Functional analysis of feedback inhibition-insensitive aspartate kinase identified in a threonine-accumulating mutant of Saccharomyces cerevisiae

• Published in: Applied and environmental microbiology Vol. 90; no. 4; p. e0015524
• Main Authors: Isogai, Shota, Nishimura, Akira, Inoue, Akiko, Sonohara, Shino, Tsugukuni, Takashi, Okada, Tomoyuki, Takagi, Hiroshi
• Format: Journal Article
• Language: English
• Published: United States American Society for Microbiology 17.04.2024
• Subjects: Accumulation; Aged; Amino acids; Animals; Appetite loss; Applied and Industrial Microbiology; Aspartate kinase; Aspartate Kinase - chemistry; Aspartate Kinase - genetics; Aspartate Kinase - metabolism; Diploids; Diploidy; Feedback; Feedback inhibition; Functional analysis; HOM3 gene; Homeostasis; Humans; Kinases; Life span; Mammals; Mutagenesis; Mutants; Nutrients; Older people; Physiology; Protein biosynthesis; Protein synthesis; Quality of Life; Saccharomyces cerevisiae; Saccharomyces cerevisiae - metabolism; Substitutes; Threonine; Yeast; Yeasts; allosteric regulation; aspartate kinase Hom3; Saccharomyces cerevisiae; threonine; yeast
• ISSN: 0099-2240; 1098-5336; 1098-5336
• DOI: 10.1128/aem.00155-24

Humans and mammals need to ingest essential amino acids (EAAs) for protein synthesis. In addition to their importance as nutrients, EAAs are involved in brain homeostasis. However, elderly people are unable to efficiently consume EAAs from their daily diet due to reduced appetite and variations in the contents of EAAs in foods. On the other hand, strains of the yeast that accumulate EAAs would enable elderly people to intakegest adequate amounts of EAAs and thus might slow down the neurodegenerative process, contributing to the extension of their healthy lifespan. In this study, we isolated a mutant (strain HNV-5) that accumulates threonine, an EAA, derived from a diploid laboratory yeast by conventional mutagenesis. Strain HNV-5 carries a novel mutation in the gene encoding the Ala462Thr variant of aspartate kinase (AK). Enzymatic analysis revealed that the Ala462Thr substitution significantly decreased the sensitivity of AK activity to threonine feedback inhibition even in the presence of 50 mM threonine. Interestingly, Ala462Thr substitution did not affect the catalytic ability of Hom3, in contrast to previously reported amino acid substitutions that resulted in reduced sensitivity to threonine feedback inhibition. Furthermore, yeast cells expressing the Ala462Thr variant showed an approximately threefold increase in intracellular threonine content compared to that of the wild-type Hom3. These findings will be useful for the development of threonine-accumulating yeast strains that may improve the quality of life in elderly people.IMPORTANCEFor humans and mammals, essential amino acids (EAAs) play an important role in maintaining brain function. Therefore, increasing the intake of EAAs by using strains of the yeast that accumulate EAAs may inhibit neurodegeneration in elderly people and thus contribute to extending healthy lifespan and improving their quality of life. Threonine, an EAA, is synthesized from aspartate. Aspartate kinase (AK) catalyzes the first step in threonine biosynthesis and is subject to allosteric regulation by threonine. Here, we isolated a threonine-accumulating mutant of by conventional mutagenesis and identified a mutant gene encoding a novel variant of AK. In contrast to previously isolated variants, the Hom3 variant exhibited AK activity that was insensitive to feedback inhibition by threonine but retained its catalytic ability. This resulted in increased production of threonine in yeast. These findings open up the possibility for the rational design of AK to increase threonine productivity in yeast.