产谷胱甘肽毕赤酵母工程菌的构建及能量调控

Construction and energy regulation of engineered glutathione-producing Pichia pastoris

谷胱甘肽(glutathione,GSH)是生物体内重要非编码且含有巯基的三肽类物质,具有调节和保护等功能,在医药、食品等领域有着广泛的应用。目前,工业上主要通过高密度发酵生产GSH,ATP的供应往往成为GSH生产的限制因素。该文以毕赤酵母GS115为出发菌株,整合串联表达来源于酿酒酵母的Scgsh1和Scgsh2基因,在添加氨基酸前体的条件下,GSH质量浓度可达(302.27±5.06)mg/L,较改造前提高2.88倍。之后优化了柠檬酸钠的添加条件,摇瓶水平最高可达(371.12±8.47)mg/L。最后对工程菌的上罐发酵,通过控制乙醇质量浓度优化葡萄糖的补料速率,实现两阶段高效合成GSH,菌体生物量OD 600最高可达257,发酵68 h时GSH产量最高可达2000 mg/L。该研究为GSH的工业化生产提供了策略参考。

Glutathione is an important non-coding sulfhydryl-containing tripeptide substance in the organism.It has functions such as regulation and protection,and has a wide range of applications in food and medical industries.Recently,glutathione is mainly produced through high cell density fermentation in the industry,and the supply of ATP becomes a limiting factor for GSH production.In this paper,Pichia pastoris GS115 was used as the original strain to heterologously express Scgsh1 and Scgsh2 genes derived from Saccharomyces cerevisiae.With the addition of amino acid precursor,the GSH concentration reached(302.27±5.06)mg/L,which was 2.88 times higher than before.And then,combined with the optimization of sodium citrate addition conditions,the highest GSH concentration in shake flask reached(371.12±8.47)mg/L.Finally,fermentation of the engineered strain was carried out in 100 L tank.By controlling the ethanol concentration to optimize the feed rate of glucose,the two-stage efficient synthesis of glutathione was realized.The maximum biomass OD 600 reached 257,and the highest concentration of GSH reached 2000 mg/L at 68 h.