A gene transfer system based on the homologous pyrG gene and efficient expression of bacterial genes in Aspergillus oryzae

Summary

A homologous transformation system for Aspergillus oryzae is described. The system is based on an A. oryzae strain deficient in orotidine-5′-phosphate decarboxylase (pyrG) and the vector pA04-2, which contains a functional A. oryzae pyrG gene as selection marker. Transformation of the A. oryzae pyrG mutant with circular PA04-2 resulted in the appearance of Pyr⁺ transformants at a frequency of up to 20 per μg of DNA, whereas with linear pA04-2 up to 200 transformants per μg DNA were obtained. In 75 % of the Pyr⁺ transformants recombination events had occurred at the pyrG locus, most of which (90%) resulted in insertion of one or two copies of the vector and the others (10%) in a replacement of the mutant allele by the wild-type allele. Vector pA04-2 is also capable of transforming a corresponding mutant of Aspergillus niger. This transformation system was used to introduce into A. oryzae the heterologous and non-selectable bacterial genes lacZ, encoding β-galactosidase, and uidA, encoding β-glucuronidase. Using the Aspergillus nidulans gpdA promoter to drive bacterial gene expression in A. oryzae, relatively high levels of activity, as well as protein per se, as judged by western blot analyses, were obtained.