Enhancing High-Level Food-Grade Expression of Glutamate Decarboxylase and Its Application in the Production of γ-Aminobutyric Acid.

IF 2.5 4区 生物学 Q3 BIOTECHNOLOGY & APPLIED MICROBIOLOGY
Kang Zhang, Huihui Lv, Xinrui Yu, Xuyang Zhu, Sheng Chen, Jing Wu
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引用次数: 0

Abstract

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 Bacillus subtilis 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, in vitro fermentation demonstrated that GABA supplementation promoted gut microbial growth and increased the relative abundance of Actinobacteriota and Bacteroidota.

谷氨酸脱羧酶在食品级的高水平表达及其在γ-氨基丁酸生产中的应用
γ -氨基丁酸(Gamma-aminobutyric acid, 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的低食品级表达阻碍了GABA的大规模生产。在本研究中,我们旨在通过辅助因子合成增强、基于crispr的宿主菌株修饰和发酵优化来提高枯草芽孢杆菌中GAD的表达。在3-L发酵罐中,优化菌株在没有抗生素选择压力的情况下获得了319.62 U/ml的GAD活性,这是迄今为止报道的最高食品级表达量。随后,酶学性质分析促进了以味精和l -谷氨酸为底物生产GABA的优化,达到100%的摩尔转化率分别为274.40 g/l和481.62 g/l,后者的生产率达到前所未有的48.16 g/l/h。最后,体外发酵实验表明,添加GABA促进了肠道微生物的生长,增加了放线菌群和拟杆菌群的相对丰度。
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来源期刊
Journal of microbiology and biotechnology
Journal of microbiology and biotechnology BIOTECHNOLOGY & APPLIED MICROBIOLOGY-MICROBIOLOGY
CiteScore
5.50
自引率
3.60%
发文量
151
审稿时长
2 months
期刊介绍: The Journal of Microbiology and Biotechnology (JMB) is a monthly international journal devoted to the advancement and dissemination of scientific knowledge pertaining to microbiology, biotechnology, and related academic disciplines. It covers various scientific and technological aspects of Molecular and Cellular Microbiology, Environmental Microbiology and Biotechnology, Food Biotechnology, and Biotechnology and Bioengineering (subcategories are listed below). Launched in March 1991, the JMB is published by the Korean Society for Microbiology and Biotechnology (KMB) and distributed worldwide.
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