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首页> 外文期刊>Journal of Biotechnology >Comparative analysis of transcriptional activities of heterologous promoters in the rare actinomycete Actinoplanes friuliensis
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Comparative analysis of transcriptional activities of heterologous promoters in the rare actinomycete Actinoplanes friuliensis

机译：稀有放线菌猕猴桃异源启动子转录活性的比较分析

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摘要

Manipulation of secondary metabolite production in the rare actinomycete Actinoplanes friuliensis, the producer of the lipopeptide antibiotic friulimicin, is hampered by the lack of sophisticated genetic tools. Since no expression vectors have been developed from endogenous Actinoplanes plasmids and expression signals, engineering of antibiotic biosynthesis relies on the use of vector systems derived from Streptomyces. While EC31 derived vectors were shown to integrate efficiently into the chromosome of Actinoplanes, information on promoter activity is missing. The manuscript describes the investigation of several different promoter systems which are widely used in Streptomyces in A. friuliensis by promoter probe experiments using eGFP as a reporter. These experiments indicated that promoter strength in A. friuliensis did not correlate to activity in Streptomyces lividans. The ermE* promoter regarded as one of the strongest promoter in Streptomyces has only low activity in A. friuliensis. In contrast, the promoter of the apramycin resistance gene aac(3)IV, originating from the Gram-negative Escherichia coli had the highest activity. By real-time RT-PCR experiments the transcription activity of ermE* promoter in comparison to a native promoter of the friulimicin biosynthetic gene cluster was analysed. This confirmed the results of the promoter probe experiments that indicated quite weak promoter activity of P-ermE* in Actinoplanes.
机译：缺乏复杂的遗传工具阻碍了稀有的放线菌猕猴桃放线菌Friuliensis的生产者次生代谢产物的生产。由于尚未从内源性放线菌质粒和表达信号开发表达载体，因此抗生素生物合成的工程依赖于链霉菌衍生的载体系统的使用。尽管显示EC31衍生的载体可有效整合到放线菌的染色体中，但缺少有关启动子活性的信息。该手稿描述了几种不同的启动子系统的研究，这些系统通过使用eGFP作为报告基因的启动子探针实验，广泛应用于弗氏链霉菌的链霉菌中。这些实验表明，A.friuliensis中的启动子强度与青紫链霉菌中的活性无关。在链霉菌中被认为是最强启动子之一的ermE *启动子在A. friuliensis中仅具有低活性。相反，来自革兰氏阴性大肠杆菌的阿霉素抗性基因aac（3）IV的启动子活性最高。通过实时RT-PCR实验，分析了与friulimicin生物合成基因簇的天然启动子相比，ermE *启动子的转录活性。这证实了启动子探针实验的结果，该实验表明放线型飞机中P-ermE *的启动子活性非常弱。

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