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[1]程毅鹏,饶志明,杨套伟,等.一种新型抗性质粒的构建及其在核黄素生产菌中的运用[J].应用与环境生物学报,2015,21(03):435-440.[doi:10.3724/SP.J.1145.2014.12003]
 CHENG Yipeng,RAO Zhiming,YANG Taowei,et al.Construction of a new resistance plasmid capable of riboflavin production in Bacillus subtilis RF1[J].Chinese Journal of Applied & Environmental Biology,2015,21(03):435-440.[doi:10.3724/SP.J.1145.2014.12003]
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一种新型抗性质粒的构建及其在核黄素生产菌中的运用()
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《应用与环境生物学报》[ISSN:1006-687X/CN:51-1482/Q]

卷:
21卷
期数:
2015年03期
页码:
435-440
栏目:
研究论文
出版日期:
2015-06-25

文章信息/Info

Title:
Construction of a new resistance plasmid capable of riboflavin production in Bacillus subtilis RF1
作者:
程毅鹏饶志明杨套伟满在伟徐美娟张显
江南大学生物工程学院,工业生物技术教育部重点实验室 无锡 214122
Author(s):
CHENG Yipeng RAO Zhiming YANG Taowei MAN Zaiwei XU Meijuan ZHANG Xian
Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, China
关键词:
枯草芽孢杆菌抗性质粒核黄素葡萄糖-6-磷酸脱氢酶
Keywords:
Bacillus subtilis resistance plasmid riboflavin glucose-6-phosphate dehydrogenase
分类号:
Q78 : Q936
DOI:
10.3724/SP.J.1145.2014.12003
文献标志码:
A
摘要:
枯草芽孢杆菌Bacillus subtilis RF1是课题组前期通过基因工程改造得到的一株核黄素高产菌,为了进一步提高核黄素的产量,需要对该菌株进行进一步的基因工程改造. 本研究首先将编码的链丝菌素乙酰基转移酶基因sat克隆到pMA5质粒上,构建具有诺瓦丝菌素Norseothricin(NTC)抗性的重组质粒pMA5-sat,实验证实该重组质粒能够用于B. subtilis RF1的抗性筛选. 随后将核黄素合成相关的关键酶葡萄糖-6-磷酸脱氢酶编码基因zwf克隆到重组质粒pMA5-sat上,获得重组质粒pMA5-sat-zwf,并成功构建重组菌B. subtilis RF1/pMA5-sat-zwf. 结果显示,重组菌胞内葡萄糖-6-磷酸脱氢酶活力比原始菌提高了近50倍,说明葡萄糖-6-磷酸脱氢酶在重组菌中成功过量表达;根据发酵特性分析,重组菌B. subtilis RF1/pMA5-sat-zwf最终核黄素产量达到12.01 g/L,比原始菌B. subtilis RF1提高了30.3%. 综上,本研究构建的新型抗性质粒能够成功运用于核黄素生产菌枯草芽孢杆菌的基因工程改造.
Abstract:
In our previous study, a genetically engineered strain Bacillus subtilis RF1 was constructed for riboflavin production. To enhance riboflavin production, further genetic modification was required to operate on B. subtilis RF1. The sat gene encoded Streptothricin acetyltransferase was inserted into expression vector pMA5, and the recombinant plasmid pMA5-sat was constructed and transformed into B. subtilis RF1. Glucose-6-phosphate dehydrogenase encoded by gene zwf is one of key enzymes involved in pentose phosphate pathway. And gene zwf harbored in the new plasmid pMA5-sat was constructed and was successfully transformed into B. subtilis RF1 to obtain recombinant strain B. subtilis RF1/pMA5-sat-zwf. The intracellular glucose-6-phosphate dehydrogenase activity of recombinant strain B. subtilis RF1/pMA5-sat-zwf was about 50-fold higher than that of strain B. subtilis RF1. The fermentation results showed that the riboflavin production by recombinant strain B. subtilis RF1/pMA5-sat-zwf reached 12.01 g/L, which was 30.3% higher than that by strain B. subtilis RF1. The results indicate that this new resistance plasmid can be successfully used in riboflavin-producing strain B. subtilis RF1.

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备注/Memo

备注/Memo:
国家重点基础研究发展计划(973计划)项目(2012CB725202)、国家自然科学基金项目(31400082)、教育部重点基金(113033A)、江苏高校优势学科建设工程资助项目和江苏省现代发酵工业协同创新中心资助 Supported by the State Key Basic R & D Program of China (973 Program, 2012CB725202), the National Natural Science Foundation of China (31400082), the Research Project of Chinese Ministry of Education (113033A), the Priority Academic Program Development of Jiangsu Higher Education Institutions, and the Collaborative Innovation Center for Advanced Industrial Fermentation of Jiangsu Province
更新日期/Last Update: 2015-06-23