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

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• 作者：Jane C. Schneider ; Annika F. Jenings ; Deborah M. Mun ; Patricia M. McGovern ; and Lawrence C. Chew
• 刊名：Biotechnology Progress
• 出版年：2005
• 出版时间：April 2005
• 年：2005
• 卷：21
• 期：2
• 页码：343 - 348
• 全文大小：103K
• 年卷期：v.21,no.2(April 2005)
• ISSN：1520-6033

文摘

The use of antibiotic-resistance genes as selectable markers in transgenic organismsis coming under increased scrutiny, for fear that they may spread to human pathogens,thereby reducing the effectiveness of antibiotic therapy. A current Pseudomonasfluorescens protein expression system uses a tetracycline resistance gene (tetR/tetA)to maintain an expression plasmid under control of a repressible promoter and akanamycin resistance gene (kanR) to maintain a plasmid carrying a repressor gene.We investigated using auxotrophic markers to replace these two antibiotic resistancegenes: pyrF (encoding orotidine-5'-phosphate decarboxylase) in place of tetR/tetA andproC (encoding pyrroline-5-carboxylate reductase) in place of kanR, complementingtheir respective precise chromosomal deletions created by allele exchange using asuicide vector carrying pyrF as a counterselectable marker. The resulting strains,devoid of antibiotic-resistance genes, were shown to achieve high productivity ofnitrilase and thermostable -amylase equal to that of the former antibiotic-resistantproduction host. The production plasmids were stable. The pyrF (uracil-dependent)background of the production host strain also allows us to sequentially alter the genometo incorporate other desired genomic changes, deletions, or insertions using 5'-fluoroorotic acid counterselection, restoring the selectable marker after each step.