PNNL

Abstract

Yarrowia lipolytica is an ascomycete yeast used in biotechnological research for its abilities to secrete high concentrations of proteins and accumulate lipids. We have developed a set of genetic and molecular tools in order to expand capabilities of Y. lipolytica for both biological research and industrial bioengineering applications. In this work, we generated a set of isogenic auxotrophic strains with decreased non-homologous recombination for targeted DNA incorporation. Genome sequencing, assembly, and annotation of this genetic background uncovers previously unidentified genes in Y. lipolytica. To complement these strains we constructed plasmids with Yarrowia-optimized superfolderGFP for targeted overexpression and fluorescent tagging. We used these constructs to build the “Yarrowia lipolytica Cell Atlas”, a collection of strains with endogenous fluorescently tagged organelles in the same genetic background, in order to define organelle morphology in live cells. These tools are useful for conditional assessment of organelle-specific protein expression, and for overexpression of lipid biosynthetic enzymes to determine cellular localization in Y. lipolytica. This work provides the Yarrowia community with tools for cell biology and metabolism research in Y. lipolytica for further development of biofuels and natural products.