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

摘要

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.Electronic supplementary materialThe online version of this article (10.1007/s00253-018-8767-1) contains supplementary material, which is available to authorized users.