Comparative analysis of transcriptional activities of heterologous promoters in the rare actinomycete Actinoplanes friuliensis

Abstract

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 ΦC31 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.