Construction of new vectors for high-level expression in actinomycetes
Construction of new vectors for high-level expression in actinomycetes
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DOI：
10.1016/s0378-1119(98)00327-8
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发表时间：
1998-08-17
期刊：
影响因子：
3.5
通讯作者：
Leadley, PF
中科院分区：
文献类型：
--
作者：
Rowe, CJ;Cortés, J;Leadley, PF
构建了一个新的整合载体（pCJR 24），用于红霉素生产菌多孢放线菌和其他放线菌。它包括来自天蓝色链霉菌的放线菌紫素生物合成基因簇的途径特异性激活基因actII-ORF 4。actI启动子和相关的核糖体结合位点位于NdeI位点（5 ′-CATATG-3 ′）的上游，NdeI位点包含actI起始密码子，如果这些信号由ActII-ORF 4激活剂忠实地介导，则允许响应营养信号以高水平产生蛋白质。将多个聚酮合酶基因克隆到pCJR 24中，并通过同源重组将其整合到染色体上后在S. coelicolor启动子/激活子在S. pCJR 24介导的重组也用于将红霉素产生聚酮合酶的整个基因组置于actI启动子的控制下。与野生型S.菜该系统的应用为合理选育产磷放线菌提供了手段。(C)1998 Elsevier Science B. V.保留所有权利。
A new integrative vector (pCJR24) was constructed for use in the erythromycin producer Saccharopolyspora erythraea and in other actinomycetes. It includes the pathway-specific activator gene actII-ORF4 from the actinorhodin biosynthetic gene cluster of Streptomyces coelicolor. The actI promoter and the associated ribosome binding site are located upstream of an NdeI site (5'-CATATG-3') which encompasses the actI start codon allowing protein(s) to be produced at high levels in response to nutritional signals if these signals are faithfully mediated by the ActII-ORF4 activator. Several polyketide synthase genes were cloned in pCJR24 and overexpressed in S, erythraea after integration of the vector into the chromosome by homologous recombination, indicating the possibility that the S. coelicolor promoter/activator functions appropriately in S. erythraea, pCJR24-mediated recombination was also used to place the entire gene set for the erythromycin-producing polyketide synthase under the control of the actI promoter. The resulting strain produced copious quantities of erythromycins and precursor macrolides when compared with wild-type S. erythraea. The use of this system provides the means for rational strain improvement of antibiotic-producing actinomycetes. (C) 1998 Elsevier Science B.V. All rights reserved.