Auxotrophic markers pyrF and proC can replace antibiotic markers on protein production plasmids in high-cell-density Pseudomonas fluorescens fermentation.
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.
Schneider
Jane C
JC
The Dow Chemical Company, 5501 Oberlin Drive, San Diego, California 92121, USA. jcschneider@dow.com
Jenings
Annika F
AF
Mun
Deborah M
DM
McGovern
Patricia M
PM
Chew
Lawrence C
LC
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*
D000900:Anti-Bacterial Agents; D015415:Biomarkers; D017931:DNA Primers