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

Appl Microbiol Biotechnol. 2018 Mar;102(6):2867-2884. doi: 10.1007/s00253-018-8767-1. Epub 2018 Feb 1.

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.

Keywords: Cre-mediated excision; Excisable enhancer; Fluorescence proteins; Intrachromosomal amplification; Random integration; Replicative transposition.

MeSH terms

  • Bacteriophage mu*
  • Chromosomes, Bacterial*
  • Corynebacterium glutamicum / genetics*
  • DNA Transposable Elements*
  • Gene Dosage
  • Gene Editing / methods*
  • Genetic Vectors
  • Genetics, Microbial / methods*
  • Plasmids
  • Recombination, Genetic

Substances

  • DNA Transposable Elements