Method for stable gene-amplification in a bacterial host cell

A method for producing a protein encoded by a gene of interest, comprising

• a) culturing a bacterial host cell comprising at least two duplicated copies of an amplification unit in its genome, the amplification unit comprising:
• i) at least one copy of the gene of interest, and
• ii) an expressible conditionally essential gene, wherein the conditionally essential gene is either promoterless or transcribed from a heterologous promoter having an activity lower than the endogenous promoter of the conditionally essential gene, and wherein the conditionally essential gene encodes an enzyme from the biosynthetic pathway of an amino acid,
• wherein the conditionally essential gene if not functional would render the cell auxotrophic for the amino acid;
• and
• b) recovering the protein.

A method for producing a bacterial cell comprising two or more amplified chromosomal copies of a gene of interest, the method comprising:

• a) providing a bacterial cell comprising at least one copy of an amplification unit, the unit comprising:
• i) at least one copy of the gene of interest, and
• ii) an expressible functional copy of a conditionally essential gene, which is either promoterless or transcribed from a heterologous promoter having an activity lower than the endogenous promoter of the conditionally essential gene, and wherein the conditionally essential gene encodes an enzyme from the biosynthetic pathway of an amino acid,
• wherein the conditionally essential gene if not functional would render the cell auxotrophic for the amino acid;
• b) cultivating the cell under conditions suitable for growth in a medium deficient of the amino acid, thereby providing a growth advantage to a cell in which the amplification unit has been duplicated in the chromosome;
• and
• c) selecting a cell wherein the amplification unit has been duplicated in the chromosome, whereby two or more amplified chromosomal copies of the gene of interest were produced.