Laboratory of Genetically Encoded Small Molecules, Howard Hughes Medical Institute, The Rockefeller University, New York, New York 10065, USA.
J Am Chem Soc. 2010 Sep 1;132(34):11902-3. doi: 10.1021/ja104550p.
Culture independent approaches for accessing small molecules produced by uncultured bacteria are often hampered by the inability to easily clone environmental DNA (eDNA) fragments large enough to capture intact biosynthetic gene clusters that can be used in heterologous expression studies. Here we show that homology screening of eDNA megalibraries for clones containing natural product biosynthetic genes, coupled with transformation-assisted recombination (TAR) in yeast, can be used to access large, functionally intact, natural product gene clusters from the environment. The eDNA derived gene cluster reported here was functionally reconstructed from two overlapping cosmid clones using TAR. The isolation and structure elucidation of three new fluostatins (F, G, and H) produced by this TAR reconstructed gene cluster is described.
利用非培养细菌产生的小分子的无细胞方法往往受到阻碍，因为难以克隆足够大的环境 DNA(eDNA)片段来捕获可用于异源表达研究的完整生物合成基因簇。在这里，我们表明，对 eDNA 巨文库中包含天然产物生物合成基因的克隆进行同源筛选，结合酵母中的转化辅助重组 (TAR)，可用于从环境中获取大型、功能完整的天然产物基因簇。本文报道的 eDNA 衍生基因簇是使用 TAR 从两个重叠的 cosmid 克隆中功能重建的。描述了该 TAR 重建基因簇产生的三个新氟他汀 (F、G 和 H) 的分离和结构阐明。
