Connecting primary and secondary metabolism: AreB, an IclR‐like protein, binds the AREccaR sequence of S. clavuligerus and modulates leucine biosynthesis and cephamycin C and clavulanic acid production
Connecting primary and secondary metabolism: AreB, an IclR‐like protein, binds the AREccaR sequence of S. clavuligerus and modulates leucine biosynthesis and cephamycin C and clavulanic acid production
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DOI：
10.1111/j.1365-2958.2007.05937.x
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发表时间：
2007-10
影响因子：
3.6
通讯作者：
I. Santamarta;M. T. López-García;R. Pérez-Redondo;B. Koekman;J. Martín;P. Liras
中科院分区：
文献类型：
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作者：
I. Santamarta;M. T. López-García;R. Pérez-Redondo;B. Koekman;J. Martín;P. Liras
先前通过DNA亲和结合的方法分离到了链霉菌clavuligerus调控ccaR基因上游的自调节元件（autoregulatory element， ARE）蛋白。编码该蛋白的areB基因位于S. clavuligerus的上游，与leuCD操纵子方向相反；它编码一个239个氨基酸的IclR家族蛋白，在N端区域有一个螺旋-转-螺旋基序。通过基因置换，构建了一个areB缺失突变体S. clavuligerusΔareB。该菌株需要亮氨酸才能在确定的培养基中获得最佳生长。在S. clavuligerusΔareB中，由于AreB结合了AreB - leuCD基因间区作为正调节因子，因此leuCD操纵子的表达被阻滞。在ΔareB突变体中，克拉维酸和头孢霉素C的产量得到了提高，但没有观察到ccaR表达的显著差异。纯重组AreB蛋白不能结合arecar序列（如EMSA所示），除非在结合反应中加入从S. clavuligerus ATCC 27064‐中过滤的含有Mr小于10 kDa分子的提取物。当从ΔareB突变体中获得经过过滤的提取物时，没有观察到与AREccaR序列的结合恢复，这表明小分子量效应物的生物合成也受到AreB的控制。
A protein binding to the autoregulatory element (ARE) upstream of the regulatory ccaR gene of Streptomyces clavuligerus was isolated previously by DNA affinity binding. The areB gene, encoding this protein, is located upstream and in opposite orientation to the leuCD operon of S. clavuligerus; it encodes a 239‐amino‐acid protein of the IclR family with a helix–turn–helix motif at the N‐terminal region. An areB‐deleted mutant, S. clavuligerusΔareB, has been constructed by gene replacement. This strain requires leucine for optimal growth in defined media. Expression of the leuCD operon is retarded in S. clavuligerusΔareB, because AreB binds the areB‐leuCD intergenic region acting as a positive modulator. Clavulanic acid and cephamycin C production are improved in the ΔareB mutant although no drastic difference in ccaR expression was observed. Pure recombinant AreB protein does not bind the AREccaR sequence (as shown by EMSA) unless filtered extracts from S. clavuligerus ATCC 27064‐containing molecules of Mr lower than 10 kDa are added to the binding reaction. Restoration of binding to the AREccaR sequence is not observed when filtered extracts are obtained from the ΔareB mutant, suggesting that biosynthesis of the small‐molecular‐weight effector is also controlled by AreB.