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[1]刘春宇,陈燕,黄金群,等.厌氧梭菌表达载体在大肠杆菌中的直接表达及活性筛选[J].应用与环境生物学报,2013,19(05):822-826.[doi:10.3724/SP.J.1145.2013.00822]
 LIU Chunyu,CHEN Yan,HUANG Jinqun,et al.Direct Construction and Screening of the Expression Vector of Anaerobic Clostridium in Escherichia coli[J].Chinese Journal of Applied & Environmental Biology,2013,19(05):822-826.[doi:10.3724/SP.J.1145.2013.00822]
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厌氧梭菌表达载体在大肠杆菌中的直接表达及活性筛选()
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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:
822-826
栏目:
研究论文
出版日期:
2013-10-25

文章信息/Info

Title:
Direct Construction and Screening of the Expression Vector of Anaerobic Clostridium in Escherichia coli
作者:
刘春宇陈燕黄金群裴建新庞浩黄日波
(1广西大学生命科学与技术学院 南宁 530005) (2广西科学院国家非粮生物质能源工程技术研究中心 南宁 530003)
Author(s):
LIU Chunyu CHEN Yan HUANG Jinqun PEI Jianxin PANG Hao HUANG Ribo
(1Life Science and Technology College, Guangxi University, Nanning 530005, China) (2National Engineering Research Center for Non-food Biorefinery, Guangxi Academy of Sciences, Nanning 530003, China)
关键词:
厌氧梭菌硫解酶启动子大肠杆菌重组表达 纤维素酶菌株筛选
Keywords:
anaerobic Clostridium thiolase promoter Escherichia coli recombinant expression cellulase strain screening
分类号:
Q936 : Q78
DOI:
10.3724/SP.J.1145.2013.00822
摘要:
厌氧微生物的代谢途径改造、重组基因表达等分子生物学操作步骤多,易造成氧气接触,从而导致厌氧微生物死亡. 针对厌氧梭菌-大肠杆菌穿梭载体的构建和筛选问题,利用纤维素内切酶基因Cphy3367作为报告基因,分析穿梭载体的硫解酶启动子在大肠杆菌中启动基因转录表达的可能性. 含有重组质粒pSOS95-Cphy3367的大肠杆菌菌株相对于对照菌株生长迟缓,生物量降低. 实时定量PCR检测及重组蛋白酶活检测结果中,发现硫解酶启动子在大肠杆菌中能启动基因转录表达,在37 ℃温度下48 h后达到高峰. 表明硫解酶启动子在大肠杆菌中可以被宿主识别和启动基因表达,基因可以翻译成为蛋白质. 在厌氧梭菌的穿梭载体构建中,含有硫解酶启动子的载体可以在大肠杆菌中进行重组载体的活性筛选这个步骤,从而降低梭菌厌氧操作的复杂度和难度. 图4 表1 参24
Abstract:
The complex procedures of molecular biological experiments with anaerobic microbes, such as reconstruction of metabolic pathway and expression of the recombinant gene, would introduce oxygen easily, which may result in death of the anaerobic microorganisms. This study was aimed at the problem that the process of construction and screening of shuttle vector in Clostridium would make strains be exposed to oxygen and die. The possibility of initializing gene transcription by the thiolase promoter in Escherichia coli was analyzed using endoglucanase gene Cphy3367 as a reporter gene. Comparing with control strain, recombinant strains contained pSOS95-Cphy3367 plasmid grown more slowly and had a biomass reduction. The results of real-time quantitative PCR and activity detection of recombinant enzyme showed that the thiolase promoter in E. coli initiated the expression of gene Cphy3367. The initiation of gene Cphy3367 peaked at 48 h incubation at 37 ℃. In conclusion, the thiolase promoter can be recognized by host cell and the gene can be translated into protein in E. coli. The anaerobic Clostridium shuttle vector containing the thiolase promoter can be actively screened in E. coli, thereby reduce complexity and difficulty of the anaerobic operation. Fig 4, Tab 1, Ref 24

参考文献/References:

1 Imlay JA. How obligatory is anaerobiosis? [J]. Mol Microbiol, 2008, 68 (4): 801-804 2 Speers AM, Cologgi DL, Reguera G. Anaerobic cell culture [J]. Curr Protoc Microbiol, 2009, Appendix 4: Appendix 4F 3 Bacic MK, Smith CJ. Laboratory maintenance and cultivation of bacteroides species [A]. In: Coico R, McBride A, Quarles J, Stevenson B, Taylor R eds. Current Protocols in Microbiology [C]. Hoboken, New Jersey: John Wiley & Sons, Inc., 2008, Chapter 13: Unit 13C 11 4 Behnken S, Hertweck C. Anaerobic bacteria as producers of antibiotics [J]. Appl Microbiol Biotechnol, 2012, 96 (1): 61-67 5 Collas F, Kuit W, Clement B, Marchal R, Lopez-Contreras AM, Monot F. Simultaneous production of isopropanol, butanol, ethanol and 2,3-butanediol by Clostridium acetobutylicum ATCC 824 engineered strains [J]. AMB Express, 2012, 2 (1): 45 6 Schiel-Bengelsdorf B, Durre P. Pathway engineering and synthetic biology using acetogens [J]. FEBS Lett, 2012, 586 (15): 2191-2198 7 龙飞, 靳艳玲, 赵云, 郜晓峰, 李宇浩, 陈谦, 赵海. 丙酮丁醇梭菌Clostridium acetobutylicum CICC 8012发酵鲜芭蕉芋生产丁醇[J]. 应用与环境生物学报, 2013, 19 (1): 54-60 [Long F, Jin Y, Zhao Y, Gao X, Li Y, Chen Q, Zhao H. Optimization of butanol production from Canna edulis Ker by Clostridium acetobutylicum using response surface methodology. Chin J Appl Environ Biol, 2013, 19 (1): 54-60] 8 Strobel HJ. Basic laboratory culture methods for anaerobic bacteria [J]. Methods Mol Biol, 2009, 581: 247-261 9 Napoli F, Olivieri G, Russo ME, Marzocchella A, Salatino P. Butanol production by Clostridium acetobutylicum in a continuous packed bed reactor [J]. J Ind Microbiol Biotechnol, 2010, 37 (6): 603-608 10 Perret S, Casalot L, Fierobe HP, Tardif C, Sabathe F, Belaich JP, Belaich A. Production of heterologous and chimeric scaffoldins by Clostridium acetobutylicum ATCC 824 [J]. J Bacteriol, 2004, 186 (1): 253-257 11 Kawasaki S, Watamura Y, Ono M, Watanabe T, Takeda K, Niimura Y. Adaptive responses to oxygen stress in obligatory anaerobes Clostridium acetobutylicum and Clostridium aminovalericum [J]. Appl Environ Microbiol, 2005, 71 (12): 8442-8450 12 Dong H, Tao W, Zhu L, Zhang Y, Li Y. CAC2634-disrupted mutant of Clostridium acetobutylicum can be electrotransformed in air [J]. Lett Appl Microbiol, 2011, 53 (3): 379-382 13 Perret S, Belaich A, Fierobe HP, Belaich JP, Tardif C. Towards designer cellulosomes in Clostridia: mannanase enrichment of the cellulosomes produced by Clostridium cellulolyticum [J]. J Bacteriol, 2004, 186 (19): 6544-6552 14 Heap JT, Pennington OJ, Cartman ST, Minton NP. A modular system for Clostridium shuttle plasmids [J]. J Microbiol Methods, 2009, 78 (1): 79-85 15 付军涛, 祁高富, 刘军, 冀志霞, 马昕, 陈守文. 两种启动子对聚γ-谷氨酸降解酶基因在地衣芽胞杆菌中的加强表达效果[J]. 应用与环境生物学报, 2012, 18 (3): 450-454 [Fu J, Qi G, Liu J, Ji Z, Ma X, Chen S. Enhancing expression of ywtD gene in Bacillus licheniformis WX-02 by two types of promoters [J]. Chin J Appl Environ Biol, 2012, 18 (3): 450-454] 16 Tomas CA, Welker NE, Papoutsakis ET. Overexpression of groESL in Clostridium acetobutylicum results in increased solvent production and tolerance, prolonged metabolism, and changes in the cell’s transcriptional program [J]. Appl Environ Microbiol, 2003, 69 (8): 4951-4965 17 Warnick TA, Methe BA, Leschine SB. Clostridium phytofermentans sp. nov., a cellulolytic mesophile from forest soil [J]. Int J Syst Evol Microbiol, 2002, 52 (Pt 4): 1155-1160 18 Tolonen AC, Chilaka AC, Church GM. Targeted gene inactivation in Clostridium phytofermentans shows that cellulose degradation requires the family 9 hydrolase Cphy3367 [J]. Mol Microbiol, 2009, 74 (6): 1300-1313 19 张颖, 贾芸, 吕军. 大肠杆菌σ70启动子的识别[J]. 生物物理学报, 2007, 23 (6): 475-481 [Zhang Y, Jia Y, Lü J. The recognition of σ70 promoters in Escherichia coli K-12 [J]. Acta Biophys Sin, 2007, 23 (6): 475-481] 20 Pribnow D. Nucleotide sequence of an RNA polymerase binding site at an early T7 promoter [J]. Proc Natl Acad Sci USA, 1975, 72 (3): 784-788 21 Seeburg PH, Nüsslein C, Schaller H. Interaction of RNA polymerase with promoters from bacteriophage fd [J]. Eur J Biochem, 1977, 74 (1): 107-113 22 Wiesenborn DP, Rudolph FB, Papoutsakis ET. Thiolase from Clostridium acetobutylicum ATCC 824 and its role in the synthesis of acids and solvents [J]. Appl Environ Microbiol, 1988, 54 (11): 2717-2722 23 Zhu L, Dong H, Zhang Y, Li Y. Engineering the robustness of Clostridium acetobutylicum by introducing glutathione biosynthetic capability [J]. Metab Eng, 2011, 13 (4): 426-434 24 Lutke-Eversloh T, Bahl H. Metabolic engineering of Clostridium acetobutylicum: recent advances to improve butanol production [J]. Curr Opin Biotechnol, 2011, 22 (5): 634-647

备注/Memo

备注/Memo:
国家自然科学基金项目(21366007)和广西科学研究与技术开发项目(1347004-1)资助 Supported by the National Natural Science Foundation of China (No. 21366007), and the Guangxi Program for Science and Technology Development (No. 1347004-1)
更新日期/Last Update: 2013-10-28