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

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

Genome engineering of the Corynebacterium glutamicum chromosome by the Dual-In/Out strategy

“…After the recombinant CRIM plasmid integration process occurred, the vector part of the plasmid bracketed by the mutant lox66 and lox71 sites could be eliminated by Cre-dependent SSR. For this purpose, a plasmid constitutively expressing the cre gene from the laboratory collection, p06-P dapA -cre (Gorshkova et al 2018), was modi ed by substitution of the Cm R marker for Km R , which resulted in the construction of a new p06-Km R -P dapA -Cre plasmid (GenBank OK651224, Fig. 2E), which was used in the current study.…”

“…Different recombineering-based strategies have already been developed for the integration of large DNA fragments (Rivero-Müller et al 2007) and second and subsequent copies introduction (Igonina et al 2020) into the chromosomes of various organisms. Similar tools, based mainly on modi ed transposons (Suzuki et al 2006) and, recently, on the phage Mu-derived system (Gorshkova et al 2018), have already been adjusted for C. glutamicum.…”

Advances and Perspectives for Genome Editing Tools of Corynebacterium glutamicum

“…It can cause interruption or inactivation of some genes by random transposon insertion (Alain et al, 1994). Many transposable elements have been identified and used in C. glutamicum, such as IS31831 (Vertes et al, 1994), miniTn31831 (Alain et al, 1994), Tn14751 (Inui et al, 2005), IS1249 (Tauch et al, 1995), Tn5 (Suzuki et al, 2006), and mini-Mu (Gorshkova et al, 2018). These transposons have different transposition efficiency, sequence preference (AT-rich regions or GC-rich regions preference), and cargo delivery capacity.…”

“…Unfortunately, transposons are rarely used for random integration of heterogenous genes, because the length of cargo fragments is usually limited (Gorshkova et al, 2018). By contrast, integrase-mediated site-directed integration of heterologous genes can integrate fragments up to tens of kb (Figure 1I) (Huang et al, 2019).…”

Genome-Reduced Corynebacterium glutamicum Fit for Biotechnological Applications

“…phenylpropanoids [49,52,101], isoprenoids [25,57,[102][103][104], alcohols [105], carboxylic acids [100,106], and proteins [107][108][109][110]. In addition, MB001 and derivatives have been used to study Mu-transposition [111], assembly of the septal cell envelope [112], infection with phages φ673 and φ674 phages [113], identification of an isoprenoid pyrophosphatedependent transcriptional regulator [114], cAMP phosphodiesterase CpdA [115] and cryptic prophages [94], as basis for ALE towards higher growth rates on glucose minimal medium [116] and to assemble bacterial microcompartments [117].…”