Liu Jiafeng, Qiao Zhina, Zhao Youxi, Xu Meijuan, Zhang Xian, Yang Taowei, Rao Zhiming
School of Biotechnology, Jiangnan University, Wuxi 214122, Jiangsu, China.
College of Biochemical Engineering, Beijing Union University, Beijing 100023, China.
Sheng Wu Gong Cheng Xue Bao. 2023 Aug 25;39(8):3273-3289. doi: 10.13345/j.cjb.230018.
L-glutamic acid is the world's largest bulk amino acid product that is widely used in the food, pharmaceutical and chemical industries. Using G01 as the starting strain, the fermentation by-product alanine content was firstly reduced by knocking out the gene encoding alanine aminotransferase (), a major by-product related to alanine synthesis. Secondly, since the α-ketoglutarate node carbon flow plays an important role in glutamate synthesis, the ribosome-binding site (RBS) sequence optimization was used to reduce the activity of α-ketoglutarate dehydrogenase and enhance the glutamate anabolic flow. The endogenous conversion of α-ketoglutarate to glutamate was also enhanced by screening different glutamate dehydrogenase. Subsequently, the glutamate transporter was rationally desgined to improve the glutamate efflux capacity. Finally, the fermentation conditions of the strain constructed using the above strategy were optimized in 5 L fermenters by a gradient temperature increase combined with a batch replenishment strategy. The glutamic acid production reached (135.33±4.68) g/L, which was 41.2% higher than that of the original strain (96.53±2.32) g/L. The yield was 55.8%, which was 11.6% higher than that of the original strain (44.2%). The combined strategy improved the titer and the yield of glutamic acid, which provides a reference for the metabolic modification of glutamic acid producing strains.
L-谷氨酸是世界上产量最大的大宗氨基酸产品，广泛应用于食品、制药和化工行业。以G01为出发菌株，首先通过敲除编码丙氨酸转氨酶（一种与丙氨酸合成相关的主要副产物）的基因，降低发酵副产物丙氨酸的含量。其次，由于α-酮戊二酸节点碳流在谷氨酸合成中起重要作用，通过核糖体结合位点（RBS）序列优化来降低α-酮戊二酸脱氢酶的活性，增强谷氨酸合成代谢流。通过筛选不同的谷氨酸脱氢酶，还增强了α-酮戊二酸向内源性谷氨酸的转化。随后，合理设计谷氨酸转运体以提高谷氨酸外排能力。最后，采用梯度升温结合分批补料策略在5 L发酵罐中对使用上述策略构建的菌株的发酵条件进行优化。谷氨酸产量达到（135.33±4.68）g/L，比原始菌株（96.53±2.32）g/L高41.2%。产率为55.8%，比原始菌株（44.2%）高11.6%。该联合策略提高了谷氨酸的产量和产率，为谷氨酸生产菌株的代谢改造提供了参考。
