Promoter constructions for efficient secretion expression in Streptomyces lividans.
Promoters from different Streptomyces genes were cloned in front of the Tendamistat gene from S. tendae, in order to study secretion-expression in S. lividans using a pIJ702 plasmid vector system. Besides the promoters we cloned a transcriptional terminator downstream of the Tendamistat gene to improve transcription efficiency. The promoters we selected were: (1) a synthetic Escherichia coli-like consensus promoter; (2) the aphI promoter of the neomycin resistance gene from S. fradiae; (3) an ermE-up promoter mutant from Saccharopolyspora erythraea; (4) the melC promoter of the tyrosinase operon from Streptomyces antibioticus. In addition, we tested the thiostrepton-inducible tipA promoter from S. lividans in our Tendamistat secretion system. The promoters were cloned upstream of the Tendamistat ribosome binding site in order to conserve the original translation initiation. The Tendamistat secretion mediated by the different promoter constructions above varied dramatically in up to 10 mg/l in the case of the synthetic promoter and the aph promoter, and up to 500 mg/l mediated by the ermE-up promoter. The melC promoter allowed about 200 mg/l Tendamistat secretion and the tipA promoter proved to be inducible from less than 0.5 mg/l up to 40 mg/l of Tendamistat secretion. Based on the amount of secreted Tendamistat and on the analysis of mRNA levels, we conclude that transcriptional activity regulates the efficiency of our secretion-expression system.
Schmitt-John
T
T
Institut für Organische Chemie, J. W. Goethe Universität, Frankfurt/Main, Federal Republic of Germany.
Engels
J W
JW
D001709:Biotechnology; D015966:Gene Expression Regulation, Fungal; D005800:Genes, Fungal; D010455:Peptides / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011994:Recombinant Proteins; D013302:Streptomyces / Q000235:genetics*; D014158:Transcription, Genetic; D000516:alpha-Amylases / Q000037:antagonists & inhibitors*
D010455:Peptides; D011994:Recombinant Proteins; D000516:alpha-Amylases; C030526:tendamistate