|本期目录/Table of Contents|

[1]郝大利,诸葛斌,方慧英,等.大肠杆菌aroG基因的定点突变及与trpBA基因的串联表达[J].应用与环境生物学报,2013,19(05):817-821.[doi:10.3724/SP.J.1145.2013.00817]
 HAO Dali,ZHUGE Bin,FANG Huiying,et al.Site-mutation of AroG Gene and Co-expression with TrpBA Gene in Escherichia coli[J].Chinese Journal of Applied & Environmental Biology,2013,19(05):817-821.[doi:10.3724/SP.J.1145.2013.00817]
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大肠杆菌aroG基因的定点突变及与trpBA基因的串联表达()
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《应用与环境生物学报》[ISSN:1006-687X/CN:51-1482/Q]

卷:
19卷
期数:
2013年05期
页码:
817-821
栏目:
研究论文
出版日期:
2013-10-25

文章信息/Info

Title:
Site-mutation of AroG Gene and Co-expression with TrpBA Gene in Escherichia coli
作者:
郝大利诸葛斌方慧英张成宗红诸葛健
(1江南大学工业生物技术教育部重点实验室 无锡 214122) (2江南大学生物工程学院工业微生物研究中心 无锡 214122)
Author(s):
HAO Dali ZHUGE Bin FANG Huiying ZHANG Cheng ZONG Hong ZHUGE Jian
(1Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, China) (2Research Center of Industrial Microorganisms, School of Biotechnology, Jiangnan University, Wuxi 214122, China)
关键词:
色氨酸大肠杆菌aroG基因trpBA基因基因定点突变
Keywords:
tryptophan Escherichia coli aroG trpBA site-directed mutation
分类号:
Q786 : Q936
DOI:
10.3724/SP.J.1145.2013.00817
摘要:
在大肠杆菌中aroG基因编码的3-脱氧-D-阿拉伯庚酮糖-7-磷酸合成酶(DAHPS)与trpBA基因编码的色氨酸合成酶(TSase)是色氨酸合成的关键酶,为了提高大肠杆菌生产色氨酸的产量,利用SOE-PCR方法对aroG基因进行632位碱基定点突变,获得抗反馈抑制的定点突变aroGfbr基因,并与扩增获得trpBA基因串联于pEtac表达载体上共表达, 获得重组菌Escherichia coli JM109/pEtac-aroGfbr-tac-trpBA,同时构建对照菌株 E. coli JM109/pEtac-aroG-tac-trpBA。两种串联表达质粒重组菌的DAHPS酶活性,分别比含空载体出发菌株相应酶的活性提高4.3倍和3.8倍,突变DAHPS酶活提高1.13倍。摇瓶发酵表明,所构建的两菌株与出发菌株色氨酸产量相比较,分别提高8.23倍和11.47倍,带突变aroGfbr基因的菌株比对照菌株色氨酸产量提高1.4倍,色氨酸产量明显提高,说明突变菌株能够有效解除苯丙氨酸的反馈抑制作用。图5 表2 参15
Abstract:
TrpBA-encoded tryptophan synthetase (TSase) and aroG-encoded 3-deoxy-D-arabino-heptulosonate-7- phosphate synthase (DAHPS) are key enzymes in tryptophan pathway in Escherichia coli. In order to improve bio-production of tryptophan through bioengineering means, a feedback inhibition resistant aroGfbr (C632T) gene was cloned by SOE-PCR and co-expressed with trpBA gene. The recombinant plasmids pEtac-aroGfbr-tac-trpBA and pEtac-aroG-tac-trpBA were constructed successfully in the recombinant strains, with the enzyme activity analysis indicating an increase of specific activities of DAHPS by 4.3 folds and 3.8 folds, respectively. The enzyme activity of DAHPS after mutation was increased by 1.13 folds. The yield of tryptophan biosynthesis in recombinant strain E. coli JM109/pEtac-aroGfbr-tac-trpBA was increased by 1.4 folds compared with that of the host strains E. coli JM109/pEtac-aroG-tac-trpBA. Fig 5, Tab 2, Ref 15

参考文献/References:

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备注/Memo

备注/Memo:
国家高技术研究发展计划(“863”计划, 2012AA021201)资助 Supported by the National High-tech Research and Development Program of China (“863”Program, No. 2012AA021201)
更新日期/Last Update: 2013-10-28