Pathway integration and expression in host cells

Abstract

Provided herein are methods for integrating a gene of interest into a chromosome of a host cell. In some embodiments, the methods include introducing into a host cell a first plasmid comprising a transposase coding sequence and a donor sequence, which includes a selectable marker coding sequence flanked by a first and a second lox site and is itself flanked by inverted repeats recognized by the transposase. Following transposase-mediated chromosomal integration of the donor sequence into the host cell, a second plasmid is introduced, which comprises the gene of interest and a second selectable marker coding sequence, both flanked by a first and a second lox site. The gene of interest is chromosomally integrated into the host cell by recombinase-mediated cassette exchange (RMCE) between the donor sequence and the second plasmid via Cre-lox recombination. Further provided herein are host cells, vectors, and methods of producing a product related thereto.

What is claimed is: 
     
         1 . A method for integrating a gene of interest into a chromosome of a bacterial host cell, the method comprising:
 (a) providing (i) a bacterial host cell comprising a landing pad comprising a first lox site and a second lox site, and a first selectable marker, and (ii) a plasmid comprising a gene of interest and a second selectable marker coding sequence, wherein the gene of interest and the second selectable marker are both flanked by a first lox site of the plasmid and a second lox site of the plasmid, wherein the first and the second lox sites of the plasmid are different; and   (b) introducing the plasmid into the bacterial host cell, wherein at least the plasmid or the bacterial host cell, or a second plasmid in the bacterial host cell, comprises a Cre recombinase coding sequence, and wherein upon introduction of the plasmid into the bacterial host cell, Cre recombinase is expressed from the Cre recombinase coding sequence, and the gene of interest is integrated into the chromosome of the bacterial host cell by the Cre recombinase through recombinase-mediated cassette exchange (RMCE) between the first lox site of the second plasmid and the first lox site of the landing pad, and between the second lox site of the plasmid and the second lox site of the donor sequence.