Supplementary data on the characterization and safety evaluation of HPPD W336, a modified 4-hydroxyphenylpyruvate dioxygenase protein, which confers herbicide tolerance, and on the compositional assessment of field grown MST-FGØ72-2 soybean expressing HPPD W336.

Supplementary data are provided which are supportive to the research article entitled "Characterization and safety evaluation of HPPD W336, a modified 4-hydroxyphenylpyruvate dioxygenase protein, and the impact of its expression on plant metabolism in herbicide-tolerant MST-FGØ72-2 soybean" (Dreesen et al., 2018) [1]. The conducted supplementary analyses include the characterization of additional Escherichia coli-produced HPPD W336 protein batches used as a surrogate in HPPD W336 safety studies, the assessment of potential glycosylation and monitoring of stability in simulated intestinal fluid and during heating of the HPPD W336 protein. Furthermore, data are provided on conducted field trials and subsequent compositional analysis in MST-FGØ72-2 soybean grain of compounds related to the tyrosine degradation pathway and the metabolism of homogentisate.

Characterization and safety evaluation of HPPD W336, a modified 4-hydroxyphenylpyruvate dioxygenase protein, and the impact of its expression on plant metabolism in herbicide-tolerant MST-FGØ72-2 soybean.

By transgenic expression technology, a modified 4-hydroxyphenylpyruvate dioxygenase enzyme (HPPD W336) originating from Pseudomonas fluorescens is expressed in MST-FGØ72-2 soybean to confer tolerance to 4-benzoyl isoxazole and triketone type of herbicides. Characterization and safety assessment of HPPD W336 were performed. No relevant sequence homologies were found with known allergens or toxins. Although sequence identity to known toxins showed identity to HPPD proteins annotated as hemolysins, the absence of hemolytic activity of HPPD W336 was demonstrated in vitro. HPPD W336 degrades rapidly in simulated gastric fluid. The absence of toxicity and hemolytic potential of HPPD W336 was confirmed by in vivo studies. The substrate spectrum of HPPD W336 was compared with wild type HPPD proteins, demonstrating that its expression is unlikely to induce any metabolic shifts in soybean. The potential effect of expression of HPPD W336 on metabolic pathways related to tyrosine was investigated by comparing seed composition of MST-FGØ72-2 soybean with non-genetically modified varieties, demonstrating that expression of HPPD W336 does not change aromatic amino acid, homogentisate and tocochromanol levels. In conclusion, HPPD W336 was demonstrated to be as safe as other food proteins. No adverse metabolic effects were identified related to HPPD W336 expression in MST-FGØ72-2 soybean.

Safety evaluation of a lipase enzyme preparation, expressed in Pichia pastoris, intended for use in the degumming of edible vegetable oil.

BD16449 lipase is the product of a phospholipid-specific lipase gene expressed in the yeast Pichia pastoris strain DVSA-PLC-004. This type C phospholipid lipase (EC 3.1.4.3) is intended for use in the degumming of edible vegetable oil. BD16449 lipase was tested as a refined test article preparation (DV16449) for its effects on genotoxicity and in acute, inhalation, and subchronic toxicity studies. Dosages ranged from 5000 microg/plate for in vitro toxicity studies to 2000 mg/kg/day for in vivo toxicity studies. The highest oral dose tested in vivo (NOAEL of 2000 mg/kg/day) resulted in a safety margin of 133,000 based on the conservative estimate of the total human consumption of BD16449 lipase of 0.015 mg/kg/day. When adjusted for total organic solids (TOS), the highest oral dose tested in vivo (NOAEL of 1680 mg TOS/kg/day) resulted in a safety margin of 18,300 based on the conservative estimate of the total human consumption of BD16449 lipase of 0.092 mg TOS/kg/day [corrected] There was no toxicity reported for any of these studies including additional safety studies. A review of the literature indicates that P. pastoris fulfills recognized safety criteria pertinent to microbial production strains used in the manufacture of food enzyme preparations. The results of the toxicity studies presented herein attest to the safety of BD16449 lipase for use in the degumming of edible vegetable oil.

T-DNA recombination and replication in maize cells.

T-DNA recombination and replication was analyzed in 'black mexican sweet' (BMS) cells transformed with T-DNAs containing the replication system from wheat dwarf virus (WDV). Upon recombination between the T-DNA ends, a promoterless marker gene (gusA) was activated. Activation of the recombination marker gene was delayed and increased exponentially over time, suggesting that recombination and amplification of the T-DNA occurred in maize cells. Mutant versions of the viral initiator gene (rep), known to be defective in the replication function, failed to generate recoverable recombinant T-DNA molecules. Circularization of T-DNA by the FLP/FRT site-specific recombination system and/or homologous recombination was not necessary to recover circular T-DNAs. However, replicating T-DNAs appeared to be suitable substrates for site-specific and homologous recombination. Among 33 T-DNA border junctions sequenced, only one pair of identical junction sites was found implying that the population of circular T-DNAs was highly heterogenous. Since no circular T-DNA molecules were detected in treatments without rep, it suggested that T-DNA recombination was linked to replication and might have been stimulated by this process. The border junctions observed in recombinant T-DNA molecules were indicative of illegitimate recombination and were similar to left-border recombination of T-DNA into the genome after Agro-mediated plant transformation. However, recombination between T-DNA molecules differed from T-DNA/genomic DNA junction sites in that few intact right borders were observed. The replicating T-DNA molecules did not enhance genomic random integration of T-DNA in the experimental configuration used for this study.