Metabolically engineered soybean seed with enhanced threonine levels: biochemical characterization and seed-specific expression of lysine-insensitive variants of aspartate kinases from the enteric bacterium Xenorhabdus bovienii.

Threonine (Thr) is one of a few limiting essential amino acids (EAAs) in the animal feed industry, and its level in feed rations can impact production of important meat sources, such as swine and poultry. Threonine as well as EAAs lysine (Lys) and methionine (Met) are all synthesized via the aspartate family pathway. Here, we report a successful strategy to produce high free threonine soybean seed via identification of a feedback-resistant aspartate kinase (AK) enzyme that can be over-expressed in developing soybean seed. Towards this goal, we have purified and biochemically characterized AK from the enteric bacterium Xenorhabdus bovienii (Xb). Site-directed mutagenesis of XbAK identified two key regulatory residues Glu-257 and Thr-359 involved in lysine inhibition. Three feedback-resistant alleles, XbAK_T359I, XbAK_E257K and XbAK_E257K/T359I, have been generated. This study is the first to kinetically characterize the XbAK enzyme and provide biochemical and transgenic evidence that Glu-257 near the catalytic site is a critical residue for the allosteric regulation of AK. Furthermore, seed-specific expression of the feedback-resistant XbAK_T359I or XbAK_E257K allele results in increases of free Thr levels of up to 100-fold in R(1) soybean seed when compared to wild-type. Expression of feedback-sensitive wild-type AK did not substantially impact seed Thr content. In addition to high Thr, transgenic seed also showed substantial increases in other major free amino acid (FAA) levels, resulting in an up to 3.5-fold increase in the total FAA content. The transgenic seed was normal in appearance and germinated well under greenhouse conditions.