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α-ketoglutarate is an important short-chain organic acid that is widely used in various fields such as food, medicine, cosmetics, and animal feed. However, the efficiency of producing α-ketoglutarate through biological fermentation remains to be improved, primarily due to the limitations in the synthetic capacity of microbial metabolic pathways. [Objective] To address the above issues, we developed an engineered Escherichia coli that can efficiently produce α-ketoglutarate, thereby providing theoretical support for the large-scale production of α-ketoglutarate in the future. [Methods] We employed an efficient approach combining rational and irrational modifications to overcome the constraints of endogenous metabolic pathways and enhance the biosynthesis efficiency of α-ketoglutarate. [Results] The oxidative TCA pathway was reconstructed to improve α-ketoglutarate production through expressing pyruvate carboxylase, citrate synthase, aconitase, and isocitrate dehydrogenase. The metabolic network for α-ketoglutarate biosynthesis was irrationally optimized and strengthened to enhance its biosynthesis capability by atmospheric pressure room temperature plasma mutagenesis. To improve the supply efficiency of the precursor for α-ketoglutarate biosynthesis, we reduced the dissipation of carbon flux in the pyruvate node by knocking out genes related to the accumulation of lactate, acetate, and formate. Furthermore, we knocked out the genes related to the degradation pathway of α-ketoglutarate to achieve the retention of carbon flux at α-ketoglutarate node and improve its production. Through the optimization of fermentation conditions, the fermentation in a 5 L fermenter with the engineered strain E. coli KA29 achieved the α-ketoglutarate titer, yield, and productivity of 28.7 g/L, 0.29 g/g, and 0.48 g/(L·h), respectively. [Conclusion] The research strategies mentioned above lay a foundation for the development and application of strains with high production of α-ketoglutarate and provide a reference for metabolic engineering to produce other organic acids.
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| 科 Family | 属数 Number of genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) | 属 Genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) |
|---|---|---|---|---|---|---|
| 鹅膏菌科Amanitaceae | 2 | 11 | 5.26 | 鹅膏菌属 Amanita | 10 | 4.78 |
| 小菇科 Mycenaceae | 2 | 12 | 5.74 | 丝盖伞属 Inocybe | 5 | 2.39 |
| 多孔菌科 Polyporaceae | 8 | 14 | 6.70 | 蜡蘑属 Laccaria | 5 | 2.39 |
| 红菇科 Russulaceae | 3 | 23 | 11.00 | 小皮伞属 Marasmius | 6 | 2.87 |
| 小菇属 Mycena | 11 | 5.26 | ||||
| 光柄菇属 Pluteus | 5 | 2.39 | ||||
| 红菇属 Russula | 17 | 8.13 | ||||
| 栓菌属 Trametes | 5 | 2.39 |
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