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Soybean seeds produce oil enriched in oxidatively unstable polyunsaturated fatty acids (PUFAs) and are also a potential biotechnological platform for synthesis of oils with nutritional omega-3 PUFAs. In this study, we engineered soybeans for seed-specific expression of a barley homogentisate geranylgeranyl transferase (HGGT) transgene alone and with a soybean γ-tocopherol methyltransferase (γ-TMT) transgene. Seeds for HGGT-expressing lines had 8- to 10-fold increases in total vitamin E tocochromanols, principally as tocotrienols, with little effect on seed oil or protein concentrations. Tocochromanols were primarily in δ- and γ-forms, which were shifted largely to α- and β-tocochromanols with γ-TMT co-expression. We tested whether oxidative stability of conventional or PUFA-enhanced soybean oil could be improved by metabolic engineering for increased vitamin E antioxidants. Selected lines were crossed with a stearidonic acid (SDA, 18:4 Δ6,9,12,15 )-producing line, resulting in progeny with oil enriched in SDA and α- or γ-linoleic acid (ALA, 18:3 Δ9,12,15 or GLA, 18:3 Δ6,9,12 ), from transgene segregation. Oil extracted from HGGT-expressing lines had ≥6-fold increase in free radical scavenging activity compared to controls. However, the oxidative stability index of oil from vitamin E-enhanced lines was ~15% lower than that of oil from non-engineered seeds and nearly the same or modestly increased in oil from the GLA, ALA and SDA backgrounds relative to controls. These findings show that soybean is an effective platform for producing high levels of free-radical scavenging vitamin E antioxidants, but this trait may have negative effects on oxidative stability of conventional oil or only modest improvement of the oxidative stability of PUFA-enhanced oil., (Copyright © 2019 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)

Human alpha-tocopherol transfer protein (alpha-TTP) plays a central role in vitamin E homeostasis: mutations in the protein are a cause of a progressive neurodegenerative disorder known as ataxia with vitamin E deficiency (AVED). Despite normal dietary intake of vitamin E, affected individuals suffer from a relative deficiency of this essential lipophilic antioxidant. Disease-associated mutations in alpha-TTP impair its ability to prevent the degradation and excretion of alpha-T. Recently, we and others solved the crystal structures of alpha-TTP bound to a molecule of (2R, 4'R, 8'R)-alpha-T, which has led to a better understanding of the molecular basis of its biochemical activity. Surprisingly, the ligand was found buried in the hydrophobic core of the protein, completely sequestered from the aqueous milieu. In this chapter, the implications of the structure of alpha-TTP bound to its ligand regarding the mechanism of alpha-T retention are discussed. A comparison to a crystal structure of the apo form of alpha-TTP indicates a possible specific conformational change that allows the entry and exit of the ligand. The effect of known disease-associated point mutations is examined in light of the crystal structure as well as recent biochemical studies. Despite the knowledge gained from these studies, the exact molecular mechanism by which alpha-TTP retains alpha-T remains enigmatic and will likely prove a fruitful area for future research.