Increasing the efficiency of heterologous promoters in actinomycetes.
Increasing the efficiency of heterologous promoters in actinomycetes.
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发表时间：
2002-07
影响因子：
1.2
通讯作者：
C. Wilkinson;Z. Hughes-Thomas;Christine J. Martin;I. Böhm;T. Mironenko;M. Deacon;M. Wheatcroft;G. Wirtz;J. Staunton;P. Leadlay
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作者：
C. Wilkinson;Z. Hughes-Thomas;Christine J. Martin;I. Böhm;T. Mironenko;M. Deacon;M. Wheatcroft;G. Wirtz;J. Staunton;P. Leadlay
构建了大肠杆菌-放线菌穿梭载体pCJW 93，该载体将克隆的基因置于来自变铅青链霉菌的硫链丝菌素诱导型顶端启动子的控制下。我们还构建了带有天蓝色链霉菌放线菌红素生物合成途径的actII-ORF 4/PactI激活子-启动子系统的表达载体。对于这两种类型的载体，表达水平在不同的放线菌菌株中变化很大，表明最佳表达所需的宿主因素的水平不同。删除actII-ORF 4激活基因从一个这样的质粒中的多孢霉的同源actI启动子的表达急剧减少，表明宿主因子是所需的激活蛋白本身的最佳生产。然而，聚酮合酶DEBS 1-TE的低拷贝表达载体pWIZ 1，其中激活基因的启动子被S.在Streptomyces cinnamonensis中，Artraea指导高效生产聚酮合酶蛋白;发现三酮内酯产物的水平比由相同质粒产生的高100倍，其中actII-ORF 4从其自身的启动子表达。确保特异性激活蛋白的适当表达应该能够在广泛的放线菌宿主中实现基因的更方便和一致的异源表达。
An Escherichia coli-actinomycete shuttle vector, pCJW93, was constructed which places cloned genes under the control of the thiostrepton-inducible tip promoter from Streptomyces lividans. We also constructed expression vectors bearing the actII-ORF4/PactI activator-promoter system of the actinorhodin biosynthetic pathway of Streptomyces coelicolor. With both types of vector, levels of expression varied widely in different actinomycete strains, indicating different levels of the host factors needed for optimal expression. Deletion of the actII-ORF4 activator gene from one such plasmid in Saccharopolyspora erythraea drastically reduced expression from the cognate actI promoter, showing that host factors are required for optimal production of the activator protein itself. However, a low copy number expression vector pWIZ1 for the polyketide synthase DEBS1-TE, in which the promoter for the activator gene was replaced by the strong heterologous ermE* promoter of S. erythraea directed highly efficient production of polyketide synthase protein in Streptomyces cinnamonensis; and the levels of triketide lactone product found were up to 100-fold greater than were produced by the same plasmid in which actII-ORF4 was expressed from its own promoter. Ensuring appropriate expression of a specific activator protein should enable more convenient and consistent heterologous expression of genes in a broad range of actinomycete hosts.