Mu-driven transposition of recombinant mini-Mu unit DNA in the Corynebacterium glutamicum chromosome

• Published in: Applied microbiology and biotechnology 2018-03, Vol.102 (6), p.2867-2884
• Main Authors: Gorshkova, Natalya V., Lobanova, Juliya S., Tokmakova, Irina L., Smirnov, Sergey V., Akhverdyan, Valerii Z., Krylov, Alexander A., Mashko, Sergey V.
• Format: Article
• Language: English
• Subjects: Amplification; Bacteria; Biomedical and Life Sciences; Biotechnology; Chromosomes; Corynebacteria; Corynebacterium glutamicum; Deoxyribonucleic acid; DNA; Fluorescence; Genes; Genetic aspects; Genomes; Gram-negative bacteria; Integration; Life Sciences; Liquid oxygen; Methods and Protocols; Microbial Genetics and Genomics; Microbiological research; Microbiology; Plasmids; Proteins; Transposition; Transposons
• ISSN: 0175-7598; 1432-0614
• DOI: 10.1007/s00253-018-8767-1

A dual-component Mu-transposition system was modified for the integration/amplification of genes in Corynebacterium . The system consists of two types of plasmids: (i) a non-replicative integrative plasmid that contains the transposing mini-Mu( LR ) unit bracketed by the L / R Mu ends or the mini-Mu( LER ) unit, which additionally contains the enhancer element, E , and (ii) an integration helper plasmid that expresses the transposition factor genes for MuA and MuB. Efficient transposition in the C. glutamicum chromosome (≈ 2 × 10 −4 per cell) occurred mainly through the replicative pathway via cointegrate formation followed by possible resolution. Optimizing the E location in the mini-Mu unit significantly increased the efficiency of Mu-driven intramolecular transposition–amplification in C. glutamicum as well as in gram-negative bacteria. The new C. glutamicum genome modification strategy that was developed allows the consequent independent integration/amplification/fixation of target genes at high copy numbers. After integration/amplification of the first mini-Mu( LER ) unit in the C. glutamicum chromosome, the E -element, which is bracketed by lox -like sites, is excised by Cre-mediated fashion, thereby fixing the truncated mini-Mu( LR ) unit in its position for the subsequent integration/amplification of new mini-Mu( LER ) units. This strategy was demonstrated using the genes for the citrine and green fluorescent proteins, yECitrine and yEGFP , respectively.