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

Piotr P Hapeta,Guillaume Menard,Govindprasad Bhutada,Rodrigo Ledesma-Amaro,Peter J Eastmond,Rupam Kumar Bhunia

doi:10.1016/j.mec.2022.e00192

Abstract

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 exclude palmitic acid from the sn-2 position. 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 lysophosphatidic acid acyltransferases with palmitoyl-Coenzyme A specificity. 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 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 be used as an ingredient in infant formulas.

Highlights

Human milk is the best source of nutrition for infants and it is their main food during the first four to six months of life (Innis 2011; Wei et al, 2019)
The fatty acids (FA) composition of TAG from WT and gsy1Δ-LPAAT2 cells grown on glucose (Fig. 3A) was similar with that of cells grown on glycerol (Fig. 2C) except that 18:0 levels were signifi cantly (P < 0.05) lower (~8% versus ~12%)
The change in TAG stereoisomeric struc ture, combined with the native FA composition found in Y. lipolytica cells cultured on glycerol or glucose, produces fat that is similar to human milk, in terms of the major molecular species of FAs (16:0, 18:1 and 18:2), and has the potential to be used as a human milk fat substitutes (HMFS) (β-palmitate) ingredient in infant formulas

Summary

Introduction

Human milk is the best source of nutrition for infants and it is their main food during the first four to six months of life (Innis 2011; Wei et al, 2019). Palmitic acid (16:0) is esterified to the middle (sn-2) position on the glycerol backbone and oleic acid (18:1) and linoleic acid (18:2) to the outer (sn-1/3) positions (Breckenridge et al, 1969; Christie and Clapperton, 1982; Giuffrida et al, 2019), giving the TAG molecules distinctive stereochemistry that clinical trials have sug gested can assist nutrient absorption in the infant gut (Innis 2011; Beghin et al, 2018) This is because sn-1/3 regioselective lipases hy drolyze TAG during digestion to produce fatty acids (FA) and 2-monoa cylglycerols (2-MAG). Mixtures of vegetable fats (plus algal and fish oils) can be blended to mimic the fatty acid (FA) composition of human milk, but not its stereoisomeric structure (Wei et al, 2019)

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Conclusion