Engineering the stereoisomeric structure of seed oil to mimic human milk fat

Human milk fat substitute (HMFS) is a class of structured lipid that is widely used as an ingredient in infant formulas. Like human milk fat, HMFS is characterised by enrichment of palmitoyl (C16:0) groups specifically at the middle (sn-2 or β) position on the glycerol backbone, and there is evidence that triacylglycerol (TAG) with this unusual stereoisomeric structure provides nutritional benefits. HMFS production currently relies on enzyme-based catalysis since there is no appropriate biological source of fat with the equivalent structure, other than humans. Most of the fat currently used in infant formulas is obtained from plants, which exclude C16:0 from the middle position. In this study we have modified the metabolic pathway for TAG biosynthesis in the model oilseed Arabidopsis thaliana to increase the percentage of C16:0 at the middle (versus outer) positions by more than 20-fold (i.e. from ~3% in wild type to >70% in our final iteration). This level of C16:0 enrichment is comparable to human milk fat. We achieved this by relocating the C16:0-specific chloroplast isoform of the enzyme lysophosphatidic acid acyltransferase (LPAT) to the endoplasmic reticulum so that it functions within the cytosolic glycerolipid biosynthetic pathway to esterify C16:0 to the middle position. We then suppressed endogenous LPAT activity to relieve competition and knocked out phosphatidylcholine:diacylglycerol cholinephosphotransferase activity to promote the flux of newly-made diacylglycerol directly into TAG. Applying this technology to oilseed crops might provide a new source of HMFS for infant formula.