Institute for Research Initiative, Nara Institute of Science and Technology, Takayama, Nara, Japan.
Plant Bio Business Unit, Musashi Seimitsu Industry Co., Ltd., Toyohashi, Aichi, Japan.
Appl Environ Microbiol. 2024 Apr 17;90(4):e0015524. doi: 10.1128/aem.00155-24. Epub 2024 Mar 8.
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.
人类和哺乳动物需要摄入必需氨基酸（EAA）来合成蛋白质。除了作为营养物质的重要性外，EAA 还参与大脑内环境的稳定。然而，由于食欲下降和食物中 EAA 含量的变化，老年人无法从日常饮食中有效地摄取 EAA。另一方面，积累 EAA 的酵母菌株可以使老年人摄入足够的 EAA，从而可能减缓神经退行性过程，有助于延长他们的健康寿命。在这项研究中，我们通过常规诱变从二倍体实验室酵母中分离出一种积累苏氨酸（EAA）的突变株（菌株 HNV-5）。菌株 HNV-5在编码天冬氨酸激酶（AK）Ala462Thr 变体的 基因中携带一个新的突变。酶分析表明，Ala462Thr 取代显着降低了 AK 活性对苏氨酸反馈抑制的敏感性，即使存在 50mM 苏氨酸也是如此。有趣的是，与先前报道的导致对苏氨酸反馈抑制的敏感性降低的氨基酸取代相反，Ala462Thr 取代不影响 Hom3 的催化能力。此外，与野生型 Hom3 相比，表达 Ala462Thr 变体的酵母细胞的细胞内苏氨酸含量增加了约三倍。这些发现将有助于开发可能改善老年人生活质量的积累苏氨酸的酵母菌株。
重要性
对于人类和哺乳动物而言，必需氨基酸（EAA）在维持大脑功能方面起着重要作用。因此，使用积累 EAA 的酵母菌株增加 EAA 的摄入量可能会抑制老年人的神经退行性变，从而有助于延长健康寿命并改善他们的生活质量。苏氨酸是一种 EAA，由天冬氨酸合成。天冬氨酸激酶（AK）催化苏氨酸生物合成的第一步，受苏氨酸的变构调节。在这里，我们通过常规诱变从 中分离出一种苏氨酸积累突变株，并鉴定出一种编码 AK 新型变体的突变基因。与以前分离的变体不同，Hom3 变体表现出对苏氨酸反馈抑制不敏感的 AK 活性，但保留了其催化能力。这导致酵母中天冬氨酸激酶的产量增加。这些发现为理性设计 AK 以提高酵母中天冬氨酸激酶的产量开辟了可能性。
