Production of human milk fat substitute by engineered strains of Yarrowia lipolytica

Human milk fat has a distinctive stereoisomeric structure where palmitic acid is esterified to the middle (sn-2) position on the glycerol backbone of the triacylglycerol and unsaturated fatty acids to the outer (sn-1/3) positions. This configuration allows for more efficient nutrient absorption in the infant gut. However, the fat used in most infant formulas originates from plants, which tend only to esterify palmitic acid to the sn-1/3 positions. Oleaginous yeasts provide an alternative source of lipids for human nutrition. However, these yeasts also exclude palmitic acid from the sn-2 position of their triacylglycerol. Here we show that Yarrowia lipolytica can be engineered to produce triacylglycerol with more than 60% of the palmitic acid in the sn-2 position, by expression of a lysophosphatidic acid phosphatase with palmitoyl-Coenzyme A specificity, such as LPAAT2 from Chlamydomonas reinhardtii. The engineered Y. lipolytica strains can be cultured on glycerol, glucose, palm oil or a mixture of substrates, under nitrogen limited condition, to produce triacylglycerol with a fatty acid composition that resembles human milk fat, in terms of the major molecular species; palmitic, oleic and linoleic acids. Culture on palm oil or a mixture of glucose and palm oil produced the highest lipid titre in shake flask culture and a triacylglycerol composition that is most similar with human milk fat. Our data show that an oleaginous yeast can be engineered to produce a human milk fat substitute (β-palmitate), that could potentially be used as an ingredient in infant formulas.