BackgroundMolecular mechanisms generating genetic variation provide the basis for evolution and long-term survival of a population in a changing environment. In stable, laboratory conditions, the variation-generating mechanisms are dispensable, as there is limited need for the cell to adapt to adverse conditions. In fact, newly emerging, evolved features might be undesirable when working on highly refined, precise molecular and synthetic biological tasks.ResultsBy constructing low-mutation-rate variants, we reduced the evolutionary capacity of MDS42, a reduced-genome E. coli strain engineered to lack most genes irrelevant for laboratory/industrial applications. Elimination of diversity-generating, error-prone DNA polymerase enzymes involved in induced mutagenesis achieved a significant stabilization of the genome. The resulting strain, while retaining normal growth, showed a significant decrease in overall mutation rates, most notably under various stress conditions. Moreover, the error-prone polymerase-free host allowed relatively stable maintenance of a toxic methyltransferase-expressing clone. In contrast, the parental strain produced mutant clones, unable to produce functional methyltransferase, which quickly overgrew the culture to a high ratio (50% of clones in a 24-h induction period lacked functional methyltransferase activity). The surprisingly large stability-difference observed between the strains was due to the combined effects of high stress-induced mutagenesis in the parental strain, growth inhibition by expression of the toxic protein, and selection/outgrowth of mutants no longer producing an active, toxic enzyme.ConclusionsBy eliminating stress-inducible error-prone DNA-polymerases, the genome of the mobile genetic element-free E. coli strain MDS42 was further stabilized. The resulting strain represents an improved host in various synthetic and molecular biological applications, allowing more stable production of growth-inhibiting biomolecules.

The ideal bacterial chassis provides a simplified, stable and predictable host environment for synthetic biological circuits. Mutability and evolution can, however, compromise stability, leading to deterioration of artificial genetic constructs. By eliminating certain sources of instability, these undesired genetic changes can be mitigated. Specifically, deletion of prophages and insertion sequences, nonessential constituents of bacterial genomes, has been shown to be beneficial in cellular and genetic stabilization. Here, we sought to establish a rapid methodology to improve the stability of microbial hosts. The novel workflow involves genome shuffling between a mobile genetic element-free strain and the target cell, and subsequent rounds of CRISPR/Cas-assisted MAGE on multiplex targets. The power and speed of the procedure was demonstrated on E. coli BL21(DE3), a host routinely used for plasmid-based heterologous protein expression. All 9 prophages and 50 insertion elements were efficiently deleted or inactivated. Together with additional targeted manipulations (e.g., inactivation of error-prone DNA-polymerases), the changes resulted in an improved bacterial host with a hybrid (harboring segments of K-12 DNA), 9%-downsized and clean genome. The combined capacity of phage-mediated generalized transduction and CRISPR/Cas-selected MAGE offers a way for rapid, large scale editing of bacterial genomes.