Abstract γ-Aminobutyrate (GABA) is an important bioactive compound synthesized through decarboxylation of L-glutamate by the glutamate decarboxylase (GAD). Biosynthesis of GABA by lactic acid bacteria (LAB) has particularly attracted attention, as LAB are generally recognized as safe organisms. In this work, based on identification of biosynthetic gene cluster for GABA in Lactococcus lactis subsp. lactis CV56, the GadB has been heterologously expressed in Escherichia coli, purified and biochemically characterized. The recombinant enzyme existed as a homodimer under native conditions with a molecular mass of 115.03 kDa, and exhibited maximal activity at 50 °C and pH 4.7. The Km value of GadB was 3.9 mM for L-glutamate, with a limiting rate (Vmax) of 15.1 U/mg. Subsequently, the genetic engineering technique has been employed to increase the levels of expression of key GAD system genes from strain CV56 in the model LAB-L. lactis subsp. cremoris NZ9000. The fermentation results revealed that the coexpression of gadC and gadB made much more contribution to the overall yield of GABA than overexpression of any individual gene. Moreover, a novel two-stage pH control strategy for the selected strain L. lactis NZ9000/pNZ8148-gadBC was developed, and 25.61 g/L of GABA was finally achieved in the batch fermentation.

中文翻译：

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基因工程乳酸乳球菌对γ-氨基丁酸生物合成的改进

摘要 γ-氨基丁酸（GABA）是一种重要的生物活性化合物，通过谷氨酸脱羧酶（GAD）使L-谷氨酸脱羧合成。乳酸菌 (LAB) 对 GABA 的生物合成尤其引起了人们的关注，因为 LAB 被普遍认为是安全的生物。在这项工作中，基于乳酸乳球菌亚种中 GABA 生物合成基因簇的鉴定。乳杆菌 CV56，GadB 已在大肠杆菌中异源表达，纯化和生化表征。重组酶在天然条件下作为同源二聚体存在，分子量为 115.03 kDa，在 50 °C 和 pH 4.7 时表现出最大活性。L-谷氨酸的 GadB 的 Km 值为 3.9 mM，极限速率 (Vmax) 为 15.1 U/mg。随后，基因工程技术已被用于提高模型 LAB-L 中来自菌株 CV56 的关键 GAD 系统基因的表达水平。乳酸亚种 克雷莫里斯 NZ9000。发酵结果表明，与任何单个基因的过表达相比，gadC 和 gadB 的共表达对 GABA 的总产量的贡献更大。此外，针对所选菌株 L. lactis NZ9000/pNZ8148-gadBC 开发了一种新的两阶段 pH 控制策略，最终在分批发酵中实现了 25.61 g/L 的 GABA。