Preliminary safety assessment of a membrane-bound delta 9 desaturase candidate protein for transgenic oilseed crops

AbstractA gene encoding delta 9 desaturase (D9DS), an integral membrane protein, is being considered for incorporation into oilseed crops to reduce saturated fatty acids and thus improve human nutritional value. Typically, a safety assessment for transgenic crops involves purifying heterologously produced transgenic proteins in an active form for use in safety studies. Membrane-bound proteins have been very difficult to isolate in an active form due to their inherent physicochemical properties. Described here are methods used to derive enriched preparations of the active D9DS protein for use in early stage safety studies. Results of these studies, in combination with bioinformatic results and knowledge of the mode of action of the protein, along with a history of safe consumption of related proteins, provides a weight of evidence supporting the safety of the D9DS protein in food and feed

Characterization of Aryloxyalkanoate Dioxygenase-12, a Nonheme Fe(II)/α-Ketoglutarate-Dependent Dioxygenase, Expressed in Transgenic Soybean and <i>Pseudomonas fluorescens</i>

Aryloxyalkanoate dioxygenase-12 (AAD-12) was discovered from the soil bacterium <i>Delftia acidovorans</i> MC1 and is a nonheme Fe­(II)/α-ketoglutarate-dependent dioxygenase, which can impart herbicide tolerance to transgenic plants by catalyzing the degradation of certain phenoxyacetate, pyridyloxyacetate, and aryloxyphenoxypropionate herbicides. The development of commercial herbicide-tolerant crops, in particular AAD-12-containing soybean, has prompted the need for large quantities of the enzyme for safety testing. To accomplish this, the enzyme was produced in <i>Pseudomonas fluorescens</i> (<i>Pf</i>) and purified to near homogeneity. A small amount of AAD-12 was partially purified from transgenic soybean and through various analytical, biochemical, and <i>in vitro</i> activity analyses demonstrated to be equivalent to the <i>Pf</i>-generated enzyme. Furthermore, results from <i>in vitro</i> kinetic analyses using a variety of plant endogenous compounds revealed activity with <i>trans</i>-cinnamate and indole-3-acetic acid (IAA). The catalytic efficiencies (<i>k</i>_cat/<i>K</i>_m) of AAD-12 using <i>trans</i>-cinnamate (51.5 M^–1 s^–1) and IAA (8.2 M^–1 s^–1) as substrates were very poor when compared to the efficiencies of plant endogenous enzymes. The results suggest that the presence of AAD-12 in transgenic soybean would not likely have an impact on major plant metabolic pathways