Zhang Kang, Lv Huihui, Yu Xinrui, Zhu Xuyang, Chen Sheng, Wu Jing
School of Biotechnology and Key Laboratory of Industrial Biotechnology Ministry of Education, Jiangnan University, 1800 Lihu Avenue, Wuxi 214122, P.R. China.
State Key Laboratory of Food Science and Resources, Jiangnan University, 1800 Lihu Avenue, Wuxi 214122, P.R. China.
J Microbiol Biotechnol. 2024 Dec 12;35:e2410013. doi: 10.4014/jmb.2410.10013.
Gamma-aminobutyric acid (GABA), a non-proteinogenic amino acid, exhibits diverse physiological functions and finds extensive applications in food, medicine, and various industries. Glutamate decarboxylase (GAD) can effectively convert L-glutamic acid (L-Glu) or monosodium glutamate (MSG) into GABA. However, the low food-grade expression of GAD has hindered large-scale GABA production. In this study, we aimed to elevate GAD expression in through cofactor synthesis enhancement, CRISPRi-based host strain modification, and fermentation optimization. In a 3-L fermenter, the optimized strain achieved a remarkable GAD activity of 319.62 U/ml without antibiotic selection pressure, representing the highest reported food-grade expression to date. Subsequently, enzymatic property analysis facilitated the optimization of GABA production using MSG and L-Glu as substrates, achieving 100% molar conversion yields of 274.40 g/l and 481.62 g/l, respectively, with the latter yielding an unprecedented productivity of 48.16 g/l/h. Finally, fermentation demonstrated that GABA supplementation promoted gut microbial growth and increased the relative abundance of and .
γ-氨基丁酸（GABA）是一种非蛋白质氨基酸，具有多种生理功能，在食品、医药和各种工业中有着广泛的应用。谷氨酸脱羧酶（GAD）能有效地将L-谷氨酸（L-Glu）或味精（MSG）转化为GABA。然而，GAD在食品级的低表达阻碍了GABA的大规模生产。在本研究中，我们旨在通过增强辅因子合成、基于CRISPRi的宿主菌株改造和发酵优化来提高GAD的表达。在3-L发酵罐中，优化后的菌株在无抗生素选择压力的情况下实现了319.62 U/ml的显著GAD活性，这是迄今为止报道的最高食品级表达。随后，酶学性质分析有助于以味精和L-谷氨酸为底物优化GABA的生产，分别实现了274.40 g/l和481.62 g/l的100%摩尔转化率，后者产生了前所未有的48.16 g/l/h的生产率。最后，发酵表明补充GABA促进了肠道微生物的生长，并增加了[具体微生物名称1]和[具体微生物名称2]的相对丰度。
