Matsuhisa Kazuho, Ogawa Katsuhiro, Komata Kento, Hirasawa Takashi
School of Life Science and Technology, Institute of Science Tokyo, 4259 Nagatsuta-cho, Midori-Ku, Yokohama, Kanagawa, 226-8501, Japan.
Appl Microbiol Biotechnol. 2025 Mar 10;109(1):61. doi: 10.1007/s00253-025-13444-y.
Cysteine, a common amino acid used in food, cosmetic, and pharmaceutical industries, has a growth inhibitory effect. This growth inhibition by cysteine poses a problem, as the production of cysteine using microbial cells results in decreased cell growth and cysteine productivity. The underlying mechanism of growth inhibition by cysteine is unclear. This study aims to understand the mechanism of growth inhibition by cysteine in Corynebacterium glutamicum. To do this, cysteine-resistant mutants of C. glutamicum were isolated based on adaptive laboratory evolution (ALE) and their characteristics were analyzed. Genome resequencing revealed that mutations in the open reading frame of the ilvN gene encoding the regulatory small subunit of acetohydroxyacid synthase (AHAS), which is involved in branched-chain amino acid biosynthesis, were found in ALE cell populations and the isolated cysteine-resistant mutants. The ilvN mutations which are responsible for increased valine production resulted in improved cell growth in the presence of cysteine. Moreover, the addition of valine to the culture medium mitigated growth inhibition by cysteine, whereas the addition of leucine and isoleucine showed a slight mitigation. Additionally, the activity of AHAS from C. glutamicum was inhibited by cysteine, whereas AHAS from the strains carrying ilvN mutations exhibited resistance to cysteine. These results indicate that growth inhibition by cysteine is caused by perturbations in the biosynthesis of branched-chain amino acids, particularly valine in C. glutamicum. Furthermore, the cysteine-resistant mutants obtained by ALE demonstrated enhanced cysteine production as production hosts, suggesting that cysteine resistance is a useful phenotype for cysteine production in C. glutamicum. KEY POINTS: • Cysteine-resistant mutants of C. glutamicum obtained by ALE were analyzed. • Perturbation of valine biosynthesis by cysteine results in growth inhibition in C. glutamicum. • Cysteine resistance is a useful phenotype for cysteine production by C. glutamicum.
半胱氨酸是食品、化妆品和制药行业常用的一种氨基酸，具有生长抑制作用。半胱氨酸引起的这种生长抑制带来了一个问题，因为利用微生物细胞生产半胱氨酸会导致细胞生长和半胱氨酸生产力下降。半胱氨酸生长抑制的潜在机制尚不清楚。本研究旨在了解谷氨酸棒杆菌中半胱氨酸生长抑制的机制。为此，基于适应性实验室进化（ALE）分离了谷氨酸棒杆菌的半胱氨酸抗性突变体，并分析了它们的特性。基因组重测序显示，在ALE细胞群体和分离出的半胱氨酸抗性突变体中，发现参与支链氨基酸生物合成的乙酰羟酸合酶（AHAS）调节小亚基的ilvN基因开放阅读框发生了突变。导致缬氨酸产量增加的ilvN突变使细胞在半胱氨酸存在下的生长得到改善。此外，向培养基中添加缬氨酸可减轻半胱氨酸对生长的抑制作用，而添加亮氨酸和异亮氨酸则显示出轻微的缓解作用。此外，谷氨酸棒杆菌的AHAS活性受到半胱氨酸的抑制，而携带ilvN突变的菌株的AHAS对半胱氨酸具有抗性。这些结果表明，半胱氨酸对生长的抑制是由支链氨基酸生物合成的扰动引起 的，特别是谷氨酸棒杆菌中的缬氨酸。此外，通过ALE获得的半胱氨酸抗性突变体作为生产宿主表现出增强的半胱氨酸生产能力，这表明半胱氨酸抗性是谷氨酸棒杆菌生产半胱氨酸的一种有用表型。要点：• 分析了通过ALE获得的谷氨酸棒杆菌半胱氨酸抗性突变体。• 半胱氨酸对缬氨酸生物合成的扰动导致谷氨酸棒杆菌生长受到抑制。• 半胱氨酸抗性是谷氨酸棒杆菌生产半胱氨酸的一种有用表型。
