Isolation and transformation of the pyruvate kinase gene of Aspergillus nidulans

Summary The Aspergillus nidulans pyruvate kinase gene was isolated by heterologous hybridization using the corresponding yeast gene as a probe. A 2.9 kb EcoRI/BamHI fragment, which exclusively hybridized to the yeast gene, was subcloned in pBR322. This clone was used to transform an A. nidulans pkiA deletion mutant to PKI⁺. The analysis of transformants with respect to the kind of integration revealed about 80% homologous integration – 55% by a double cross-over event (type III integration), 25% by a single cross-over event (type I integration). Type II transformants (20%) that arise by non-homologous integration have not been further characterized with respect to the sites of integration.A direct correlation between the number of copies of the gene integrated into the genome and the measured pyruvate kinase activity was found after growth on a glycolytic carbon source. From this, it was concluded that the 2.9 kb EcoRI/BamHI fragment contains the complete pyruvate kinase structural gene, including the promoter region.However, after growth on a gluconeogenic carbon source, the regulation of gene expression was found to be disturbed. On acetate an increase in activity per gene copy (0.2 IU) was found in the transformants, as compared with wild-type levels. It is suggested that the pyruvate kinase gene is regulated by negative control, and that some sequences involved in this regulation are missing in the cloned fragment.Abbreviations ATP Adenosinetriphosphate - MM Minimal medium - PEP Phosphoenolpyruvate