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

Abstract

SummaryThreonine (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 feedbackresistant 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 XbA-K_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.

Transgenic soybean plants overexpressing O-acetylserine sulfhydrylase accumulate enhanced levels of cysteine and Bowman–Birk protease inhibitor in seeds

“…A recent study showed that seed-specific expression of the feedback-resistant variants of aspartate kinases could result in up to 100-fold increase in free threonine in transgenic soybean seeds (Qi et al 2011). Interestingly, these soybean plants also accumulated increased levels of methionine, lysine, and isoleucine resulting in an upto 3.5-fold increase in the total free amino acid content; however, this study did not examine if there was also any increase in the levels of protein-bound essential amino acids.…”

Crystal structure of Clostridium acetobutylicum aspartate kinase (CaAk): An important allosteric enzyme for amino acids production

“…This eventually helps to manipulating the biosynthesis of amino acid to increase the amino acid content and nutritive value in crops. Recently, much work has been done to metabolically engineered crops and grains with enhanced amino acid levels [42], [43]. Thirdly, the mechanism of structural transition to tetramer assembly is similar way to the other three different crystallographic environments.…”

Advancements in Transgenic Soy: From Field to Bedside

“…In this case, a >100-fold increase of free lysine and 5-fold increase of total seed lysine content was observed. More recently, soybeans showing enhanced threonine levels have been engineered using seed-specific expression of lysine-insensitive variants of aspartate kinases from bacteria [58]. This strategy produced transgenic soybeans with a 100-fold increase in threonine levels and 3.5-fold increase in total free amino acid content without negative impacts on seed morphology or germination.…”