Substrate-induced gene-expression screening of environmental metagenome libraries for isolation of catabolic genes

被引:220
作者
Uchiyama, T
Abe, T
Ikemura, T
Watanabe, K
机构
[1] Marine Biotechnol Inst, Appl Microbiol Lab, Kamaishi, Iwate 0260001, Japan
[2] Natl Inst Genet, Dept Populat Genet, Shizuoka 4118540, Japan
[3] Grad Univ Adv Studies, Shizuoka 4118540, Japan
[4] Xanagen Inc, Nakano Ku, Tokyo 1640003, Japan
关键词
D O I
10.1038/nbt1048
中图分类号
Q81 [生物工程学(生物技术)]; Q93 [微生物学];
学科分类号
071005 ; 0836 ; 090102 ; 100705 ;
摘要
Recent awareness that most microorganisms in the environment are resistant to cultivation has prompted scientists to directly clone useful genes from environmental metagenomes(1). Two screening methods are currently available for the metagenome approach, namely, nucleotide sequence-based screening(2) and enzyme activity-based screening(3). Here we have introduced and optimized a third option for the isolation of novel catabolic operons, that is, substrate-induced gene expression screening (SIGEX). This method is based on the knowledge that catabolic-gene expression is generally induced by relevant substrates and, in many cases, controlled by regulatory elements situated proximate to catabolic genes. For SIGEX to be high throughput, we constructed an operon-trap gfp-expression vector available for shotgun cloning that allows for the selection of positive clones in liquid cultures by fluorescence-activated cell sorting. The utility of SIGEX was demonstrated by the cloning of aromatic hydrocarbon-induced genes from a groundwater metagenome library and subsequent genome-informatics analysis.
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收藏
页码:88 / 93
页数:6
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