Identification of biosynthetic gene clusters from metagenomic libraries using PPTase complementation in a Streptomyces host
Identification of biosynthetic gene clusters from metagenomic libraries using PPTase complementation in a Streptomyces host
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DOI：
10.1093/femsle/fnx155
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发表时间：
2017-08-01
影响因子：
2.1
通讯作者：
Brady, Sean F.
中科院分区：
文献类型：
--
作者：
Bitok, J. Kipchirchir;Lemetre, Christophe;Brady, Sean F.
大多数环境细菌不容易在实验室中培养，使得它们使用依赖于培养的发现方法无法获得天然产物。从环境样本中提取的DNA（环境DNA，eDNA）的克隆和异源表达提供了一种绕过这一发现瓶颈的方法。为了便于鉴定含有生物合成基因簇的克隆，我们开发了一个模型异源表达报告菌株白色链霉菌：：bpsA Delta PPTase。该菌株在PPTase缺失背景中携带4 '-磷酸泛酰巯基乙胺基转移酶（PPTase）依赖性蓝色色素合成酶A基因bpsA。表达功能性PPT酶的cDNA克隆恢复了蓝色素靛蓝的产生。由于PPTase基因通常出现在生物合成基因簇（BGC）中，靛蓝的产生可用于鉴定含有BGC的eDNA克隆。我们筛选了一个土壤cDNA文库。albus：：bpsA Delta PPTase，并鉴定了含有非核糖体肽合成酶（NRPS）、聚酮化合物合成酶（PKS）和混合NRPS/PKS生物合成基因簇的克隆。一个NRPS基因簇被证明赋予粘螯素A到S的生产。albus：：bpsA Delta PPTase。
The majority of environmental bacteria are not readily cultured in the lab, leaving the natural products they make inaccessible using culture-dependent discovery methods. Cloning and heterologous expression of DNA extracted from environmental samples (environmental DNA, eDNA) provides a means of circumventing this discovery bottleneck. To facilitate the identification of clones containing biosynthetic gene clusters, we developed a model heterologous expression reporter strain Streptomyces albus::bpsA Delta PPTase. This strain carries a 4 '-phosphopantetheinyl transferase (PPTase)-dependent blue pigment synthase A gene, bpsA, in a PPTase deletion background. eDNA clones that express a functional PPTase restore production of the blue pigment, indigoidine. As PPTase genes often occur in biosynthetic gene clusters (BGCs), indigoidine production can be used to identify eDNA clones containing BGCs. We screened a soil eDNA library hosted in S. albus::bpsA Delta PPTase and identified clones containing non-ribosomal peptide synthetase (NRPS), polyketide synthase (PKS) and mixed NRPS/PKS biosynthetic gene clusters. One NRPS gene cluster was shown to confer the production of myxochelin A to S. albus::bpsA Delta PPTase.