Glutathione is a bioactive tripeptide composed of glycine, L-cysteine, and L-glutamate, and has been widely used in pharmaceutical, food, and healthy products. The current metabolic studies of glutathione were mainly focused on the native producing strains with precursor amino acid supplementation. In the present work, Corynebacterium glutamicum, a workhorse for industrial production of a series of amino acids, was engineered to produce glutathione. First, the introduction of glutathione synthetase gene gshF from Streptococcus agalactiae fulfilled the ability of glutathione production in C. glutamicum and revealed that L-cysteine was the limiting factor. Then, considering the inherent capability of L-glutamate synthesis and the availability of external addition of low-cost glycine, L-cysteine biosynthesis was enhanced using a varieties of pathway engineering methods, such as disrupting the degradation pathways of L-cysteine and L-serine, and removing the repressor responsible for sulfur metabolism. Finally, the simultaneously introduction of gshF and enhancement of cysteine formation enabled C. glutamicum strain to produce glutathione greatly. Without external addition of L-cysteine and L-glutamate, 756 mg/L glutathione was produced. This is first time to demonstrate the potential of the glutathione non-producing strain C. glutamicum for glutathione production and provide a novel strategy to construct glutathione-producing strains.

中文翻译：

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在谷氨酸棒杆菌中通过多途径工程高效生产谷胱甘肽。

谷胱甘肽是由甘氨酸、L-半胱氨酸和L-谷氨酸组成的具有生物活性的三肽，广泛应用于医药、食品和保健品中。目前谷胱甘肽的代谢研究主要集中在补充前体氨基酸的天然生产菌株上。在目前的工作中，谷氨酸棒杆菌是工业生产一系列氨基酸的主力，被设计用于生产谷胱甘肽。首先，从无乳链球菌中引入谷胱甘肽合成酶基因gshF，实现了谷氨酸棒杆菌产生谷胱甘肽的能力，并揭示了L-半胱氨酸是限制因素。然后，考虑到L-谷氨酸合成的固有能力和外部添加低成本甘氨酸的可用性，L-半胱氨酸生物合成使用多种途径工程方法得到增强，例如破坏 L-半胱氨酸和 L-丝氨酸的降解途径，以及去除负责硫代谢的阻遏物。最后，同时引入 gshF 和增强半胱氨酸形成使谷氨酸棒杆菌菌株能够大量产生谷胱甘肽。在没有外部添加 L-半胱氨酸和 L-谷氨酸的情况下，产生了 756 mg/L 谷胱甘肽。这是首次证明谷胱甘肽非生产菌株C. glutamicum 生产谷胱甘肽的潜力，并为构建谷胱甘肽生产菌株提供了一种新策略。同时引入gshF和增强半胱氨酸形成使谷氨酸棒杆菌菌株大量产生谷胱甘肽。在没有外部添加 L-半胱氨酸和 L-谷氨酸的情况下，产生了 756 mg/L 谷胱甘肽。这是首次证明谷胱甘肽非生产菌株C. glutamicum 生产谷胱甘肽的潜力，并为构建谷胱甘肽生产菌株提供了一种新策略。同时引入gshF和增强半胱氨酸形成使谷氨酸棒杆菌菌株大量产生谷胱甘肽。在没有外部添加 L-半胱氨酸和 L-谷氨酸的情况下，产生了 756 mg/L 谷胱甘肽。这是首次证明谷胱甘肽非生产菌株C. glutamicum 生产谷胱甘肽的潜力，并为构建谷胱甘肽生产菌株提供了一种新策略。