The Key Laboratory of Systematic Mycology and Lichenology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China.
Fungal Genet Biol. 2012 Feb;49(2):114-22. doi: 10.1016/j.fgb.2011.12.004. Epub 2011 Dec 19.
Glutathione is a ubiquitous thiol in eukaryotic cells, and its high intracellular ratio of reduced form (GSH) to oxidized form (GSSG) is largely maintained by glutathione reductase (GR) using NADPH as electron donor. glrA, a glutathione reductase encoding gene, was found and cloned from Acremonium chrysogenum by searching its genomic sequence based on similarity. Its deduced protein exhibits high similarity to GRs of other eukaryotic organisms. Disruption of glrA resulted in lack of GR activity and accumulation of a high level of GSSG in A. chrysogenum. Overexpression of glrA dramatically enhanced GR activity and the ratio of GSH/GSSG in this fungus. The spore germination and hyphal growth of glrA disruption mutant was strongly reduced in chemical defined medium. Meanwhile, the mutant was more sensitive to hydrogen peroxide than the wild-type strain. We found that the glrA mutant recovered normal germination and growth by adding exogenous methionine (Met). Exogenous Met also enhanced the antioxidative ability of both the mutant and wild-type strain. GSH determination indicated that the total GSH and ratio of GSH/GSSG in the mutant or wild-type strain were significantly increased when addition of Met into the medium. The glrA mutant grew poorly and could not produce detectable cephalosporin in the fermentation medium without Met. However, its growth and cephalosporin production was restored with addition of exogenous Met. These results indicate that glrA is required for the normal growth and protection against oxidative damage in A. chrysogenum, and its absence can be complemented by exogenous Met.
谷胱甘肽是真核细胞中普遍存在的巯基化合物，其还原形式（GSH）与氧化形式（GSSG）的高细胞内比例主要通过谷胱甘肽还原酶（GR）利用 NADPH 作为电子供体来维持。通过基于相似性搜索其基因组序列，从产黄青霉中发现并克隆了一个编码谷胱甘肽还原酶的基因 glrA。其推导的蛋白质与其他真核生物的 GR 具有高度相似性。glrA 缺失导致 GR 活性缺失和 GSSG 在产黄青霉中积累水平升高。glrA 的过表达显著增强了该真菌中的 GR 活性和 GSH/GSSG 比值。glrA 缺失突变体在化学限定培养基中的孢子萌发和菌丝生长受到强烈抑制。同时，该突变体对过氧化氢比野生型菌株更敏感。我们发现 glrA 突变体通过添加外源性蛋氨酸（Met）可以恢复正常的萌发和生长。外源性 Met 还增强了突变体和野生型菌株的抗氧化能力。GSH 测定表明，当向培养基中添加 Met 时，突变体或野生型菌株的总 GSH 和 GSH/GSSG 比值均显著增加。在不含 Met 的发酵培养基中，glrA 突变体生长不良且不能产生可检测的头孢菌素。然而，通过添加外源性 Met，其生长和头孢菌素的产生得到恢复。这些结果表明，glrA 是产黄青霉正常生长和抵御氧化损伤所必需的，其缺失可以通过外源性 Met 来补充。
