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

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.

Red Fluorescent Protein (RFP) in the pK7WGR2 vector (Vlaams Institute for Biotechnology). The PCR product was cloned in the pENTR TM /D-TOPO TM vector (Thermo Fisher Scientific), sequenced (Fig. S1) and recombined into the pK7WGR2 vector using the Gateway TM LR Clonase TM II Enzyme mix (Thermo Fisher Scientific). ∆CTS-LPAT1 was cloned in the pBinGlyRed3 vector in between the soybean glycinin-1 (GLY) promoter and terminator for seed specific expression (3). ∆CTS-LPAT1 was PCR-amplified from the pENTR-D-TOPO vector using KOD DNA polymerase and primer pair P3+P4. The PCR product was gel purified and digested with EcoRI and XbaI. The pBinGlyRed3 vector was also digested with EcoRI and XbaI, alkaline phosphatase treated (Promega), gel purified and ∆CTS-LPAT1 was ligated into the vector using T4 DNA ligase (NEB). Heat shock was used to transform the vectors into Agrobacterium tumefaciens strain GV3101 and Arabidopsis transformation was then carried out using the floral-dip method (4). T1 seeds expressing the selectable marker were identified under a Leica M205 FA microscope using the DsRed filter.

Transient expression in Nicotiana benthamiana and imaging
Transient expression in Nicotiana benthamiana leaves was carried out as described by Wood et al., (5) using A. tumefaciens cultures transformed with vectors harbouring Pro35S:RFP-ΔCTS-LPAT1, Pro35S:m-GFP5-ER or Pro35S:p19. Cultures were hand-infiltrated into leaves and the inoculated plants were left for 48 h. N. benthamiana leaves were then mounted in water on a Zeiss LSM 780 laser scanning confocal microscope under an Apochromat 63x/1.20 W Korr M27 objective. GFP was excited at a wavelength of 488 nm and RFP at 561 nm. Filters with an emission band at 473-551 nm were used for detection.

Lipid analysis
Total lipids were extracted from seeds and seedlings and TAG was purified as described previously (6). TAG stereochemical analysis was performed by lipase digestion following the method described previously (7), except that 2-monoacylglycerols were separated by thin layer chromatography (Silica gel 60, 20 x 20 cm; Sigma-Aldrich/Merck) using hexane:diethylether:acetic acid (35:70:1.5, v/v/v) (8). Fatty acyl groups present in whole seeds and purified lipid fractions were trans-methylated and quantified by gas chromatography (GC) coupled to flame ionization detection, as described previously (9), using a 7890A GC system fitted with DB-23 columns (30 m x 0.25 mm i.d. x 0.25 µm) (Agilent Technologies). TAG and PC molecular species composition were analysed by high resolution / accurate mass (HR/AM) lipidomics (10-12) using a Vanquish -Q Exactive Plus UPLC-MS/MS system (Thermo Fisher Scientific). Work flow consisted of using total lipids purified at 3 µg/µl and diluted 1 in 100 in chloroform:methanol (1:1, v/v). Internal tripalmitin standard (0.857µM) was added and 20µl injected into the UPLC. Lipids were separated using a Accucore C18 (2.1 x 150 mm, 2.6 mm) column (Thermo Fisher Scientific) at 35 o C with autosampler tray temperature, 10 o C flow rate at 400 µl min -1 . Mobile phase: A = 10 mM ammonium formate in 50% acetonitrile + 0.1% formic acid, B = 2 mM ammonium formate in acetonitrile:propan-2-ol:water (10:88:2 v/v) + 0.02% formic acid. Elution gradient ran for 28 minutes from 35% B at start to 100% at 24 mins. Thermo Q Exactive HESI II probe conditions, sweep plate in use probe position in C. Conditions were adjusted for separate positive and negative runs, running samples in a single polarity resulted in more identifications. LC/MS at 140K resolution and data-independent HCD MS2 experiments (35K resolution) were performed in positive and negative ion modes. Full Scan @ 140,000 resolution m/z 150-1200 Top 15 most abundant MS/MS @ 35,000 resolution using an isolation window of 1 m/z, maximum integration time of 75 ms and dynamic exclusion window of 8s. The stepped collision energy was 25, 30, 40 eV replacing 25 with 30 eV negative ion mode. Sheath gas set to 60, Aux gas 20, sweep gas 1 spray voltage 3.2 KV in positive ion mode with small adjustments in negative ion mode, capillary temperature 320 and aux gas heater set to 370 o C. LipidSearch 4.2 experimental workflow (Thermo Fisher Scientific) was used for lipid characterization and potential lipid species were identified separately from positive or negative ion adducts. The data for each biological replicate were aligned within a chromatographic time window by combining the positive and negative ion annotations and merging these into a single lipid annotation.

Seed germination and establishment assays
Around 50 seeds from each plant were surface sterilized, sown on a ½ MS agar plate, stratified at 4°C for two days and transferred to a growth chamber set to 20 o C or 10 o C, 16h light/8h dark, PPFD = 150 µmol m -2 s -1 . Germination (radicle emergence), expanded cotyledons and expanded true leaves were scored visually under a dissecting stereomicroscope. Seeds and seedlings were also collected at zero and four days after stratification for lipid analysis.

Statistical analysis
All experiments were carried out using between three and six biological replicates and the data are presented as the mean values ± standard error of the mean (SE). For statistical analysis we either used one-way analysis of variance (ANOVA) with post-hoc Tukey HSD (Honestly Significant Difference) tests, or two-tailed Student's t-tests.