Phosphinothricin resistance in Aspergillus niger and its utility as a selectable transformation marker

Abstract

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 (KI, 54 μM) 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. © 2008 Elsevier Inc. All rights reserved.