轮状链霉菌 ATCC15003 中博来霉素生物合成基因簇的体内操作揭示了对其生物合成途径的新见解。,Journal of Biological Chemistry

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轮状链霉菌 ATCC15003 中博来霉素生物合成基因簇的体内操作揭示了对其生物合成途径的新见解。
Journal of Biological Chemistry ( IF 4.0 ) Pub Date : 2008 Oct 17 , DOI: 10.1074/jbc.m804971200
Ute Galm ₁ , Liyan Wang , Evelyn Wendt-Pienkowski , Runying Yang , Wen Liu , Meifeng Tao , Jane M Coughlin , Ben Shen

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博来霉素 (BLM) 是一种重要的临床抗肿瘤化合物，其类似物很难通过化学合成制备。生产者菌株中生物合成途径的基因工程将提供一种在体内产生这种复杂分子的新衍生物的有效和方便的方法。然而，近 20 年来，产生 BLM 的轮状链霉菌 ATCC15003 一直无法通过所有方法将质粒 DNA 引入其细胞。在克隆和鉴定博来霉素生物合成基因簇数年后，该研究首次证明了通过属间大肠杆菌-链霉菌共轭对该药学相关生产菌株的遗传可及性。基因替换和框内缺失突变体是通过 lambdaRED 介导的 PCR 靶向诱变产生的，所得突变体的次级代谢物谱证实了 BLM 生物合成基因簇的身份并确定了其边界。最终，框内 blmD 缺失突变株 S. verticillus SB5 导致产生博莱霉素中间体。阐明了该化合物 decarbamoyl-BLM 的结构，并将其 DNA 裂解活性与母体化合物进行了比较。

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In vivo manipulation of the bleomycin biosynthetic gene cluster in Streptomyces verticillus ATCC15003 revealing new insights into its biosynthetic pathway.

Bleomycin (BLM), an important clinically used antitumor compound, and its analogs are challenging to prepare by chemical synthesis. Genetic engineering of the biosynthetic pathway in the producer strain would provide an efficient and convenient method of generating new derivatives of this complex molecule in vivo. However, the BLM producing Streptomyces verticillus ATCC15003 has been refractory to all means of introducing plasmid DNA into its cells for nearly two decades. Several years after cloning and identification of the bleomycin biosynthetic gene cluster, this study demonstrates, for the first time, genetic accessibility of this pharmaceutically relevant producer strain by intergeneric Escherichia coli-Streptomyces conjugation. Gene replacement and in-frame deletion mutants were created by lambdaRED-mediated PCR targeting mutagenesis, and the secondary metabolite profile of the resultant mutants confirmed the identity of the BLM biosynthetic gene cluster and established its boundaries. Ultimately, the in-frame blmD deletion mutant strain S. verticillus SB5 resulted in the production of a bleomycin intermediate. The structure of this compound, decarbamoyl-BLM, was elucidated, and its DNA cleavage activity was compared with the parent compounds.

更新日期：2017-01-31

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