State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai 200237, China.
J Biotechnol. 2011 Jul 20;154(4):261-8. doi: 10.1016/j.jbiotec.2011.06.001. Epub 2011 Jun 12.
Glutathione (GSH) is one of the most ubiquitous non-protein thiols that is involved in numerous cellular activities. The gene coding for a novel bifunctional enzyme catalyzing the reaction for glutathione synthesis, gshF, was cloned from Streptococcus thermophilus SIIM B218 and expressed in Escherichia coli JM109. In the presence of the precursor amino acids and ATP, the induced cells of E. coli JM109 (pTrc99A-gshF) could accumulate 10.3 mM GSH in 5 h. The S. thermophilus GshF was insensitive to feedback inhibition caused by GSH even at 20 mM. At elevated concentrations of the precursor amino acids and ATP, E. coli JM109 (pTrc99A-gshF) produced 36 mM GSH with a molar yield of 0.9 mol/mol based on added cysteine and of 0.45 mol/mol based on added ATP. When ATP was replaced with glucose, E. coli JM109 (pTrc99A-gshF) produced 7 mM in 3h. Saccharomyces cerevisiae was used to generate ATP for GSH production. In the presence of glucose and the pmr1 mutant of S. cerevisiae BY4742, JM109 (pTrc99A-gshF) produced 33.9 mM GSH in 12h with a yield of 0.85 mol/mol based on added l-cysteine. It is shown that the S. thermophilus GshF can be successfully used for GSH production.
谷胱甘肽 (GSH) 是一种广泛存在的非蛋白巯基，参与多种细胞活动。来自嗜热链球菌 SIIM B218 的编码一种新的双功能酶的基因，该酶催化谷胱甘肽合成反应，被克隆并在大肠杆菌 JM109 中表达。在存在前体氨基酸和 ATP 的情况下，诱导的大肠杆菌 JM109 (pTrc99A-gshF) 细胞可以在 5 小时内积累 10.3 mM GSH。即使在 20 mM 的 GSH 浓度下，嗜热链球菌 GshF 也对 GSH 的反馈抑制不敏感。在高浓度的前体氨基酸和 ATP 下，大肠杆菌 JM109 (pTrc99A-gshF) 以摩尔产率 0.9 mol/mol（基于添加的半胱氨酸）和 0.45 mol/mol（基于添加的 ATP）产生 36 mM GSH。当用葡萄糖代替 ATP 时，大肠杆菌 JM109 (pTrc99A-gshF) 在 3 小时内产生 7 mM。酿酒酵母用于产生 ATP 以用于 GSH 生产。在葡萄糖和酿酒酵母 pmr1 突变体 BY4742 的存在下，JM109 (pTrc99A-gshF) 在 12 小时内产生 33.9 mM GSH，摩尔产率为 0.85 mol/mol（基于添加的 l-半胱氨酸）。结果表明，嗜热链球菌 GshF 可成功用于 GSH 生产。
