title: Auxotrophic markers pyrF and proC can replace antibiotic markers on protein production plasmids in high-cell-density Pseudomonas fluorescens fermentation.
abstract: The use of antibiotic-resistance genes as selectable markers in transgenic organisms is coming under increased scrutiny, for fear that they may spread to human pathogens, thereby reducing the effectiveness of antibiotic therapy. A current Pseudomonas fluorescens protein expression system uses a tetracycline resistance gene (tetR/tetA) to maintain an expression plasmid under control of a repressible promoter and a kanamycin resistance gene (kanR) to maintain a plasmid carrying a repressor gene. We investigated using auxotrophic markers to replace these two antibiotic resistance genes: pyrF (encoding orotidine-5'-phosphate decarboxylase) in place of tetR/tetA and proC (encoding pyrroline-5-carboxylate reductase) in place of kanR, complementing their respective precise chromosomal deletions created by allele exchange using a suicide vector carrying pyrF as a counterselectable marker. The resulting strains, devoid of antibiotic-resistance genes, were shown to achieve high productivity of nitrilase and thermostable alpha-amylase equal to that of the former antibiotic-resistant production host. The production plasmids were stable. The pyrF (uracil-dependent) background of the production host strain also allows us to sequentially alter the genome to incorporate other desired genomic changes, deletions, or insertions using 5'-fluoroorotic acid counterselection, restoring the selectable marker after each step.
authors.affiliation: The Dow Chemical Company, 5501 Oberlin Drive, San Diego, California 92121, USA. jcschneider@dow.com
authors.forename: Jane C
authors.initials: JC
authors.lastname: Schneider
authors.forename: Annika F
authors.initials: AF
authors.lastname: Jenings
authors.forename: Deborah M
authors.initials: DM
authors.lastname: Mun
authors.forename: Patricia M
authors.initials: PM
authors.lastname: McGovern
authors.forename: Lawrence C
authors.initials: LC
authors.lastname: Chew
mesh_terms: D000900:Anti-Bacterial Agents; D001483:Base Sequence; D015415:Biomarkers; D017931:DNA Primers; D005285:Fermentation; D017353:Gene Deletion; D005798:Genes, Bacterial; D010957:Plasmids; D011551:Pseudomonas fluorescens / Q000378:metabolism*
chemical_list: D000900:Anti-Bacterial Agents; D015415:Biomarkers; D017931:DNA Primers