Auxotrophic Markers pyrF and proC Can Replace Antibiotic Markers on Protein Production Plasmids in High-Cell-Density Pseudomonas fluorescens Fermentation

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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 α‐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.