利用双报道基因筛选系统改造产烃基因工程细菌(英文)  

Engineering Alkane-Producing Bacteria through A Dual-Reporter Screening System

作　　者：岳海兵[1] 赵云[1] 杭海英[1] 

机构地区：[1]中国科学院生物物理研究所,生物大分子国家重点实验室,蛋白质与多肽药物所重点实验室,北京100101

出　　处：《生物物理学报》2014年第6期424-432,共9页Acta Biophysica Sinica

基　　金：supported by grants from The "973" Program(2011CBA00906);The National Special Projects for Key Scientific Instruments and Equipment Development of Ministry of Science and Technology(2011YQ03013404);The State Key Laboratory for Microbial Resources,Institute of Microbiology,Chinese Academy of Sciences(SKLMR-20130605)~~

摘　　要：自然界中所有的蓝藻都能产生烷烃,但这些烷烃都比汽油（C5~C12）的链长更长。两个源于蓝藻的产烃基因已经在大肠杆菌细胞中成功表达。迄今为止,从头合成的最短烷烃为C9。本研究在大肠杆菌细胞中设计了一个人工进化系统,该系统能生产短链烷烃,并可以提高烷烃的产量。该进化系统由两大部分元件组成：产烃基因和胞内烷烃响应系统。烷烃是报道基因氯霉素抗性基因（Cm R）和16S r DNA的一种主要诱导物。携带有Cm R的细胞能在含有氯霉素（Cm）的培养基中存活下来,而烷烃诱导产生更多的16S RNA,能加快细胞的生长和繁殖。将这些基因组装在一种易突变的大肠杆菌菌株中,并成功地筛选出了具有生长优势和高产烷烃能力的细菌。最后,用这些细菌实现了辛烷（C8）的生产——在不加入产烃底物的情况下,创造了在人工生物系统中产生最短链长烷烃的记录。以上结果说明,在大肠杆菌中生产比C9更短的烷烃不存在机理方面的限制。All the natural cyanobacteria can produce alkanes that are longer than the major constituents of gasoline （C5-C12）. Two alkane-producing genes from cyanobacteria have been heterologously expressed in E. coli. Thus far, the shortest alkane de novo generated by engineered bacterial systems is C9. In this study, the authors designed an artificial evolution system in E. coil to produce short chain alkanes and to improve alkane yield. The evolution system consists of two alkane-producing genes and an intracelluler octane-responsive system. Alkane is a key inducer of the reporter genes chloramphenicol resistance gene （CmR） and 16S rDNA; CmR supports the survival of the CmR-carrying bacteria in medium containing chloramphenicol （Cm）, and a high level of 16S RNA induces the bacterial proliferation. The genes were assembled in a mutator-bacterial strain, and mutant bacteria with growth-advantage and high yield of alkanes were successfully screened. Finally, octane （C8） was successfully produced using these bacteria-a record of the shortest alkane generated by an artificial biosystem without adding substrates in medium. These data suggest that there is no mechanistic limitation to produce alkanes shorter than C9 in E. coli.

关 键 词：自进化 16S RDNA 烷烃诱导 微生物烷烃 

分 类 号：Q319.33[生物学—遗传学]