γ-氨基丁酸(γ-aminobutyric acid,GABA)广泛应用于食品和医药等领域,目前主要利用大肠杆菌(Escherichia coli)作为宿主菌,由酶催化方法生产。在全细胞转化过程中,GABA会进入三羧酸循环进行降解,导致转化率低下;通过敲基因阻碍此途径又会严重影响宿主菌的生长。该研究揭示了一条新的GABA的消耗途径:GABA在胞质非特异性二肽酶(cytosolic nonspecific dipeptidase,pepD)的作用下缩合形成二肽。该文构建了2个工程菌株:敲除E.coli BL21(DE3)基因组上的pepD基因,构建了ΔpepD菌株;在E.coli BL21(DE3)中过表达了pepD基因,构建了OEpepD菌株。以GABA和谷氨酸钠(monosodium glutamate,MSG)为底物,进行全细胞转化反应。结果表明,该酶不能催化MSG发生反应,但是能催化GABA发生反应。通过研究2个菌株消耗GABA的历程,发现OEpepD的GABA消耗量比ΔpepD菌株高出17.5%,消耗量在2 h之后都不再增加。利用LC-MS鉴定纯酶催化反应的产物,证实产物是GABA形成的二肽。该研究构建的菌株可以在不影响大肠杆菌生长的情况下有效提高GABA的产量,为GABA的工业化生产提供有益参考。
γ-Aminobutyric acid (GABA), widely used in the fields of food and medicine, is currently primarily produced using Escherichia coli (E.coli) as the host through enzyme-catalyzed methods.In the whole-cell conversion process, GABA enters the TCA cycle for degradation, leading to a low conversion rate.Inhibiting this pathway by knocking out genes severely affects the growth of the host.This study reveals a new GABA consumption pathway:GABA is catalyzed by the nonspecific dipeptidase (cytosolic nonspecific dipeptidase,pepD) enzyme to form dipeptides.This study constructed two engineered strains:ΔpepD strain, in which the pepD gene on the E.coli BL21(DE3) genome was knocked out, and OEpepD strain, in which the pepD gene was overexpressed in E.coli BL21(DE3).Using GABA and monosodium glutamate (MSG) as substrates, the whole-cell conversion reactions were conducted.Results indicated that MSG cannot be catalyzed by the pepD, but GABA could be catalyzed by the pepD.It was found that the GABA consumption in the OEpepD strain was 17.5% higher than that in the ΔpepD strain during the GABA consumption process, and the consumption did not increase after 2 hours.LC-MS analysis of the products catalyzed by the pure pepD confirmed that a dipeptide was formed from GABA.The results of this study provide an engineered strains that effectively increases GABA production without affecting the growth of E.coli, and provide valuable insights for the industrial production of GABA.
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