A Serine Integrase-Based ‘Landing Pad’ System for Chromosomal Integrations of Heterologous Genes in E. coli

Serine integrases represent a powerful and flexible tool for synthetic biology and the genomic integration of heterologous genes or pathways into host organisms. Integrases function naturally to incorporate bacteriophage DNA into the host genome, binding to attachment sites on the bacterial and phage genomes to facilitate integration and later excision of the prophage. They have been shown to have several characteristics which make them ideally suited to synthetic biology applications, including high site-specificity, controllable directionality and short minimum attachment sites compared to other recombinases. In the system described here, serine integrase enzymes are used to recombine two pairs of attachment sites located on a chromosomally-integrated target sequence and an introduced donor cassette, resulting in efficient integration of a target gene into a known locus. Escherichia coli strains have been created with genome-based ‘landing pads’ for highly efficient gene cassette exchange reactions mediated by φC31 integrase. The work is further expanded to include a list of alternative integrases, with the aim of creating a toolkit for multiple simultaneous integrations into the E. coli genome. This system has the potential for multiplexed, markerless integrations of whole pathways into E. coli and other industrially relevant organisms, while additionally enabling rapid switching of genes for pathway optimisation and fine-tuning of expression from a library of chromosomal sites.