大肠杆菌中核黄素的生物合成途径改造及生产

摘要

利用PCR技术从枯草芽孢杆菌(Bacillus subtilis 168)中扩增出3.5 kb的核黄素操纵子,将其分别连接到不同拷贝数的表达载体pSC101、p15A、pBR322,得到重组载体pSC101-BSrib、p15A-BSrib和pBR322-BSrib,并分别转化到大肠杆菌(Escherichia coli K-12 MG1655)。对含有核黄素操纵子的重组大肠杆菌进行摇瓶发酵,结果表明其核黄素合成能力随着质粒拷贝数的增加而增强。随后对E. coli K-12 MG1655 ECX3菌株的诱导剂IPTG浓度和发酵温度进行了优化。结果显示,0.1 mmol·L^-1 IPTG和42 ℃为核黄素生产的最适宜条件。在此条件下,菌株ECX3在LB培养基中核黄素产量达到251.4 mg·L^-1。最后,通过无痕基因操作技术,减弱了工程菌株ECX3核黄素激酶/黄素腺嘌呤二核苷酸氨酰转移酶(ribF)的表达以减少核黄素转化为FMN和FAD,摇瓶中工程菌株ECX4的核黄素产量提高到292.3 mg·L^-1。

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	徐志博
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关键词：核黄素大肠杆菌枯草芽孢杆菌 rib操纵子核黄素激酶/黄素腺嘌呤二核苷酸氨酰转移酶

Abstract：

A 3.5 kb rib operon of Bacillus subtilis 168 was amplified by PCR and cloned into different copy number of plasmids, including pSC101, p15A and pBR322. The resulting plasmids, pSC101-BSrib, p15A-BSrib and pBR322-BSrib, were transformed into Escherichia coli K-12 MG1655. The results showed that, in shaking-flask fermentation, the riboflavin titer was higher as the plasmid copy number increased. Then the fermentation conditions of E. coli K-12 MG1655 ECX3 were optimized. It was founded that the optimum culturing conditions for producing riboflavin were adding 0.1 mmol·L^-1 IPTG and incubating at 42 ℃. Under above condition, strain ECX3 produced 251.4 mg·L^-1 riboflavin in LB medium with 10 g·L^-1 glucose. Finally, we decreased bifunctional riboflavin kinase/FMN adenylyltransferase (encoded by ribF gene) activity to reduce the conversion of riboflavin to FMN and FAD by a scarless gene manipulation method. The final riboflavin production strain ECX4 produced 292.3 mg·L^-1 riboflavin in shaking-flask experiments.

Keywords： riboflavin； Escherichia coli； Bacillus subtilis； rib operon； riboflavin kinase/FMN adenylyltransferase

Received 2014-04-01;

Fund:

国家重点基础研究发展计划(2011CBA00804, 2012CB725203),国家高技术研究发展计划(2012AA02A702, 2012AA022103)。

Corresponding Authors: 陈涛,E-mail:chentao@tju.edu.cn。 Email: chentao@tju.edu.cn

About author: 徐志博(1987-),女,硕士研究生,主要从事代谢工程方向的研究。

引用本文:

徐志博, 林振泉, 陈涛.大肠杆菌中核黄素的生物合成途径改造及生产[J]. 化学工业与工程, 2016,33(2): 85-90

Xu Zhibo, Lin Zhenquan, Chen Tao.Engineering Riboflavin Biosynthesis Pathway in Escherichia coli for Riboflavin Production[J]. Chemcial Industry and Engineering, 2016,33(2): 85-90

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