Phosphinothricin resistance in Aspergillus niger and its utility as a selectable transformation marker.
Aspergillus niger is moderately susceptible to inhibition by phosphinothricin (PPT)-a potent inhibitor of glutamine synthetase. This growth inhibition was relieved by L-glutamine. PPT inhibited A. niger glutamine synthetase in vitro (K(I), 54 microM) and the inhibition was competitive with L-glutamate. The bar gene, imparting resistance to PPT, was successfully exploited as a dominant marker to transform this fungus. Very high PPT concentrations were required in the overlay for selection. Apart from bar transformants, colonies spontaneously resistant to PPT were frequently encountered on selection media. Reasons for such spontaneous resistance, albeit of moderate growth phenotype, were sought using one such isolate (SRPPT). The SRPPT isolate showed a 2-3-fold decrease in its glutamate uptake rate. Elevated external glutamate levels further suppressed the PPT-induced growth inhibition. Cellular entry of PPT could be through the L-glutamate uptake system thereby accounting for the observed spontaneous resistant phenotype. These results were useful in the fine-tuning of bar-selection in A. niger.
Ahuja
Manmeet
M
Biotechnology Group, School of Bioscience and Bioengineering, Indian Institute of Technology Bombay, Mumbai 400076, India.
Punekar
Narayan S
NS
D000613:Aminobutyrates / Q000378:metabolism / Q000494:pharmacology*; D001234:Aspergillus niger / Q000187:drug effects* / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism; D025141:Drug Resistance, Fungal; D004791:Enzyme Inhibitors / Q000378:metabolism / Q000494:pharmacology; D005656:Fungal Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D005819:Genetic Markers; D005974:Glutamate-Ammonia Ligase / Q000037:antagonists & inhibitors / Q000378:metabolism; D018698:Glutamic Acid / Q000378:metabolism; D007700:Kinetics; D014170:Transformation, Genetic
D000613:Aminobutyrates; D004791:Enzyme Inhibitors; D005656:Fungal Proteins; D005819:Genetic Markers; D018698:Glutamic Acid; C003121:phosphinothricin; D005974:Glutamate-Ammonia Ligase