Re-engineering of genetic circuit for 2-deoxystreptamine (2-DOS) biosynthesis in Escherichia coli BL21 (DE3) |
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Authors: | Amit Kumar Chaudhary Je Won Park Yeo Joon Yoon Byung-Gee Kim Jae Kyung Sohng |
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Institution: | 1. Institute of Biomolecule Reconstruction, SunMoon University, 100, Kalsan-ri, Tangjeonmyun, Chungnam, Asansi, 336-708, Republic of Korea 2. Department of Chemistry and Nanoscience, Ewha Womans University, Seoul, Republic of Korea 3. Institute of Molecular Biology and Genetics, School of Chemical and Biological Engineering, Seoul National University, Seoul, 151-744, Republic of Korea
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Abstract: | Various approaches for monocistronic constructions of genetic circuits have been designed for metabolite production but there has been no attempt to apply such methodology for aminoglycosides biosynthesis. Here, a simple and commercially available bio-part, despite the current trend focusing on the standardized BioBricks bio-parts available in the registry, is used. A 181-bp nucleotide fragment was designed for the efficient construction of an expression vector for monocistronic assembly of genes. Furthermore, a single vector with multi-monocistronic assembled genes for 2-deoxystreptamine (2-DOS) synthesis was constructed for production in engineered Escherichia coli. The working efficiency of model vector was concluded by reporter assay whereas the expressions of biosynthesis genes were confirmed by RT-PCR and SDS-PAGE. Production of 2-DOS was confirmed by TLC, LC-ELSD, and ESI–MS/MS. |
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