Cloning large natural product gene clusters from the environment: piecing environmental DNA gene clusters back together with TAR.
Cloning large natural product gene clusters from the environment: piecing environmental DNA gene clusters back together with TAR.
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DOI：
10.1002/bip.21450
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发表时间：
2010-09
期刊：
影响因子：
2.9
通讯作者：
Brady, Sean F.
中科院分区：
文献类型：
--
作者：
Kim, Jeffrey H.;Feng, Zhiyang;Bauer, John D.;Kallifidas, Dimitris;Calle, Paula Y.;Brady, Sean F.
一克土壤可能含有数千种独特的细菌，其中只有一小部分是在实验室中定期培养的。虽然培养微生物的发酵提供了许多具有生物活性的次生代谢物，但用这些相同的方法，不可能表征由未培养的大多数编码的天然产物。从环境样本中直接提取的DNA （eDNA）克隆的生物合成基因簇的异源表达有可能提供未培养细菌基因组中编码的化学多样性。这种方法面临的挑战之一是，许多天然产物生物合成基因簇太大，难以在克隆eDNA的单个片段上捕获。从较小的重叠克隆集合中重组大型edna衍生的天然产物基因簇代表了这个问题的一个潜在解决方案。不幸的是，从多个重叠克隆中组装大DNA序列的传统方法在技术上具有挑战性。在这里，我们提出了一个通用的实验框架，该框架允许在重叠的土壤来源的eDNA cosmid克隆上恢复大型天然产物生物合成基因簇，并在酿酒酵母中使用转化相关重组（TAR）对这些大型基因簇进行重组。从重叠的eDNA克隆集合中快速组装生物合成基因簇的实用方法的发展是能够从未培养的细菌中功能性研究更大的天然产物基因簇的重要一步。©2010 Wiley期刊公司生物工程学报（英文版），2010。
A single gram of soil can contain thousands of unique bacterial species, of which only a small fraction is regularly cultured in the laboratory. Although the fermentation of cultured microorganisms has provided access to numerous bioactive secondary metabolites, with these same methods it is not possible to characterize the natural products encoded by the uncultured majority. The heterologous expression of biosynthetic gene clusters cloned from DNA extracted directly from environmental samples (eDNA) has the potential to provide access to the chemical diversity encoded in the genomes of uncultured bacteria. One of the challenges facing this approach has been that many natural product biosynthetic gene clusters are too large to be readily captured on a single fragment of cloned eDNA. The reassembly of large eDNA-derived natural product gene clusters from collections of smaller overlapping clones represents one potential solution to this problem. Unfortunately, traditional methods for the assembly of large DNA sequences from multiple overlapping clones can be technically challenging. Here we present a general experimental framework that permits the recovery of large natural product biosynthetic gene clusters on overlapping soil-derived eDNA cosmid clones and the reassembly of these large gene clusters using transformation-associated recombination (TAR) in Saccharomyces cerevisiae. The development of practical methods for the rapid assembly of biosynthetic gene clusters from collections of overlapping eDNA clones is an important step toward being able to functionally study larger natural product gene clusters from uncultured bacteria. © 2010 Wiley Periodicals, Inc. Biopolymers 93: 833–844, 2010.
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影响因子：
14.8
作者：
Gietz, R. Daniel;Schiestl, Robert H.
通讯作者：
Schiestl, Robert H.
影响因子：
14.9
作者：
Gibson DG
通讯作者：
Gibson DG
影响因子：
14.8
作者：
Brady, Sean F.
通讯作者：
Brady, Sean F.
影响因子：
14.8
作者：
Kouprina, Natalay;Larionov, Vladimir
通讯作者：
Larionov, Vladimir
DOI：
10.1073/pnas.0807564105
发表时间：
2008-11-11
影响因子：
11.1
作者：
Banik, Jacob J.;Brady, Sean F.
通讯作者：
Brady, Sean F.