Substrate-induced gene-expression screening of environmental metagenome libraries for isolation of catabolic genes
Substrate-induced gene-expression screening of environmental metagenome libraries for isolation of catabolic genes
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DOI：
10.1038/nbt1048
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发表时间：
2005-01-01
影响因子：
46.9
通讯作者：
Watanabe, K
中科院分区：
文献类型：
--
作者：
Uchiyama, T;Abe, T;Watanabe, K
最近人们意识到环境中的大多数微生物对培养具有抗性，这促使科学家直接从环境宏基因组中克隆有用的基因(1)。目前宏基因组方法有两种筛选方法，即基于核苷酸序列的筛选(2)和基于酶活性的筛选(3)。在这里，我们引入并优化了用于分离新型分解代谢操纵子的第三种选择，即底物诱导基因表达筛选（SIGEX）。该方法基于以下知识：分解代谢基因表达通常由相关底物诱导，并且在许多情况下由位于分解代谢基因附近的调节元件控制。为了使 SIGEX 具有高通量，我们构建了一种可用于鸟枪式克隆的操纵子陷阱 gfp 表达载体，该载体允许通过荧光激活细胞分选来选择液体培养物中的阳性克隆。从地下水宏基因组库中克隆芳香烃诱导的基因以及随后的基因组信息学分析证明了 SIGEX 的实用性。
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.