Expression of Umbelopsis ramanniana DGAT2A in seed increases oil in soybean.

Oilseeds are the main source of lipids used in both food and biofuels. The growing demand for vegetable oil has focused research toward increasing the amount of this valuable component in oilseed crops. Globally, soybean (Glycine max) is one of the most important oilseed crops grown, contributing about 30% of the vegetable oil used for food, feed, and industrial applications. Breeding efforts in soy have shown that multiple loci contribute to the final content of oil and protein stored in seeds. Genetically, the levels of these two storage products appear to be inversely correlated with an increase in oil coming at the expense of protein and vice versa. One way to overcome the linkage between oil and protein is to introduce a transgene that can specifically modulate one pathway without disrupting the other. We describe the first, to our knowledge, transgenic soy crop with increased oil that shows no major impact on protein content or yield. This was achieved by expressing a codon-optimized version of a diacylglycerol acyltransferase 2A from the soil fungus Umbelopsis (formerly Mortierella) ramanniana in soybean seed during development, resulting in an absolute increase in oil of 1.5% (by weight) in the mature seed.

Elevation of seed alpha-tocopherol levels using plant-based transcription factors targeted to an endogenous locus.

Synthetic zinc finger transcription factors (ZFP-TFs) were designed to upregulate the expression of the endogenous Arabidopsis gamma-tocopherol methyltransferase (GMT) gene. This gene encodes the enzyme responsible for the conversion of gamma-tocopherol to alpha-tocopherol, the tocopherol species with the highest vitamin E activity. Five three-finger zinc finger protein (ZFP) DNA binding domains were constructed and proven to bind tightly to 9 bp DNA sequences located in either the promoter or coding region of the GMT gene. When these ZFPs were fused to a nuclear localization signal and the maize C1 activation domain, four of the five resulting ZFP-TFs were able to upregulate the expression of the GMT gene in leaf protoplast transient assays. Seed-specific expression of these ZFP-TFs in transgenic Arabidopsis produced several lines with a heritable elevation in seed alpha-tocopherol. These results demonstrate that engineered ZFP-TFs comprised of plant-derived elements are capable of modulating the expression of endogenous genes in plants.

Engineering vitamin E content: from Arabidopsis mutant to soy oil.

We report the identification and biotechnological utility of a plant gene encoding the tocopherol (vitamin E) biosynthetic enzyme 2-methyl-6-phytylbenzoquinol methyltransferase. This gene was identified by map-based cloning of the Arabidopsis mutation vitamin E pathway gene3-1 (vte3-1), which causes increased accumulation of delta-tocopherol and decreased gamma-tocopherol in the seed. Enzyme assays of recombinant protein supported the hypothesis that At-VTE3 encodes a 2-methyl-6-phytylbenzoquinol methyltransferase. Seed-specific expression of At-VTE3 in transgenic soybean reduced seed delta-tocopherol from 20 to 2%. These results confirm that At-VTE3 protein catalyzes the methylation of 2-methyl-6-phytylbenzoquinol in planta and show the utility of this gene in altering soybean tocopherol composition. When At-VTE3 was coexpressed with At-VTE4 (gamma-tocopherol methyltransferase) in soybean, the seed accumulated to >95% alpha-tocopherol, a dramatic change from the normal 10%, resulting in a greater than eightfold increase of alpha-tocopherol and an up to fivefold increase in seed vitamin E activity. These findings demonstrate the utility of a gene identified in Arabidopsis to alter the tocopherol composition of commercial seed oils, a result with both nutritional and food quality implications.