated area can lead to increased local reagent concentration, directly benefiting the kinetics of many reactions. It is possible to carry out such compartmentalization in eukaryotes by exploiting their peroxisomes: small, isolated organelles normally used to perform specific native reactions, such as β-oxidation. Peroxisomes are separated from the cytoplasm by a true membrane and possess dedicated transport mechanisms for passage into and out of the organelle. The oleaginous yeast Yarrowia lipolytica has great potential as a platform for oleochemical production. By engineering the peroxisome of this yeast, we demonstrate improved production efficiencies of heterologous products, including fatty alcohols and polyhydroxyalkanoates. Transport of fatty acid substrates into the peroxisome can be improved by overexpression of the enzyme pair PXA1/PXA2. Key enzymes are able to enter the peroxisome through mediated import by PEX5 and PEX20 and overexpression of these factors aids in rapid localization. Once inside, product degradation and undesired competitive substrate consumption were reduced through selective knockouts of key β-oxidation enzymes, such as MFE and POT1. Finally, factors affecting peroxisome proliferation are investigated in an effort to improve the size, number, and biogenesis of peroxisomes available for reaction. The conserved nature of the eukaryotic peroxisome should allow these modifications to benefit production in a broad range of species.