Safety assessment of membrane-associated protoporphyrinogen IX oxidase from <i>Enterobacter cloacae</i> expressed in genetically modified crops

Crops tolerant to protoporphyrinogen IX oxidase (PPO)-inhibiting herbicides were developed by expressing an herbicide insensitive PPO, a membrane-associated protein from <i>Enterobacter cloacae</i>. <i>E. cloacae</i> is commonly found in the environment, and PPO is present in thousands of species, including probiotic organisms with a history of safe use. Comparisons with current allergen and protein toxin databases revealed no sequence similarities between PPO and known allergens or toxins. To validate the use of <i>Escherichia coli</i>-produced PPO in safety studies, physicochemical and functional characterization demonstrated that the PPO produced by genetically modified (GM) soybean has comparable immunoreactivity and functional activity to that produced from <i>E. coli</i>, with neither being glycosylated. PPO was fully digested after exposure to pepsin and pancreatin for 2 and 5 minutes, respectively, and its activity is completely lost at temperatures of 55°C or higher. Mice dosed orally with PPO at a level of 5000 mg protein per kg body weight showed no adverse effects, as indicated in body weight gains, food consumption, and clinical observations. This comprehensive safety assessment indicates that PPO protein from GM crops is safe for food and feed consumption. Additionally, we present methods to demonstrate the functional equivalence of a membrane-associated protein from <i>E. coli</i> and plant, along with a novel process for formulating PPO in gram quantities at 76.3 mg/ml.