Tri-Hydroxy-Triacylglycerol is Efficiently Produced by Position-Specific Castor Acyltransferases.

Understanding the biochemistry of triacylglycerol (TAG) assembly is critical in tailoring seed oils to produce high-value products. Hydroxy-fatty acid (HFA) is one such valuable modified fatty acid, which can be produced at low levels in Arabidopsis (Arabidopsis thaliana) seed through transgenic expression of the castor (Ricinus communis) hydroxylase. The resulting plants have low seed oil content and poor seedling establishment, indicating that Arabidopsis lacks efficient metabolic networks for biosynthesis and catabolism of hydroxy-containing TAG. To improve utilization of such substrates, we expressed three castor acyltransferase enzymes which incorporate HFA at each stereochemical position during TAG synthesis. This produced abundant tri-HFA TAG and concentrated 44% of seed HFA moieties into this one TAG species. Ricinoleic acid was more abundant than any other fatty acid in these seed, which had 3-fold more HFA by weight than that in seeds following simple hydroxylase expression, the highest yet measured in a non-native plant. Efficient utilization of hydroxy-containing lipid substrates increased the rate of TAG synthesis two-fold, leading to complete relief of the low oil phenotype. Partition of HFA into specific TAG molecules increased the storage lipid available for mobilization during seedling development, resulting in a 1.9-fold increase in seedling establishment. Expression of a complete acyltransferase pathway to efficiently process HFA establishes a benchmark in the quest to successfully produce modified oils in plants.