Kong Min, Wang Fengjuan, Tian Liuying, Tang Hui, Zhang Liping
Engineering Laboratory of Microbial Breeding and Preservation of Hebei Province; Key Laboratory of Microbial Diversity Research and Application of Hebei Province; College of Life Sciences, Hebei University, Baoding, 071002, China.
Naturwissenschaften. 2017 Dec 15;105(1-2):4. doi: 10.1007/s00114-017-1520-2.
Glutathione (GSH) fulfills a variety of metabolic functions, participates in oxidative stress response, and defends against toxic actions of heavy metals and xenobiotics. In this study, GSH was detected in Rhodosporidium diobovatum by high-performance liquid chromatography (HPLC). Then, two novel enzymes from R. diobovatum were characterized that convert glutamate, cysteine, and glycine into GSH. Based on reverse transcription PCR, we obtained the glutathione synthetase gene (GSH2), 1866 bp, coding for a 56.6-kDa protein, and the glutamate cysteine ligase gene (GSH1), 2469 bp, coding for a 90.5-kDa protein. The role of GSH1 and GSH2 for the biosynthesis of GSH in the marine yeast R. diobovatum was determined by deletions using the CRISPR-Cas9 nuclease system and enzymatic activity. These results also showed that GSH1 and GSH2 were involved in the production of GSH and are thus being potentially useful to engineer GSH pathways. Alternatively, pET-GSH constructed using vitro recombination could be used to detect the function of genes related to GSH biosynthesis. Finally, the fermentation parameters determined in the present study provide a reference for industrial GSH production in R. diobovatum.
谷胱甘肽（GSH）具有多种代谢功能，参与氧化应激反应，并抵御重金属和外源性物质的毒性作用。在本研究中，通过高效液相色谱法（HPLC）检测了卵形红酵母中的GSH。然后，对卵形红酵母中两种将谷氨酸、半胱氨酸和甘氨酸转化为GSH的新酶进行了表征。基于逆转录PCR，我们获得了1866 bp的谷胱甘肽合成酶基因（GSH2），其编码一个56.6 kDa的蛋白质，以及2469 bp的谷氨酸半胱氨酸连接酶基因（GSH1），其编码一个90.5 kDa的蛋白质。利用CRISPR-Cas9核酸酶系统通过缺失和酶活性确定了GSH1和GSH2在海洋酵母卵形红酵母中GSH生物合成中的作用。这些结果还表明，GSH1和GSH2参与了GSH的产生，因此可能有助于构建GSH途径。另外，利用体外重组构建的pET-GSH可用于检测与GSH生物合成相关基因的功能。最后，本研究确定的发酵参数为卵形红酵母中GSH的工业化生产提供了参考。
