Molecular cloning and heterologous expression of the dehydrophos biosynthetic gene cluster.
Molecular cloning and heterologous expression of the dehydrophos biosynthetic gene cluster.
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DOI：
10.1016/j.chembiol.2010.03.007
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发表时间：
2010-04-23
影响因子：
--
通讯作者：
Metcalf WW
中科院分区：
文献类型：
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作者：
Circello BT;Eliot AC;Lee JH;van der Donk WA;Metcalf WW
Dehydrophos是一种乙烯基膦酸酯三肽，由Streptomycesluridus产生，具有广谱抗生素活性。为了鉴定这种不寻常化合物生物合成所必需的基因，我们筛选了S. luridus是因为存在磷酸烯醇式丙酮酸酯酶基因，这是大多数膦酸盐生物合成所必需的。将一个这样的fosmid克隆整合到变铅青链霉菌的染色体中导致异源生产脱氢磷。该克隆的缺失分析允许鉴定最小连续脱氢磷簇，其包含17个开放阅读框（ORF）。这些ORF的生物信息学分析与从磷酸烯醇丙酮酸产生脱氢磷的拟议生物合成途径一致。该途径的早期步骤得到了阻断突变体积累的中间产物分析和体外生化实验的支持。
Dehydrophos is a vinyl phosphonate tripeptide produced by Streptomyces luridus with demonstrated broad spectrum antibiotic activity. To identify genes necessary for biosynthesis of this unusual compound we screened a fosmid library of S. luridus for the presence of the phosphoenolpyruvate mutase gene, which is required for biosynthesis of most phosphonates. Integration of one such fosmid clone into the chromosome of Streptomyces lividans led to heterologous production of dehydrophos. Deletion analysis of this clone allowed identification of the minimal contiguous dehydrophos cluster, which contained 17 open reading frames (ORFs). Bioinformatic analyses of these ORFs are consistent with a proposed biosynthetic pathway that generates dehydrophos from phosphoenolpyruvate. The early steps of this pathway are supported by analysis of intermediates accumulated by blocked mutants and in vitro biochemical experiments.