Enhanced Production of Lipids from Glycerol By Engineered Yeast Yarrowia Lipolytica

Glycerol is an important renewable feedstock as it is the main side-product of the biodiesel production process, which is nowadays applied on a large commercial scale. The world biodiesel market is predicted to reach a 37 billion gallons by 2016. It was estimated that approximately 1 kg of crude glycerol is generated for every 10 kg of biodiesel produced. Furthermore, glycerol is produced by several other industries, such as fat saponification and alcoholic beverage production units. Taking into consideration the huge quantities of glycerol produced by above mentioned industries and its low costs, it is of urgency to find an alternative ways to convert this substrate into value added products. Unfortunately, crude glycerol may contains many impurities that significantly decrease its value and the purification process of crude glycerol is time and energy consuming. Despite the high contamination, crude glycerol might be easily utilized by yeast Yarrowia lipolytica, a well-known oleaginous yeast. Y. lipolytica is one of the most extensively studied “non-conventional” yeasts due to its biotechnological potential. This unconventional yeast, which has been classified as a GRAS organism, is able to metabolize this renewable feedstock and has a huge biotechnological potential to produce citric acid and other organic acids (pyruvic, α-ketoglutaric), single cell oils (SCO), erythritol, mannitol, coca-butter-like lipid or proteins. To enhance production of SCO from glycerol we over-expressed the GUT1 (YALI0F00484g) gene coding GK and to direct carbon flow into lipid production additional we over-expressed the SCT1 (YALI0C00209g) gene encoding G3P- acyltransferase. The modified strains have been tested for glycerol consumption rate, lipid content, fatty acid composition and biomass production. Subsequently, to improve the SCO production, we additionally over-expressed DGA1 (YALI0E32769g) gene encoding DAG-acyltransferase involved in the last step of triglycerides synthesis. All genes were under control of a TEF promoter and enhanced TEF promoter (with 16 times repeated upstream activation sequence). Finally, we achieved strain of Y. lipolytica with enhanced production of lipids from glycerol.

This work was financially supported by the Ministry of Science and Higher Education of Poland – project no. IP2012 008972