Recombineering (recombination-mediated genetic engineering) is a genetic and molecular biology technique based on homologous recombination systems, as opposed to the older/more common method of using restriction enzymes and ligases to cut and glue DNA sequences. It has been developed in E. coli and now is expanding to other bacteria species and is used to modify DNA in a precise and simple manner.
Benefits of recombineering vs other genetic engineering techniques
The biggest advantage of recombineering is that it obviates the need for conveniently positioned restriction sites, whereas in conventional genetic engineering, DNA modification is often compromised by the availability of unique restriction sites. In engineering large constructs of over 100 kb, such as the Bacterial Artificial Chromosomes (BACs), or chromosomes, recombineering has become a necessity. Recombineering can generate the desired modifications without leaving any ‘footprints’ behind. It also forgoes multiple cloning stages for generating intermediate vectors and therefore is used to modify DNA constructs in a relatively short time-frame. The homology required is short enough that it can be generated in synthetic oligonucleotides and recombination with short oligonucleotides themselves is incredibly efficient. Recently, recombineering has been developed for high throughput DNA engineering applications termed ‘recombineering pipelines’. Recombineering pipelines support the large scale production of BAC transgenes and gene targeting constructs for functional genomics programs such as EUCOMM (European Conditional Mouse Mutagenesis Consortium) and KOMP (Knock-Out Mouse Program). Recombineering has also been automated, a process called “MAGE” -Multiplex Automated Genome Engineering, in the Church lab.
The ability to efficiently generate targeted point mutations in the chromosome without the need for antibiotics, or other means of selection, is a powerful strategy for genome engineering. Although oligonucleotide-mediated recombineering (ssDNA recombineering) has been utilized in Escherichia coli for over a decade, the successful adaptation of ssDNA recombineering to Gram-positive bacteria has not been reported. Here we describe the development and application of ssDNA recombineering in lactic acid bacteria. Mutations were incorporated in the chromosome of Lactobacillus reuteri and Lactococcus lactis without selection at frequencies ranging between 0.4% and 19%. Whole genome sequence analysis showed that ssDNA recombineering is specific and not hypermutagenic. To highlight the utility of ssDNA recombineering we reduced the intrinsic vancomymycin resistance of L. reuteri over 100-fold. By creating a single amino acid change in the d-Ala-d-Ala ligase enzyme we reduced the minimum inhibitory concentration for vancomycin from over 256 to 1.5 µg/ml, well below the clinically relevant minimum inhibitory concentration. Recombineering thus allows high efficiency mutagenesis in lactobacilli and lactococci, and may be used to further enhance beneficial properties and safety of strains used in medicine and industry. We expect that this work will serve as a blueprint for the adaptation of ssDNA recombineering to other Gram-positive bacteria.
Multiplex automated genome engineering (MAGE) uses short oligonucleotides to scarlessly modify genomes; however, insertions over 10 bases are still inefficient but can be improved substantially by selection of highly modified chromosomes. Here we describe ‘coselection’ MAGE (CoS-MAGE) to optimize biosynthesis of aromatic amino acid derivatives by combinatorially inserting multiple T7 promoters simultaneously into 12 genomic operons. Promoter libraries can be quickly generated to study gain-of-function epistatic interactions in gene networks.
Recombineering has allowed researchers to rewire genomes much faster than traditional cloning techniques. With the advent of multiplex recombineering, genome-wide editing has become possible, allowing large scale reprogramming of cellular machinery. Despite these successes in large scale genome-wide reprogramming, the overall efficiency of recombineering still remains low, especially for changes over 6bp. Wang et al. have recently reported much higher efficiencies in recombineering by the use of co-selection, which enriches for cells that are highly recombinogenic. Therefore, we have designed a strain that has co-selection markers located around the genome and are easily screened/selected for, to enable high efficiency recombineering at any location. We have also developed a method to create large libraries of proteins with trackable, targeted changes in just a few days, further enabling genome editing and optimization