Abstract
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 characterized 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 is currently made by in vitro enzyme-based catalysis because there is no appropriate biological alternative to human milk fat. 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 (vs. 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 source of HMFS for infant formula.
Highlights
Human milk fat substitute (HMFS) is a class of structured lipid that is widely used as an ingredient in infant formulas
Using transient expression in Nicotiana benthamiana leaves, we found that when 101 amino acid residues containing the chloroplast targeting signal (CTS) are deleted from Brassica napus LPAT1 [17] (SI Appendix, Fig. S1) and replaced with a red fluorescent protein (RFP) marker, the RFP-ΔCTS-LPAT1 fusion protein localizes to the endoplasmic reticulum (ER) (Fig. 2A)
In this study we show that the TAG biosynthetic pathway in plants can be engineered so that the stereoisomeric structure of seed storage oil is altered to mimic that of human milk fat (HMF), with >70% of C16:0 concentrated at the middle position on the glycerol backbone
Summary
Human milk fat substitute (HMFS) is a class of structured lipid that is widely used as an ingredient in infant formulas. 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 (vs 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. The tension between price and quality is one factor that has likely restricted the use of HMFS and despite mounting clinical evidence that this ingredient is beneficial [1, 3, 6], it is currently only found in around 10% of infant formula, premium products formulated and marketed for ease-of-digestion Even in these products, there remains a substantial gap in C16:0 enrichment at the sn-2 position versus HMF [1]. The aim of this study was to explore whether the stereoisomeric structure of vegetable fat can be altered by iterative
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