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Engineering Escherichia coli for efficient glutathione production
Metabolic Engineering ( IF 6.8 ) Pub Date : 2024-07-03 , DOI: 10.1016/j.ymben.2024.07.001 Hiroki Mori 1 , Misato Matsui 1 , Takahiro Bamba 2 , Yoshimi Hori 2 , Sayaka Kitamura 3 , Yoshihiro Toya 3 , Ryota Hidese 2 , Hisashi Yasueda 4 , Tomohisa Hasunuma 5 , Hiroshi Shimizu 3 , Naoaki Taoka 1 , Shingo Kobayashi 1
Metabolic Engineering ( IF 6.8 ) Pub Date : 2024-07-03 , DOI: 10.1016/j.ymben.2024.07.001 Hiroki Mori 1 , Misato Matsui 1 , Takahiro Bamba 2 , Yoshimi Hori 2 , Sayaka Kitamura 3 , Yoshihiro Toya 3 , Ryota Hidese 2 , Hisashi Yasueda 4 , Tomohisa Hasunuma 5 , Hiroshi Shimizu 3 , Naoaki Taoka 1 , Shingo Kobayashi 1
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Glutathione is a tripeptide of excellent value in the pharmaceutical, food, and cosmetic industries that is currently produced during yeast fermentation. In this case, glutathione accumulates intracellularly, which hinders high production. Here, we engineered for the efficient production of glutathione. A total of 4.3 g/L glutathione was produced by overexpressing and , which encode cysteine glutamate ligase and glutathione synthetase, respectively, and most of the glutathione was excreted into the culture medium. Further improvements were achieved by inhibiting degradation (Δ and Δ); deleting (Δ), which encodes glutathione oxide reductase; attenuating glutathione uptake (Δ); and enhancing cysteine production (P). The engineered strain KG06 produced 19.6 g/L glutathione after 48 h of fed-batch fermentation with continuous addition of ammonium sulfate as the sulfur source. We also found that continuous feeding of glycine had a crucial role for effective glutathione production. The results of metabolic flux and metabolomic analyses suggested that the conversion of -acetylserine to cysteine is the rate-limiting step in glutathione production by KG06. The use of sodium thiosulfate largely overcame this limitation, increasing the glutathione titer to 22.0 g/L, which is, to our knowledge, the highest titer reported to date in the literature. This study is the first report of glutathione fermentation without adding cysteine in . Our findings provide a great potential of fermentation process for the industrial production of glutathione.
中文翻译：
改造大肠杆菌以高效生产谷胱甘肽
谷胱甘肽是一种在制药、食品和化妆品行业具有卓越价值的三肽，目前是在酵母发酵过程中产生的。在这种情况下，谷胱甘肽在细胞内积累，从而阻碍高产量。在这里，我们设计了谷胱甘肽的高效生产。过表达分别编码半胱氨酸谷氨酸连接酶和谷胱甘肽合成酶的 和 总共产生了 4.3 g/L 谷胱甘肽，并且大部分谷胱甘肽被分泌到培养基中。通过抑制降解实现了进一步的改进（Δ和Δ）；删除编码谷胱甘肽氧化还原酶的(Δ)；减弱谷胱甘肽的摄取（Δ）；并增强半胱氨酸的产生（P）。工程菌株KG06在连续添加硫酸铵作为硫源的情况下，分批补料发酵48小时后，产生了19.6 g/L的谷胱甘肽。我们还发现，连续饲喂甘氨酸对于有效生产谷胱甘肽具有至关重要的作用。代谢流和代谢组学分析的结果表明，β-乙酰丝氨酸向半胱氨酸的转化是KG06产生谷胱甘肽的限速步骤。硫代硫酸钠的使用很大程度上克服了这一限制，将谷胱甘肽滴度提高至 22.0 g/L，据我们所知，这是迄今为止文献报道的最高滴度。本研究是首次报道不添加半胱氨酸的谷胱甘肽发酵。我们的研究结果为谷胱甘肽的工业生产提供了发酵工艺的巨大潜力。
更新日期：2024-07-03
中文翻译：
改造大肠杆菌以高效生产谷胱甘肽
谷胱甘肽是一种在制药、食品和化妆品行业具有卓越价值的三肽，目前是在酵母发酵过程中产生的。在这种情况下，谷胱甘肽在细胞内积累，从而阻碍高产量。在这里，我们设计了谷胱甘肽的高效生产。过表达分别编码半胱氨酸谷氨酸连接酶和谷胱甘肽合成酶的 和 总共产生了 4.3 g/L 谷胱甘肽，并且大部分谷胱甘肽被分泌到培养基中。通过抑制降解实现了进一步的改进（Δ和Δ）；删除编码谷胱甘肽氧化还原酶的(Δ)；减弱谷胱甘肽的摄取（Δ）；并增强半胱氨酸的产生（P）。工程菌株KG06在连续添加硫酸铵作为硫源的情况下，分批补料发酵48小时后，产生了19.6 g/L的谷胱甘肽。我们还发现，连续饲喂甘氨酸对于有效生产谷胱甘肽具有至关重要的作用。代谢流和代谢组学分析的结果表明，β-乙酰丝氨酸向半胱氨酸的转化是KG06产生谷胱甘肽的限速步骤。硫代硫酸钠的使用很大程度上克服了这一限制，将谷胱甘肽滴度提高至 22.0 g/L，据我们所知，这是迄今为止文献报道的最高滴度。本研究是首次报道不添加半胱氨酸的谷胱甘肽发酵。我们的研究结果为谷胱甘肽的工业生产提供了发酵工艺的巨大潜力。
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