Patent ID: 8288127

Claim:
A process for the production of a recombinant polypeptide, the process comprising: a) providing an auxotrophic Pseudomonas fluorescens host cell, wherein said host cell comprises a lacI gene inserted into the chromosome; b) introducing into said host cell a nucleic acid construct comprising: i) a first nucleotide sequence encoding said recombinant polypeptide, wherein said first nucleotide sequence is operably linked to a promoter capable of directing expression of the first nucleotide sequence in said Pseudomonas fluorescens host cell; and ii) a second nucleotide sequence encoding an auxotrophic selection marker wherein said second nucleotide sequence is operably linked to a promoter, wherein the auxotrophic selection marker is a polypeptide that restores prototrophy to the auxotrophic host cell; and c) growing the auxotrophic Pseudomonas fluorescens host cell comprising the first and the second nucleotide sequences on a medium that lacks a metabolite for which the host cell is auxotrophic, wherein the auxotrophic selection marker encoded by the second nucleotide sequence and the recombinant polypeptide encoded by the first nucleotide sequence are expressed, and wherein the auxotrophic Pseudomonas fluorescens host cell is: auxotrophic for uracil, wherein the polypeptide that restores prototrophy is orotidine-5â€²-phosphate decarboxylase; auxotrophic for proline, wherein the polypeptide that restores prototrophy is Î” 1 -pyrroline-5-carboxylate reductase; or both; and: wherein when the auxotrophic Pseudomonas fluorescens host cell is auxotrophic for uracil, the metabolite is uracil; wherein when the auxotrophic Pseudomonas fluorescens host cell is auxotrophic for proline, the metabolite is proline; and wherein when the auxotrophic Pseudomonas fluorescens host cell is auxotrophic for uracil and proline, the metabolites are uracil and proline; wherein the auxotrophic Pseudomonas fluorescens host cell expressing the first and the second nucleotide sequences is grown to a density of at least about 20 grams per liter to about 150 grams per liter.