Lyu Changjiang, Yao Lili, Zhu Qi, Mei Jiaqi, Cao Yucheng, Hu Sheng, Zhao Weirui, Huang Jun, Mei Lehe, Yao Shanjing, Du Guocheng
School of Biotechnology, Jiangnan University, Wuxi, 214122, China.
School of Biological and Chemical Engineering, Zhejiang University of Science and Technology, Hangzhou, 310023, China.
Appl Microbiol Biotechnol. 2021 May;105(10):4127-4140. doi: 10.1007/s00253-021-11328-5. Epub 2021 May 15.
Gamma-aminobutyric acid (GABA), an important bioactive compound, is synthesized through the decarboxylation of L-glutamate (L-Glu) by glutamate decarboxylase (GAD). The use of lactic acid bacteria (LAB) as catalysts opens interesting avenues for the biosynthesis of food-grade GABA. However, a key obstacle involved in the improvement of GABA production is how to resolve the discrepancy of optimal pH between the intracellular GAD activity and cell growth. In this work, a potential GAD candidate (LpGadB) from Lactobacillus plantarum was heterologously expressed in Escherichia coli. Recombinant LpGadB existed as a homodimer under the native conditions with a molecular mass of 109.6 kDa and exhibited maximal activity at 40°C and pH 5.0. The K value and catalytic efficiency (k/K) of LpGadB for L-Glu was 21.33 mM and 1.19 mMs, respectively, with the specific activity of 26.67 μM/min/mg protein. Subsequently, four C-terminally truncated LpGadB mutants (GadB, GadB, GadB, GadB) were constructed based on homology modeling. Among them, the mutant GadB with highest catalytic activity at near-neutral pH values was selected. In further, the GadB and Glu/GABA antiporter (GadC) of Lactococcus lactis were co-overexpressed in the host L. lactis NZ3900. Finally, after 48 h of batch fermentation, the engineered strain L. lactis NZ3900/pNZ8149-gadBC yielded GABA concentration up to 33.52 g/L by applying a two-stage pH control strategy. Remarkably, this is the highest yield obtained to date for GABA from fermentation with L. lactis as a microbial cell factory.Key points• The GadB from L. plantarum was heterologously expressed in E. coli and biochemically characterized.• Deletion of the C-plug in GadB shifted its pH-dependent activity toward a higher pH.• Reconstructing the GAD system of L. lactis is an effective approach for improving its GABA production.
γ-氨基丁酸（GABA）是一种重要的生物活性化合物，它由谷氨酸脱羧酶（GAD）催化L-谷氨酸（L-Glu）脱羧合成。利用乳酸菌（LAB）作为催化剂为食品级GABA的生物合成开辟了有趣的途径。然而，提高GABA产量的一个关键障碍是如何解决细胞内GAD活性与细胞生长的最佳pH差异。在这项工作中，从植物乳杆菌中筛选出一种潜在的GAD候选基因（LpGadB），并在大肠杆菌中进行了异源表达。重组LpGadB在天然条件下以同源二聚体形式存在，分子量为109.6 kDa，在40°C和pH 5.0时表现出最大活性。LpGadB对L-Glu的K值和催化效率（k/K）分别为21.33 mM和1.19 mMs，比活性为26.67 μM/min/mg蛋白。随后，基于同源建模构建了四个C末端截短的LpGadB突变体（GadB、GadB、GadB、GadB）。其中，选择了在近中性pH值下具有最高催化活性的突变体GadB。进一步地，将乳酸乳球菌的GadB和Glu/GABA反向转运体（GadC）在宿主乳酸乳球菌NZ3900中共同过量表达。最后，经过48小时的分批发酵，通过应用两阶段pH控制策略，工程菌株乳酸乳球菌NZ3900/pNZ8149-gadBC产生的GABA浓度高达33.52 g/L。值得注意的是，这是迄今为止以乳酸乳球菌作为微生物细胞工厂发酵生产GABA获得的最高产量。
关键点
• 植物乳杆菌的GadB在大肠杆菌中异源表达并进行了生化特性分析。
• 删除GadB中的C-插塞使其pH依赖性活性向更高pH转移。
• 重建乳酸乳球菌的GAD系统是提高其GABA产量的有效方法。
