Significance In human milk fat, saturated fatty acids are esterified to the middle position on the glycerol backbone giving the triacylglycerol molecules an unusual stereochemistry that assists nutrient absorption in the infant gut. However, the fat used in most infant formulas is derived from plants, which esterify saturated fatty acids to the outer positions. Here, we have engineered the metabolism of an oilseed plant so that it accumulates triacylglycerol with more than 70% of the saturated fatty acid palmitate in the middle position, thereby mimicking human milk fat stereoisomeric structure. Applying this technology to oilseed crops (or oleaginous microorganisms) might provide a source of human milk fat substitute for infant nutrition.

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.

Infant formula is a manufactured food designed to substitute for human breast milk. Around half the calories in human milk are provided by fat (triacylglycerol; TAG) and in infant formula this fat is mainly sourced from plants (1). Although blended vegetable fats can replicate the fatty acyl composition of human milk fat (HMF), which mainly comprises palmitate (C16:0) and oleate (C18:1), the arrangement of acyl groups esterified to the glycerol backbone (i.e., the stereoisomeric structure) is profoundly different (2, 3). In vegetable fats, saturated long-chain fatty acyl groups such as C16:0 occupy the outer stereospecific numbering (sn) positions (sn-1/3) and are virtually excluded from the middle (sn-2 or β) position (4, 5); whereas in HMF, more than 70% of the C16:0 is present at the sn-2 position, with unsaturated fatty acyl groups (mainly C18:1) occupying the outer sn-1/3 positions (2, 3).

Multiple clinical trials on infants have suggested that the unusual stereoisomeric structure of HMF is important for nutrient absorption in the neonatal gut (1, 3, 6). The proposed mechanism is as follows. During the intestinal phase of digestion, lipases attack ingested fat at the sn-1/3 positions yielding 2-monoacylglycerols, which are easily absorbed (1, 3, 6). When unsaturated fatty acids are released from sn-1/3 positions, they are also absorbed easily, but release of long-chain saturated fatty acids such as C16:0 presents a problem. Their melting point is higher than body temperature and, at intestinal pH, they are prone to form hydrated fatty acid soaps with minerals such as calcium and magnesium (1, 3, 6). The arrangement of C16:0 at the sn-1/3 positions of vegetable fats thus means that they are more poorly absorbed than HMF (1, 3). There is evidence that the formation of C16:0 soaps also reduces calcium absorption, thus impairing early bone development, and accumulation of these soaps in the intestine also disrupts transit, causing infants discomfort (1, 3, 6).

To mimic the stereoisomeric structure of HMF, several companies have developed HMF substitutes (HMFS) (1). HMFS are made by enzyme-catalyzed acidolysis (or alcoholysis and esterification) using tripalmitin, unsaturated free fatty acids (mainly C18:1) together with an immobilized recombinant sn-1/3-regioselective lipase (1). The price of HMFS is substantially higher than that of conventional vegetable fat blends, primarily reflecting the added cost of enzyme-based catalysis, which also generates organic solvent waste (7). Different grades of HMFS are also available, providing a complete fat phase with between ∼40 and ∼70% of C16:0 at the sn-2 position. True HMF mimetics (with >70% of C16:0 at sn-2) are most expensive to produce because they require a 2-step catalytic process and a pure tripalmitin feedstock derived from palm oil by special fractionation procedures and chemical randomization (1, 7). 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, particularly 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 metabolic engineering, so that it mimics HMF. To our knowledge, no land plant (Embryophyta) produces TAG enriched in C16:0 at the sn-2 (verses sn-1/3 positions) and C16:0 is largely excluded from this position in virtually all cases (4, 5, 8). Even in palm oil that contains ∼48% C16:0 in total, only 9% of this occupies the sn-2 position (5). Here, we describe a method for modifying TAG biosynthesis, in the model oilseed Arabidopsis thaliana, that results in a stereoisomeric redistribution of acyl groups such that the amount of C16:0 at the sn-2 position increases more than 20-fold to over 70% of the total; a level of enrichment that is comparable to HMF. Applying this technology to oilseed crops might provide a source of HMFS for infant formula.

Conclusions 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 HMF, with >70% of C16:0 concentrated at the middle (sn-2 or β) position on the glycerol backbone. There is mounting evidence that this configuration is beneficial for infant nutrition (1, 3, 6), but it has not been found to occur naturally in vegetable fats where C16:0 is virtually excluded from the sn-2 position (4, 5, 9). Many infant formulas contain HMFS that are made by restructuring vegetable fats using enzyme-based catalysis, but they are relatively costly to produce; particularly for the manufacture of true mimetics with >70% of C16:0 at the sn-2 position (1, 7). Translation of our technology from the model species Arabidopsis to an oilseed crop might conceivably provide a cheaper and more sustainable source of HMFS for infant formula, but further research would be required to test this supposition. If HMFS could be obtained directly from a vegetable source, this would abrogate the need for enzyme-based catalysis. The infant formula market is currently estimated to use nearly half a million metric tons of vegetable-derived fat per year. Several oilseed crops may be considered as possible hosts for HMFS production, and it is noteworthy that conventional sunflower (Helianthus annus) and genetically modified oilseed rape (B. napus) varieties have already been developed that have the appropriate fatty acyl composition (37, 38). Even an oilseed crop with more modest C16:0 enrichment at the sn-2 position than we have achieved here may still be desirable since clinical trials have reported benefits with as little as 43% of C16:0 at the sn-2 position (1, 3, 6) and product surveys have found that this level of enrichment is common in infant formulas that are supplemented with HMFS (1).

Materials and Methods Detailed descriptions of plant material and growth conditions, cloning, and Agrobacterium-mediated transformation, microscopy, mutant genotyping, lipid analysis, qRT-PCR analysis of gene expression, germination and seedling establishment assays, and statistical analysis are provided in SI Appendix, SI Materials and Methods. Primers used are listed in SI Appendix, Table S5.

Acknowledgments We thank Prof. John Browse for pdct seeds and Prof. Edgar Cahoon for the pBinGlyRed3 vector. This work was funded by the UK Biotechnology and Biological Sciences Research Council through Grant BB/P012663/1.

Footnotes Author contributions: P.J.E. designed research; H.v.E., F.M.B., J.M.-M., L.V.M., G.B., and P.J.E. performed research; H.v.E., L.V.M., and P.J.E. analyzed data; and H.v.E. and P.J.E. wrote the paper.

The authors declare no competing interest.

This article is a PNAS Direct Submission.

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