产乙醇大肠杆菌的构建及其活性检测方法的改进

中国生物工程杂志

2011, Vol. 31

Issue (8): 73-78

技术与方法

产乙醇大肠杆菌的构建及其活性检测方法的改进

姜进举^1,3, 张宏宇^1,3, 任宝永^2,3, 施斐^1,3, 崔玉琳^2,3, 秦松

1. 中国科学院烟台海岸带研究所烟台 264003;
2. 中国科学院海洋研究所青岛 266071;
3. 中国科学院研究生院北京 100049

Construction of Alcohol-producing Escherichia coli strains and Improvement of Biological Activity Detection Method for the Engineered Strains

JIANG Jin-ju^1,3, ZHANG Hong-yu^1,3, REN Bao-yong^2,3, SHI Fei^1,3, CUI Yu-lin^2,3, QIN Song

1. Yantai Institute of Coastal Zone Research,Chinese Academy of Sciences,Yantai 264003,China;
2. Institute of Oceanology,Chinese Academy of Sciences,Qingdao 266071,China;
3. Graduate University of Chinese Academy of Sciences,Beijing 100049,China

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摘要：

adhB和pdc是运动发酵单胞菌产乙醇途径的关键基因,分别编码乙醇脱氢酶和丙酮酸脱羧酶,将添加有聚球藻PCC7942rbcLS基因RBS序列的adhB和pdc基因插入pUC18载体,经双重菌液PCR检验和酶切检验得到分别含有pUC-adhB、pUC-pdc和pUC-adhB-pdc载体的3个重组菌株。活性检测实验表明聚球藻PCC7942的rbcLS基因的RBS序列能有效介导运动发酵单胞菌的adhB和pdc基因在大肠杆菌中表达,摇瓶发酵实验表明重组大肠杆菌的产乙醇能力较出发菌株大幅提升。鉴于乙醛指示平板法存在着对希夫试剂的要求较高、易产生较强的背景色等缺点,对定性检测丙酮酸脱羧酶和乙醇脱氢酶表达菌株的方法做了改进,即:将菌液诱导表达,然后分别添加对应于两种酶的底物,让酶与底物反应0.5至1小时,之后再加希夫试剂进行显色反应,结果表明改进后的方法比乙醛指示平板法更加简便、快速、可靠。

关键词： 生物乙醇; 丙酮酸脱羧酶; 乙醇脱氢酶; 乙醛指示平板; 运动发酵单胞菌; 蓝藻

Abstract:

The genes of adhB and pdc,coding ADHII and PDC respectively,are key genes of alcohol production pathway in Zymomonas mobilis.The following vectors,pUC-adhB,pUC-pdc and pUC-adhB-pdc,were created by inserting adhB and pdc into the plasmid pUC18. Both adhB and pdc have a ribosome binding site of rbcLS of Synechococcus sp.PCC7942. The results of biological activity detections showed that ribosome binding sites of rbcLS of PCC7942 can effectively induce the expression of adhB and pdc in E.coli. Jar fermentation showed that the recombined E.coli's ability of ethanol production was obviously improved compared with the initial strain. Aldehyde indicator plate was displaced by a new method for Biological activity detection because of its defects.The new method was as follows. Induced expression of the genes by IPTG,add substrates for the enzymes ADHII and PDC,cultivate for 0.5h to 1h at 37℃ and then add Schiff reagent.The results show that the improved method is more simple and reliable than Aldehyde indicator plate.

Key words: Fuel alcohol Pyruvate decarboxylase(PDC) Alcohol Dehydrogenase(ADH) Aldehyde indicator plate Zymomonas mobilis Synechococcus sp. PCC7942

收稿日期: 2011-05-11 出版日期: 2011-08-25

ZTFLH:

Q812

基金资助:

国家自然科学基金(40876082)、山东省自然科学杰出青年基金(JQ200914)资助项目

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引用本文:

姜进举, 张宏宇, 任宝永, 施斐, 崔玉琳, 秦松. 产乙醇大肠杆菌的构建及其活性检测方法的改进[J]. 中国生物工程杂志, 2011, 31(8): 73-78.

JIANG Jin-ju, ZHANG Hong-yu, REN Bao-yong, SHI Fei, CUI Yu-lin, QIN Song. Construction of Alcohol-producing Escherichia coli strains and Improvement of Biological Activity Detection Method for the Engineered Strains. China Biotechnology, 2011, 31(8): 73-78.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/ 或 https://manu60.magtech.com.cn/biotech/CN/Y2011/V31/I8/73

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