Using Purified Tyrosine Site-Specific Recombinases In Vitro to Rapidly Construct and Diversify Metabolic Pathways

: The site-specific recombinase Cre was previously reported to have in vitro activity. Here, we describe the method of purifying two new tyrosine site-specific recombinases VCre and Dre along with Cre by nickel affinity chromatography. We proved the in vitro function of the VCre and Dre on their respective conditional recombination sites. We also developed a methodology to one-step construct and optimize the productivity of a biosynthetic pathway through the combinatorial integration of promoters into a plasmid-encoded pathway by simply incubating a DNA mixture with recombinase system at 37 °C in vitro.