Identification and utility of FdmR1 as a Streptomyces antibiotic regulatory protein activator for fredericamycin production in Streptomyces griseus ATCC 49344 and heterologous hosts
Identification and utility of FdmR1 as a Streptomyces antibiotic regulatory protein activator for fredericamycin production in Streptomyces griseus ATCC 49344 and heterologous hosts
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DOI：
10.1128/jb.00592-08
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发表时间：
2008-08-01
影响因子：
3.2
通讯作者：
Shen, Ben
中科院分区：
文献类型：
--
作者：
Chen, Yihua;Wendt-Pienkowski, Evelyn;Shen, Ben
弗雷迪霉素（FDM）是一个生物合成基因簇，先前从灰色链霉菌（Streptomyces griseus） ATCC 49344中克隆出来，包含三个推测的调控基因fdmR、fdmR和fdmR2。他们推断的基因产物与链霉菌抗生素调节蛋白（SARP）家族成员（FdmR1）或mar样调节蛋白（FdmR和FdmR2）高度相似。在这里，我们提供了支持FdmR1作为sarp型激活剂的实验数据。fdmR1的失活终止了FDM的生物合成，fdmR1::aac(3)IV突变体可以通过反式表达fdmR]来恢复FDM的生产。逆转录- pcr转录分析显示，在fdm基因簇的28个基因中，除fdmR和fdm T2外，多达26个基因直接或间接受到FdmR1的阳性控制。过表达fdmR1后，稻瘟病菌的FDM滴度比野生型稻瘟病菌提高了5.6倍（约1.36 g/l）。将完整的fdm集群克隆到一个整合质粒中并随后在异种宿主中表达，结果表明，在白色链霉菌中可以产生相当数量的fdm，而在lividans链霉菌中则不能。然而，通过fdmR的组成表达，可以对S. lividans宿主进行工程改造以产生FDMs；lividans中FDM的产生可以通过过度表达fdmC进一步增强，fdmC编码一种推测的酮还原酶，并伴随fdmR1。综上所述，这些研究证明了工程FDM生物合成和提高FDM滴度在本地生产者S. griseus和异种宿主（如S. albus和S. lividans）中的可行性。该方法利用FdmR1， FDM生物合成机制的关键激活剂。
The fredericamycin (FDM) A biosynthetic gene cluster, cloned previously from Streptomyces griseus ATCC 49344, contains three putative regulatory genes, fdmR, fdmR], and fdmR2. Their deduced gene products show high similarity to members of the Streptomyces antibiotic regulatory protein (SARP) family (FdmR1) or to MarR-like regulators (FdmR and FdmR2). Here we provide experimental data supporting FdmR1 as a SARP-type activator. Inactivation of fdmR1 abolished FDM biosynthesis, and FDM production could be restored to the fdmR1::aac(3)IV mutant by expressing fdmR] in trans. Reverse transcription-PCR transcriptional analyses revealed that up to 26 of the 28 genes within the fdm gene cluster, with the exception of fdmR and fdm T2, were under the positive control of FdmR1, directly or indirectly. Overexpression of fdmR1 in S. griseus improved the FDM titer 5.6-fold (to about 1.36 g/liter) relative to that of wild-type S. griseus. Cloning of the complete fdm cluster into an integrative plasmid and subsequent expression in heterologous hosts revealed that considerable amounts of FDMs could be produced in Streptomyces albus but not in Streptomyces lividans. However, the S. lividans host could be engineered to produce FDMs via constitutive expression of fdmR; FDM production in S. lividans could be enhanced further by over expressing fdmC, encoding a putative ketoreductase, concomitantly with fdmR1. Taken together, these studies demonstrate the viability of engineering FDM biosynthesis and improving FDM titers in both the native producer S. griseus and heterologous hosts, such as S. albus and S. lividans. The approach taken capitalizes on FdmR1, a key activator of the FDM biosynthetic machinery.