Expression of heterologous sigma factors enables functional screening of metagenomic and heterologous genomic libraries.
Expression of heterologous sigma factors enables functional screening of metagenomic and heterologous genomic libraries.
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DOI：
10.1038/ncomms8045
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发表时间：
2015-05-06
影响因子：
16.6
通讯作者：
Papoutsakis ET
中科院分区：
文献类型：
--
作者：
Gaida SM;Sandoval NR;Nicolaou SA;Chen Y;Venkataramanan KP;Papoutsakis ET
在功能基因组学和基因组工程中使用异源基因组或宏基因组文库的一个关键限制是异源基因在筛选宿主（如大肠杆菌）中表达量低。为克服这一限制，我们在此通过表达异源σ因子构建了能够识别异源启动子的大肠杆菌菌株。在测试的七种σ因子中，植物乳杆菌（Lpl）的RpoD似乎能够启动来自所有DNA来源的转录。利用启动子绿色荧光蛋白捕获概念，我们成功筛选了几个异源和宏基因组DNA文库，从而扩大了可在大肠杆菌中进行功能取样的基因组空间。作为一个应用实例，我们表明在一个染色体整合了植物乳杆菌rpoD的大肠杆菌菌株中筛选基于fosmid的植物乳杆菌基因组文库，能够鉴定赋予大肠杆菌强乙醇耐受性的植物乳杆菌遗传决定因素。转录组分析证实了工程菌株中异源基因表达的增加。
筛选基因组或宏基因组文库以寻找有价值的产物或活性常常因异源宿主中基因表达不佳而受阻。在此作者表明，一种乳杆菌σ因子的表达极大地增强了大肠杆菌中异源和环境DNA的转录。
A key limitation in using heterologous genomic or metagenomic libraries in functional genomics and genome engineering is the low expression of heterologous genes in screening hosts, such as Escherichia coli. To overcome this limitation, here we generate E. coli strains capable of recognizing heterologous promoters by expressing heterologous sigma factors. Among seven sigma factors tested, RpoD from Lactobacillus plantarum (Lpl) appears to be able of initiating transcription from all sources of DNA. Using the promoter GFP-trap concept, we successfully screen several heterologous and metagenomic DNA libraries, thus enlarging the genomic space that can be functionally sampled in E. coli. For an application, we show that screening fosmid-based Lpl genomic libraries in an E. coli strain with a chromosomally integrated Lpl rpoD enables the identification of Lpl genetic determinants imparting strong ethanol tolerance in E. coli. Transcriptome analysis confirms increased expression of heterologous genes in the engineered strain. Screening genomic or metagenomic libraries for interesting products or activities is often hampered by poor gene expression in a heterologous host. Here the authors show that the expression of a Lactobacillus sigma factor greatly enhances transcription of heterologous and environmental DNA in E. coli.
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影响因子：
2.7
作者：
Couto, JA;Pina, C;Hogg, T
通讯作者：
Hogg, T
影响因子：
3
作者：
Knoshaug, Eric P.;Zhang, Min
通讯作者：
Zhang, Min
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Jarmer, H;Larsen, TS;Knudsen, S
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Knudsen, S
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McClure R;Balasubramanian D;Sun Y;Bobrovskyy M;Sumby P;Genco CA;Vanderpool CK;Tjaden B
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Tjaden B
影响因子：
2.1
作者：
G-Alegría, E;López, I;Ruiz-Larrea, F
通讯作者：
Ruiz-Larrea, F