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Efficient Production of γ-GABA Using Recombinant E. coli Expressing Glutamate Decarboxylase (GAD) Derived from Eukaryote Saccharomyces cerevisiae

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Abstract

γ-Aminobutyric acid (γ-GABA) is a non-proteinogenic amino acid, which acts as a major regulator in the central nervous system. Glutamate decarboxylase (namely GAD, EC 4.1.1.15) is known to be an ideal enzyme for γ-GABA production using L-glutamic acid as substrate. In this study, we cloned and expressed GAD gene from eukaryote Saccharomyces cerevisiae (ScGAD) in E. coli BL21(DE3). This enzyme was further purified and its optimal reaction temperature and pH were 37 °C and pH 4.2, respectively. The cofactor of ScGAD was verified to be either pyridoxal 5′-phosphate (PLP) or pyridoxal hydrochloride. The optimal concentration of either cofactor was 50 mg/L. The optimal medium for E. coli-ScGAD cultivation and expression were 10 g/L lactose, 5 g/L glycerol, 20 g/L yeast extract, and 10 g/L sodium chloride, resulting in an activity of 55 U/mL medium, three times higher than that of using Luria–Bertani (LB) medium. The maximal concentration of γ-GABA was 245 g/L whereas L-glutamic acid was near completely converted. These findings provided us a good example for bio-production of γ-GABA using recombinant E. coli expressing a GAD enzyme derived from eukaryote.

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Acknowledgements

We gratefully acknowledge the financial support from the National Natural Science Foundation of China (31400053 to Z. Xu), Natural Science Foundation of Jiangsu Province (BK20140933 to Z. Xu), Jiangsu Province Science and Technology Support Plan Project (BE2015366 to H. Xu), National Nature Science Foundation of China (21476112 to H. Xu), and National Basic Research Program of China (973) (2013CB733603 to H. Xu).

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Author notes

  1. Qiang Xiong and Zheng Xu contributed equally to this work

Authors and Affiliations

  1. College of Food Science and Light Industry, Nanjing Tech University, 211816, Nanjing, People’s Republic of China

    Qiang Xiong, Zheng Xu, Lu Xu, Zhong Yao, Sha Li & Hong Xu

  2. State Key Laboratory of Materials-Oriented Chemical Engineering, 210009, Nanjing, People’s Republic of China

    Qiang Xiong, Zheng Xu, Lu Xu, Zhong Yao, Sha Li & Hong Xu

  3. Jiangsu National Synergetic Innovation Center for Advanced Materials (SICAM), Nanjing, People’s Republic of China

    Qiang Xiong, Zheng Xu, Lu Xu, Zhong Yao, Sha Li & Hong Xu

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  1. Qiang Xiong
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  2. Zheng Xu
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  3. Lu Xu
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  4. Zhong Yao
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  5. Sha Li
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  6. Hong Xu
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Correspondence to Hong Xu.

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Xiong, Q., Xu, Z., Xu, L. et al. Efficient Production of γ-GABA Using Recombinant E. coli Expressing Glutamate Decarboxylase (GAD) Derived from Eukaryote Saccharomyces cerevisiae . Appl Biochem Biotechnol 183, 1390–1400 (2017). https://doi.org/10.1007/s12010-017-2506-4

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  • DOI: https://doi.org/10.1007/s12010-017-2506-4

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