Lysine biofortification in rice by modulating feedback inhibition of aspartate kinase and dihydrodipicolinate synthase

Abstract

Summary Lysine is the main limiting essential amino acid (EAA) in the rice seeds, which is a major energy and nutrition source for humans and livestock. In higher plants, the rate‐limiting steps in lysine biosynthesis pathway are catalysed by two key enzymes, aspartate kinase (AK) and dihydrodipicolinate synthase (DHDPS), and both are extremely sensitive to feedback inhibition by lysine. In this study, two rice AK mutants (AK1 and AK2) and five DHDPS mutants (DHDPS1–DHDPS5), all single amino acid substitution, were constructed. Their protein sequences passed an allergic sequence‐based homology alignment. Mutant proteins were recombinantly expressed in Escherichia coli, and all were insensitive to the lysine analog S‐(2‐aminoethyl)‐l‐cysteine (AEC) at concentrations up to 12 mm. The AK and DHDPS mutants were transformed into rice, and free lysine was elevated in mature seeds of transgenic plants, especially those expressing AK2 or DHDPS1, 6.6‐fold and 21.7‐fold higher than the wild‐type (WT) rice, respectively. We then engineered 35A2D1L plants by simultaneously expressing modified AK2 and DHDPS1, and inhibiting rice LKR/SDH (lysine ketoglutaric acid reductase/saccharopine dehydropine dehydrogenase). Free lysine levels in two 35A2D1L transgenic lines were 58.5‐fold and 39.2‐fold higher than in WT and transgenic rice containing native AK and DHDPS, respectively. Total free amino acid and total protein content were also elevated in 35A2D1L transgenic rice. Additionally, agronomic performance analysis indicated that transgenic lines exhibited normal plant growth, development and seed appearance comparable to WT plants. Thus, AK and DHDPS mutants may be used to improve the nutritional quality of rice and other cereal grains.

Comparative Transcriptome and Proteome Analysis Provides New Insights Into the Mechanism of Protein Synthesis in Kenaf (Hibiscus cannabinus L.) Leaves

“…Amino acids can also play the role of intermediates of final metabolites in some metabolic pathways and be engaged in regulating various metabolic pathways and other physiological and biochemical pathways, thereby influencing protein synthesis. The Aspartic acid (Asp) participates in protein synthesis and provides raw materials for protein synthesis ( Yang et al, 2020 ).…”

Mining of Potential Gene Resources for Breeding Nutritionally Improved Maize

“…Aspartate kinase (AK) catalyzes the first step, which is a rate-limiting step that requires ATP, and this step is also regulated by subsequent steps in a feedback manner. Single amino acid substitution mutants of ak are insensitive to the feedback inhibition of lysine synthesis and elevate lysine content [ 52 ]. Asparaginyl-tRNA synthetase (SYNC) mediates the process of linking amino acids to tRNAs, and over-accumulation of the SYNC has been shown to increase lysine levels [ 53 ].…”

Biofortification of Cereals and Pulses Using New Breeding Techniques: Current and Future Perspectives

“…RNAi-based repression of AK and DHDPS enzymes was carried out in rice and resultantly 6.6-and 21.7-fold more lysine was accumulated in mutant lines, respectively. When both mutations were combined in one genotype, the lysine level was 58.5-fold more in wild types (130). Maize endosperm is deficient in provitamin A and β-carotene leading to vitamin A deficiency in masses.…”