Pathway integration and expression in host cells

A method for integrating a gene of interest into a chromosome of a bacterial host cell, the method comprising:

• (a) providing a bacterial donor cell comprising a donor plasmid, the donor plasmid comprising:
• a transposase coding sequence;
• and a donor sequence, wherein the donor sequence is flanked by inverted repeats recognized by the transposase, wherein the donor sequence comprises a first selectable marker coding sequence and a first and a second lox site, and wherein the first selectable marker coding sequence is flanked by the first and the second lox sites;
• (b) introducing the donor plasmid into a bacterial host cell by conjugation with the bacterial donor cell, wherein upon introduction of the donor plasmid into the bacterial host cell, transposase is expressed from the transposase coding sequence, and the donor sequence is integrated into a chromosome of the bacterial host cell between the inverted repeats by the transposase;
• (c) providing a second plasmid comprising the 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 second plasmid and a second lox site of the second plasmid, wherein the first and the second lox sites of the second plasmid are different;
• and
• (d) introducing the second plasmid into the bacterial host cell,
• wherein the donor sequence comprises a Cre recombinase coding sequence, and wherein upon introduction of the second 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 donor sequence, and between the second lox site of the second plasmid and the second lox site of the donor sequence.