Artificial RNA aptamer production by the marine bacterium Rhodovulum sulfidophilum: Improvement of the aptamer yield using a mutated transcriptional promoter

• Published in: Journal of bioscience and bioengineering 2011-11, Vol.112 (5), p.458-461
• Main Authors: Suzuki, Hiromichi, Umekage, So, Tanaka, Terumichi, Kikuchi, Yo
• Format: Article
• Language: English
• Subjects: Aptamers, Nucleotide - genetics; Aptamers, Nucleotide - metabolism; Bacteria; Base Sequence; Biological and medical sciences; Biotechnology; culture media; Culture Media - chemistry; Functional RNA; Fundamental and applied biological sciences. Psychology; gene expression; Molecular Sequence Data; Mutation; oligonucleotides; photosynthetic bacteria; Phototrophic bacterium; plasmid vectors; Plasmids; Promoter; Promoter Regions, Genetic; Rhodovulum - genetics; Rhodovulum - metabolism; Rhodovulum sulfidophilum; ribonucleases; Ribonucleases - metabolism; RNA; RNA aptamer; RNA, Bacterial - genetics; RNA, Bacterial - metabolism; streptavidin; Streptavidin - genetics; Transcription; transcription (genetics); Transcription, Genetic
• ISSN: 1389-1723; 1347-4421
• DOI: 10.1016/j.jbiosc.2011.07.025

Noncoding small RNAs and artificial RNA aptamers are now expected to be potential candidates for RNA therapeutic agents. We previously proposed a unique method for economical production of these RNAs using the marine phototrophic bacterium Rhodovulum sulfidophilum. This bacterium does not produce any ribonucleases but does produce extracellular nucleic acids in the culture medium in nature. Using this bacterium and an engineered plasmid containing the rrn promoter for the RNA expression, we developed a method for production of the streptavidin RNA aptamer in the culture medium. However, the yield of this RNA product in the culture medium by this method was not enough for practical use. In the present paper, we improved the yield of this product by modification of the − 35 region of the rrn promoter so as to escape from the Fis protein control and the use of a new vector plasmid. Using this system, the extracellular RNA aptamer of approximately 200 ng and the total RNA aptamer (both extra- and intracellular form) of about 20 μg from 1 L culture were accomplished by constitutive expression of the gene.