Engineering Autoinducible Lysis in the Oleaginous Yeast Yarrowia lipolytica

• Michael Machewirth, Chemical Engineering, University of Delaware

• Mark Blenner, Chemical Engineering, University of Delaware

Oleaginous (oil producing) yeasts offer sustainable means of producing commodity lipids such as food-based products, biofuels, and pharmaceuticals. Their products are backed by a rising market projected to reach over 800 million dollars by 2034. Yarrowia lipolytica is an oleaginous yeast with current lipid production titers of 115 g/L, demonstrated industrial production of fatty esters and organic acids, and the ability to use cheap feedstocks including crude glycerol and recycled cooking oil. Extracting intracellular lipids from Y. lipolytica accounts for 40-80% of the production costs due to difficulties lysing its thick, chitinous cell wall. Traditional lysis methods for oleaginous yeasts include bead beating and acid lysis, but these face challenges in cost and logistics for industrial scale-up. This project aims to create a low-cost, scalable, and efficient mechanism for release and recovery of intracellular lipids through the development of autoinducible cell lysis in Y. lipolytica. To achieve this, we are constructing strains to express toxins and other lysins under inducible promoters. We are validating toxins with the known copper inducible MT-2 promoter,  and validating dynamic range of induction with the fluorescent protein mturquoise and with known Y. lipolytica toxins HSK and HMK. Once inducer-lysin complexes are validated, we will scale-up lipid production from our strains in a bioreactor, determine lipid titers, and compare cost of lipid recovery with our autoinduction system to the standard bead-beating procedure. If successful, this project will identify autoinduction signals and lysins that establish cheap and energy-efficient autoinduction of lysis of Yarrowia lipolytica, economizing industrial fermentation processes and offering more optimal methods of yeast-derived lipid production.