Abstract
Bacteriophage serine integrases are extensively used in biotechnology and synthetic biology for assembly and rearrangement of DNA sequences. Serine integrases promote recombination between two different DNA sites, attP and attB, to form recombinant attL and attR sites. The ‘reverse’ reaction requires another phage-encoded protein called the recombination directionality factor (RDF) in addition to integrase; RDF activates attL × attR recombination and inhibits attP × attB recombination. We show here that serine integrases can be fused to their cognate RDFs to create single proteins that catalyse efficient attL × attR recombination in vivo and in vitro, whereas attP × attB recombination efficiency is reduced. We provide evidence that activation of attL × attR recombination involves intra-subunit contacts between the integrase and RDF moieties of the fusion protein. Minor changes in the length and sequence of the integrase–RDF linker peptide did not affect fusion protein recombination activity. The efficiency and single-protein convenience of integrase–RDF fusion proteins make them potentially very advantageous for biotechnology/synthetic biology applications. Here, we demonstrate efficient gene cassette replacement in a synthetic metabolic pathway gene array as a proof of principle.
Highlights
The rapidly advancing fields of biotechnology and synthetic biology demand ever more sophisticated and efficient tools for precise manipulation of DNA
We noted that the extreme C-terminus of the Listeria innocua phage (LI) integrase lies quite near the coiled-coil domain in the solved structures, so an recombination directionality factor (RDF) moiety attached here via a short flexible peptide linker might be able to interact with the coiledcoil domain of its own fusion protein molecule
The C31 and Bxb1 integrase–RDF fusion proteins that we constructed promote efficient attL × attR recombination, in contrast to their parent integrases
Summary
Bacteriophage integrases catalyse recombination between an attP site in the circularized phage DNA and an attB site in the chromosome of the bacterial host cell, leading to an integrated prophage flanked by recombinant attL and attR sites [2] (Figure 1A). The prophage persists in this integrated state until lysogenic induction, when the integrase promotes recombination between the attL and attR sites, excising the phage genome as a DNA circle. Because integrases promote recombination between two sites with different sequences (unlike many other SSRs), the recombinant sites are different from the non-recombinant sites and each other These differences can be exploited by the integrase so as to favour integration at lysogeny and excision at lysogenic induction, a property called directionality. The two families are mechanistically and evolutionarily unrelated [2,4]
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