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

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.

Application of the bacteriophage Mu-driven system for the integration/amplification of target genes in the chromosomes of engineered Gram-negative bacteria--mini review.

The advantages of phage Mu transposition-based systems for the chromosomal editing of plasmid-less strains are reviewed. The cis and trans requirements for Mu phage-mediated transposition, which include the L/R ends of the Mu DNA, the transposition factors MuA and MuB, and the cis/trans functioning of the E element as an enhancer, are presented. Mini-Mu(LR)/(LER) units are Mu derivatives that lack most of the Mu genes but contain the L/R ends or a properly arranged E element in cis to the L/R ends. The dual-component system, which consists of an integrative plasmid with a mini-Mu and an easily eliminated helper plasmid encoding inducible transposition factors, is described in detail as a tool for the integration/amplification of recombinant DNAs. This chromosomal editing method is based on replicative transposition through the formation of a cointegrate that can be resolved in a recombination-dependent manner. (E-plus)- or (E-minus)-helpers that differ in the presence of the trans-acting E element are used to achieve the proper mini-Mu transposition intensity. The systems that have been developed for the construction of stably maintained mini-Mu multi-integrant strains of Escherichia coli and Methylophilus methylotrophus are described. A novel integration/amplification/fixation strategy is proposed for consecutive independent replicative transpositions of different mini-Mu(LER) units with "excisable" E elements in methylotrophic cells.

Phage Mu-driven two-plasmid system for integration of recombinant DNA in the Methylophilus methylotrophus genome.

A phage Mu-driven two-plasmid system for DNA integration in Escherichia coli genome has been adjusted for Methylophilus methylotrophus. Constructed helper plasmids with broad-host-range replicons carry thermo-inducible genes for transposition factors MuA and MuB. Integrative plasmids that are only replicated in E. coli could be mobilized to M. methylotrophus and contained mini-Mu unit with a short terminus of Mu DNA, Mu-attL/R. Mini-Mu unit was integrated in the M. methylotrophus genome via mobilization of the integrative plasmid to the cells carrying the helper in conditions of thermo-induced expression of MuA and MuB. In this system, mini-Mu unit was mainly integrated due to replicative transposition, and the integrated copy could be amplified in the M. methylotrophus chromosome in the presence of helper plasmid. A kan-gene flanked by FRT sites was inserted in one of the mini-Mu units, and it could be readily excised by yeast FLP recombinase that is encoded by the designed plasmid. The multiple Mu-driven gene insertion was carried out by integration of the Bacillus amyloliquefaciens alpha-amylase gene followed by curing the KmR marker before integration of the second mini-Mu unit with Pseudomonas putida xylE gene encoding catechol 2,3-dioxygenase (C23O).