利用双启动子载体构建产异丁醇大肠杆菌

中国生物工程杂志

2013, Vol. 33

Issue (8): 67-74

技术与方法

利用双启动子载体构建产异丁醇大肠杆菌

郑丽娟, 陈少云, 徐刚, 吴坚平, 杨立荣

浙江大学化学工程与生物工程学系杭州 310027

Engineering E.coli for Isobutanol Production by Two-promoter Vectors

ZHENG Li-juan, CHEN Shao-yun, XU Gang, WU Jian-ping, YANG Li-rong

Zhejiang University, Hangzhou 310027, China

全文: PDF(538 KB) HTML

摘要： 为了实现异丁醇代谢途径中关键酶的高效共表达,选择具有双T7启动子的质粒载体pCDFDuet、pRSFDuet和pACYCDuet,将基因kivd和alsS分别克隆至pCDFDuet的两个启动子下,yqhD和ilvCD分别克隆至pRSFDuet或pACYCDuet的两个启动子下,构建成两种双质粒共表达系统,获得产异丁醇重组大肠杆菌Eco(CDF+RSF)和Eco(CDF+ACYC)。其中重组菌Eco(CDF+RSF)发酵产异丁醇达2.7g/L;而重组菌Eco(CDF+ACYC)产异丁醇能力较强,达3.5g/L。对两种重组菌目标蛋白的表达量及活力进行比较分析,发现重组菌Eco(CDF+ACYC)中实现了关键酶AHAS(alsS基因编码)和KDCA(kivd基因编码)的高效表达,对提高异丁醇的产量有重要的影响。利用双启动子载体成功构建了具有较高产异丁醇能力的重组菌Eco(CDF+ACYC),为后续改造以适应工业化生产打下了较好的基础。

关键词： 异丁醇; 大肠杆菌; 双T7启动子载体; 共表达

Abstract: In order to engineer E.coli for efficient isobutanol production, the two-promoter plasmids, pCDFDuet, pRSFDuet and pACYCDuet were utilized to coexpress the isobutanol biosynthetic enzymes. These plasmids are compatible, and each has two T7 promoters. The kivd and alsS genes were coexpressed using the plasmid pCDFDuet. The yqhD and ilvCD genes were coexpressed using the plamid pRSFDuet or pACYCDuet. Thus, two coexpression systems were constructed, resulting in recombinant E. coli named as Eco(CDF+RSF) and Eco(CDF+ACYC), respectively. Eco(CDF+RSF) produced isobutanol 2.7g/L, while Eco(CDF+ACYC) produced more, up to 3.5g/L. The analysis of expression level and activity of each enzyme showed that AHAS and KDCA were expressed more efficiently in Eco(CDF+ACYC), suggesting their important roles in enhanced isobutanol production. In conclusion, the recombinant E. coli Eco(ACYC+CDF) was successfully constructed using two-promoter vectors. It had high isobutanol productivity and was suitable for further engineering for industrial application.

Key words: Isobutanol E.coli two-T7 promoter vector Coexpression

收稿日期: 2013-04-11 出版日期: 2013-08-25

ZTFLH:

Q785

基金资助: 国家"973"计划(2011CB710800)资助项目

通讯作者: 吴坚平,E-mail:wjp@zju.edu.cn E-mail: wjp@zju.edu.cn

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引用本文:

郑丽娟, 陈少云, 徐刚, 吴坚平, 杨立荣. 利用双启动子载体构建产异丁醇大肠杆菌[J]. 中国生物工程杂志, 2013, 33(8): 67-74.

ZHENG Li-juan, CHEN Shao-yun, XU Gang, WU Jian-ping, YANG Li-rong. Engineering E.coli for Isobutanol Production by Two-promoter Vectors. China Biotechnology, 2013, 33(8): 67-74.

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https://manu60.magtech.com.cn/biotech/CN/ 或 https://manu60.magtech.com.cn/biotech/CN/Y2013/V33/I8/67

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