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

Abstract

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 pAO4-2, which contains a functional A. oryzae pyrG gene as selection marker. Transformation of the A. oryzae pyrG mutant with circular pAO4-2 resulted in the appearance of Pyr+ transformants at a frequency of up to 20 per micrograms of DNA, whereas with linear pAO4-2 up to 200 transformants per micrograms 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 beta-galactosidase, and uidA, encoding beta-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.

Two Novel, Putatively Cell Wall-Associated and Glycosylphosphatidylinositol-Anchored α-Glucanotransferase Enzymes ofAspergillus niger

“…The amplified PCR fragments were digested with NotI and XbaI or XbaI and BamHI, respectively, and cloned in a three-way ligation into NotIand BamHI-digested pBluescript II SK to obtain plasmid pAgtAF53. Subsequently, pAgtAF53 was digested with XbaI and ligated with the 2.7-kb XbaI fragment encoding the Aspergillus oryzae pyrG gene, obtained from plasmid pAO4-13 (18), which resulted in the agtA disruption plasmid p⌬agtA. Before transformation into strain MA70.15, p⌬agtA was linearized with NotI.…”

Database mining and transcriptional analysis of genes encoding inulin-modifying enzymes of Aspergillus niger

“…Subsequently, pF3 was digested with BamHI and KpnI, and the fragment obtained was ligated into BamHI-and KpnI-digested pF5 to give pF53. pF53 was digested with SalI and BamHI and inserted with the SalI-BamHI fragment containing the Aspergillus oryzae pyrG gene, obtained from plasmid pAO4-13 (de Ruiter-Jacobs et al, 1989), resulting in the creA disruption plasmid pXY1.1. Plasmid pXY1.1 was linearized with NotI and transformed into AB4.1.…”

Molecular genetic analysis of vesicular transport in Aspergillus niger reveals partial conservation of the molecular mechanism of exocytosis in fungi

“…The respective 59 region KpnI-XhoI fragments, 39 HindIII-NotI fragments and a 1.7 kb HindIII-XhoI fragment from pAO4-13 (de Ruiter- Jacobs et al, 1989) containing the AopyrG gene were cloned into the pBluscript-SK + backbone prepared by digestion with KpnI and NotI. In the case of sncA, 3.1 kb of the hygromycin-resistance cassette isolated from pAN7-1 (Punt et al, 1987) was used to replace the sncA ORF.…”