Coupled high-throughput functional screening and next generation sequencing for identification of plant polymer decomposing enzymes in metagenomic libraries.
Coupled high-throughput functional screening and next generation sequencing for identification of plant polymer decomposing enzymes in metagenomic libraries.
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DOI：
10.3389/fmicb.2013.00282
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发表时间：
2013
影响因子：
5.2
通讯作者：
Brodie EL
中科院分区：
文献类型：
--
作者：
Nyyssönen M;Tran HM;Karaoz U;Weihe C;Hadi MZ;Martiny JB;Martiny AC;Brodie EL
测序技术的最新进展对环境微生物的功能作用产生了新的预测和假设。然而，在我们能够以与我们产生预测的能力相匹配的规模测试这些预测之前，它们中的大多数仍将是假设。基于功能的元基因组文库挖掘可以提供基因、代谢性状和微生物类群之间的直接联系，从而弥合序列数据生成和功能预测之间的差距。在这里，我们从环境宏基因组文库中开发了高通量筛选方法，用于基于功能的植物聚合物分解活性的表征。多重检测使用荧光和显色底物，结合自动液体处理和使用转基因表达宿主，能够同时筛选12,160个克隆，在总共170,240个反应中进行14个活性。利用该平台，从凋落叶分解制备的fosmid文库中鉴定出374个（0.26%）纤维素、半纤维素、几丁质、淀粉、磷酸盐和蛋白质水解克隆。在Illumina MiSeq平台上进行测序，随后对95个fosmid克隆子集进行组装和基因预测，鉴定出广泛的细菌门，包括放线菌门，拟杆菌门，多个变形菌门亚门以及一些真菌。对20个不同糖苷水解酶（GH）家族的糖活性酶基因进行了检测。利用fosmid序列的四核苷酸频率（TNF）分组，来自不同fosmid的多种酶活性被联系起来，证明了环境宏基因组中生物化学确认的功能性状如何归因于特定生物群体。总体而言，我们的研究结果表明，宏基因组文库的功能筛选可以用于将微生物功能与群落组成联系起来，从而补充大规模宏基因组测序工作。
Recent advances in sequencing technologies generate new predictions and hypotheses about the functional roles of environmental microorganisms. Yet, until we can test these predictions at a scale that matches our ability to generate them, most of them will remain as hypotheses. Function-based mining of metagenomic libraries can provide direct linkages between genes, metabolic traits and microbial taxa and thus bridge this gap between sequence data generation and functional predictions. Here we developed high-throughput screening assays for function-based characterization of activities involved in plant polymer decomposition from environmental metagenomic libraries. The multiplexed assays use fluorogenic and chromogenic substrates, combine automated liquid handling and use a genetically modified expression host to enable simultaneous screening of 12,160 clones for 14 activities in a total of 170,240 reactions. Using this platform we identified 374 (0.26%) cellulose, hemicellulose, chitin, starch, phosphate and protein hydrolyzing clones from fosmid libraries prepared from decomposing leaf litter. Sequencing on the Illumina MiSeq platform, followed by assembly and gene prediction of a subset of 95 fosmid clones, identified a broad range of bacterial phyla, including Actinobacteria, Bacteroidetes, multiple Proteobacteria sub-phyla in addition to some Fungi. Carbohydrate-active enzyme genes from 20 different glycoside hydrolase (GH) families were detected. Using tetranucleotide frequency (TNF) binning of fosmid sequences, multiple enzyme activities from distinct fosmids were linked, demonstrating how biochemically-confirmed functional traits in environmental metagenomes may be attributed to groups of specific organisms. Overall, our results demonstrate how functional screening of metagenomic libraries can be used to connect microbial functionality to community composition and, as a result, complement large-scale metagenomic sequencing efforts.
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影响因子：
3.7
作者：
Chow J;Kovacic F;Dall Antonia Y;Krauss U;Fersini F;Schmeisser C;Lauinger B;Bongen P;Pietruszka J;Schmidt M;Menyes I;Bornscheuer UT;Eckstein M;Thum O;Liese A;Mueller-Dieckmann J;Jaeger KE;Streit WR
通讯作者：
Streit WR
大肠杆菌K-12的构造框架，单基因敲除突变体：Keio Collection。
影响因子：
9.9
作者：
通讯作者：
--
影响因子：
4.4
作者：
Berlemont, Renaud;Martiny, Adam C.
通讯作者：
Martiny, Adam C.
DOI：
10.1073/pnas.120163297
发表时间：
2000-06-06
影响因子：
11.1
作者：
Datsenko, KA;Wanner, BL
通讯作者：
Wanner, BL
影响因子：
4.4
作者：
Chung, Eu Jin;Lim, He Kyoung;Lee, Seon-Woo
通讯作者：
Lee, Seon-Woo