A gene transfer system based on the homologouspyrGgene and efficient expression of bacterial genes inAspergillus oryzae

✍ Scribed by Yolanda M. J. T. Ruiter-Jacobs; Martien Broekhuijsen; Sheila E. Unkles; Edward I. Campbell; James R. Kinghorn; Roland Contreras; Peter H. Pouwels; Cees A. M. J. J. Hondell

• Publisher: Springer-Verlag
• Year: 1989
• Tongue: English
• Weight: 421 KB
• Volume: 16
• Category: Article
• ISSN: 0172-8083
• DOI: 10.1007/bf00391472

✦ Synopsis

A homologous transformation system for Aspergillus oryzae is described. The system is based on an A. owzae strain deficient in orotidine-5'-phosphate decarboxylase (pyrG) and the vector pAO4-2, which contains a functional A. oryzae pyrG gene as selection marker. Transformation of the A. owzae pyrG mutant with circular pAO4-2 resulted in the appearance of Pyr + transformants at a frequency of up to 20 per lag of DNA, whereas with linear pAO4-2 up to 200 transformants per btg 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 pAO4-2 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 13-galactosidase, and uidA, encoding 13-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.