Abstract
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.
Highlights
Since its discovery in 1957, as an L-glutamate-producing nonpathogenic Gram-positive soil bacterium from the Actinomyces branch and its classification as a “generally recognized as safe” (GRAS) organism, Corynebacterium glutamicum has become a workhorse for the large-scale industrial production of amino acids, chemicals, materials, fuels, and various proteins (Becker and Wittmann 2012)
The integration helper plasmid pVK9-lacIQ-Ptac-MuAB (Fig. 1a) was constructed for the expression of the MuA and MuB transposition factor genes in C. glutamicum cells. This plasmid was designed on the basis of the pVK9-GmR vector for rather stable maintenance in C. glutamicum cells, but with the ability to be cured under non-selective conditions
Expression of the transposition factor genes MuAB can be induced by IPTG addition via the introduction of Ptac/Olac-promoter/operator region with the lacIQ unit as their control element, which has been repeatedly used in C. glutamicum (Eggeling and Bott 2005; Kirchner and Tauch 2003; Nešvera and Pátek 2011; Ravasi et al 2012)
Summary
Since its discovery in 1957, as an L-glutamate-producing nonpathogenic Gram-positive soil bacterium from the Actinomyces branch and its classification as a “generally recognized as safe” (GRAS) organism, Corynebacterium glutamicum has become a workhorse for the large-scale industrial production of amino acids, chemicals, materials, fuels, and various proteins (Becker and Wittmann 2012). There are many approaches for chromosomal editing of C. glutamicum. Most of these techniques are based on the application of various types of plasmids (Nešvera and Pátek 2008; Tauch 2005), which allow the deletion, substitution, and overexpression of target genes (Kirchner and Tauch 2003; Nešvera and Pátek 2011). To our knowledge, a perfect analogue of the λRed/RecET-based recombineering approach for the high-efficiency integration of double-stranded PCR products with rather short homologous arms into targeted loci of the bacterial chromosome (reviewed in (Court et al 2002))—a method developed for. The already published RecT-dependent (Binder et al 2013; Cho et al 2017; Jiang et al 2017) or annealing protein-independent (Krylov et al 2014) recombination approaches between short singlestranded oligonucleotides and a targeted locus in the C. glutamicum chromosome are good starting points. Integrative plasmid vectors have been constructed based on various corynephages, and these carry DNA elements that enable phage-governed site-specific recombinant DNA integration (Moreau et al 1999; Oram et al 2007)
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