Effect of gadB and gdh Co-expression on Bacterial γ-aminobutyric Acid Production
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摘要: 为构建一株无外源L-谷氨酸（L-Glu）条件下可直接转化葡萄糖生产γ-氨基丁酸（GABA）的安全基因工程菌，将植物乳杆菌GABA合成途径的关键酶——谷氨酸脱羧酶基因gadB与L-Glu合成途径中的关键酶谷氨酸脱氢酶基因gdh进行串联表达，导入产谷氨酸菌株谷氨酸棒杆菌SF016中，检测其对L-Glu及GABA产量的影响。经摇瓶发酵40 h重组菌SF016-pgg谷氨酸脱羧酶的酶活达0.63 mol·min-1·g-1，而谷氨酸脱氢酶的酶活达0.131 mol·min-1·g-1，比原始菌株提高了约2.0倍。重组菌SF016-pgg以葡萄糖为碳源，通过40 h发酵罐发酵可积累产生23.12 g·L-1的GABA，说明串联表达gadB和gdh基因有效实现重组菌在以葡萄糖为单一碳源、无外源L-Glu存在的培养基中直接发酵生产GABA。该生产方式的成本明显降低，且产品的安全性高，可用于食品、饲料或医药等行业。
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关键词:
- 谷氨酸脱羧酶 /
- 谷氨酸脱氢酶 /
- 谷氨酸生产菌SF016 /
- 基因串联表达
Abstract: This study aimed to create a genetically engineered bacterium that could produce γ-aminobutyric acid (GABA) directly from glucose without exogenous L-glutamic acid (L-Glu).The gadB and gdh genes responsible for the generation of two critical enzymes, glutamic acid decarboxylase (GAD) and glutamate dehydrogenase (GDH), respectively, in the synthesis of plant lactobacillus GABA were co-expressed in a Glu-producing strain of Corynebacterium glutamicum, SF016.The resulting effects on GABA production by the recombinational strain, SF016-pgg, were analyzed. The activities of GAD and GDH in SF016-pggnearly doubled after incubation in a shaking flask for 40 h reaching 0.63 mol·min-1·g-1 and 0.131 mol·min-1·g-1, respectively. Furthermore, the SF016-pgg production of GABA from glucose as a single carbon source totaled 23.12 g·L-1 in a 40 h fermentation in a tank. The results seemed to clearly demonstrate the effectiveness of the gadB and gdh co-expressed recombinant strain on producing GABA by converting glucose without exogenous addition of L-Glu. As a result, the GABA production cost was significantly reduced making the industrialization of food, feed and/or pharmaceutical applications exceedingly promising. -
图 1 重组质粒pZ8-gadB-tacgdh酶切图谱
注：M：DNA marker；1:经EcoRI酶切的pZ8-gadB质粒；2:经EcoRI酶切的pZ8-gadB-tacgdh阳性质粒；3:经EcoRI酶切的pZ8-gadB-tacgdh方向连接的阴性质粒。
Figure 1. Restriction map of pZ8-gadB-tacgdh
图 2 原始菌SF016和重组菌SF016-pgg发酵过程
注:OD562nm=测定值×稀释倍数。
Figure 2. Recombinant SF016-pgg and original strain fermentation processes
表 1 PCR引物
Table 1. Primers for PCR
引物 序列和酶切位点(5′-3′) P1gadB CGGAATTCATGGCAATGTTATACGGTAAAC(EcoRⅠ) P2gadB GCGTCGACTCAGTGTGTGAATAGGTATTTC(SalⅠ) P1gdh CGGAATTCATGACAGTTGATGAGCAGGTCT(EcoRⅠ) P2gdh GCGTCGACTTAGATGACGCCCTGTGCCAGCA(SalⅠ) P1tacgdh GCGTCGACTGACAATTAATCATCGGCTCGTA(SalⅠ) 注：下划线表示酶切位点。 
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表 2 GAD和GDH酶活力的测定
Table 2. GAD and GDH activities
菌株名称 酶活力/(mol·min-1·g-1) GABA/
(g·L-1)L-Glu/
(g·L-1)GAD GDH SF016 0 0.065 0 15.76 SF016-pgg 0.63 0.131 11.54 2.4
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表 3 重组菌SF016-pgg与原始菌的发酵参数
Table 3. Conditions for 40 h fermentation of recombinant SF016-pgg and original strain
菌株 OD562nm L-Glu/
(g·L-1)GABA/
(g·L-1)SF016 59.42±0.64 53.82±0.52 0 SF016-pgg 43.21±0.71 2.51±0.23 23.12±0.35
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[1] WONG C G, BOTTIGLIERI T. GABA, gamma-hydroxybu-tyric acid, and neurological disease[J].Annals of Neurology, 2003, 54(S6):3-12. doi: 10.1002/(ISSN)1531-8249 [2] 杨宏芳.微生物发酵法制备γ-氨基丁酸的研究进展[J].安徽农业科学, 2017, 45(24):76-78. doi: 10.3969/j.issn.0517-6611.2017.24.027 [3] 王辉, 项丽丽, 张锋华.γ-氨基丁酸(GABA)的功能性及在食品中的应用[J].食品工业, 2013, 34(6):186-189. http://kns.cnki.net/KCMS/detail/detail.aspx?filename=SPGY201306065&dbname=CJFD&dbcode=CJFQ [4] 李良铸, 李明晔.最新生化药物制备技术[M].北京:中国医药科技出版社, 2001:79-80. [5] 王金玲, 袁军, 刘登才.γ-氨基丁酸的合成[J].化学与生物工程, 2010, 27(3):40-42. doi: 10.3969/j.issn.1672-5425.2010.03.011 [6] 赵景联.固定化大肠杆菌细胞生产γ-氨基丁酸的研究[J].生物工程学报, 1989, 5(2):124-128. http://www.wanfangdata.com.cn/details/detail.do?_type=perio&id=QK000004558805 [7] XIA J, MEI L H, HUANG J, et al.Screening and mutagenesis of Lactobacillus brevis for biosynthesis of γ-aminobutyric[J].Journal of Nuclear Agricultural Sciences, 2006, 20(5):379-382. http://www.wanfangdata.com.cn/details/detail.do?_type=perio&id=hnxb200605007 [8] 田灵芝, 徐美娟, 饶志明.一株重组大肠杆菌/pET-28a-lpgad的构建及其高效生产γ-氨基丁酸转化条件的优化[J].生物工程学报, 2012, 28(1):65-75. http://www.wanfangdata.com.cn/details/detail.do?_type=perio&id=swgcxb201201008 [9] HUANG J, MEI L H, XIA J. Application of artificial neural network coupling particle swarm optimization algorithm to biocatalytic production of GABA[J]. Biotechnol Bioeng, 2007, 96(5):924-931. doi: 10.1002/(ISSN)1097-0290 [10] 孙红梅, 饶志明, 李秀鹏, 等.以葡萄糖为底物一步法合成γ-氨基丁酸整合型重组钝齿棒杆菌的构建[J].微生物学报, 2013, 53(8):817-824. http://d.old.wanfangdata.com.cn/Periodical/wswxb201308005 [11] KINOSHITA S, UDAKA S, SHIMONO M. Studies on the amino acid fermentation. Part 1. Production of L-Glutamic acid by various microorganisms[J]. J Gen Appl Microbiol, 1957, 50:193-205. [12] WENDISCH V F, BOTT M, EIKMANNS B J. Metabolic engineering of Escherichia coli and Corynebacterium glutamicum for biotechnological production of organic acids and aminoacids[J]. Curr Opin Microbiol, 2006(9):268-274. https://www.sciencedirect.com/science/article/pii/S1369527406000464 [13] KALINOWSKI J, BATHE B, BARTELS D, et al. The complete Corynebacterium glutamicum ATCC 13032 genome sequence and its impact on the production of L-aspartate-derived amino acids and vitamins[J]. J Biotechnol, 2003, 104:5-25. doi: 10.1016/S0168-1656(03)00154-8 [14] 余秉琦, 沈微, 王正祥, 等.谷氨酸棒杆菌的乙醛酸循环与谷氨酸合成[J].生物工程学报, 2005, 21(2):270-276. doi: 10.3321/j.issn:1000-3061.2005.02.019 [15] VANDER R, LANGE C, MOLENAAR D. A heat shock following electroporation induces highly efficient transformation of Corynebacterium glutamicum with xenogenetic plasmid DNA[J]. App Microbiol Biotech, 1999, 52(4):541-545. doi: 10.1007/s002530051557 [16] CHAO Y P, LO T E, LUO N S. Selective production of L-aspartic acid and L-phenylalanine by coupling reactions of aspartase and aminotranferase in Escherichia coli[J]. Enzyme MicrobTech, 2000, 27(2):19-25. https://www.sciencedirect.com/science/article/pii/S0141022900001496 [17] KITAOKA S, NAKANO Y.Colorimetric determination of omega-amino acids[J]. Journal of Biochemistry, 1969, 66(1):87-94. doi: 10.1093/oxfordjournals.jbchem.a129124 [18] GOURDON P, NICHOLAS D. Metabolic Analysis of Glutamate Production by Corynebacterium glutamicum[J]. Metab Eng, 1999, 1(3):224-231. doi: 10.1006/mben.1999.0122 [19] SHI F, JIANG J, LI Y, et al. Enhancement of γ-aminobutyric acid production in recombinant Corynebacterium glutamicum by co-expressing two glutamate decarboxylase genes from Lactobacillus brevis[J]. Journal of industrial microbiology & biotechnology, 2013, 40(11):1285-1296. doi: 10.1007/s10295-013-1316-0 -
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