The NADP-dependent glutamate dehydrogenase gene from Penicillium chrysogenum and the construction of expression vectors for filamentous fungi

The gdhA gene encoding the NADP-dependent glutamate dehydrogenase activity from Penicillium chrysogenum has been isolated and characterized for its us e in gene expression. The nucleotide sequence of a 2816-bp genomic fragment was determined, showing an open reading frame of 1600 bp interrupted by tw o introns, of 160 bp and 57 bp respectively, with fungal consensus splice-s ite junctions. The predicted amino acid sequence revealed a high degree of identity to glutamate dehydrogenase enzymes, especially to those from the f ungi Aspergillus nidulans (82%) and Neurospora crassa (78%). The gdhA gene was found to be present in a single copy in the genome of several P. chryso genum strains with different penicillin productivity. The use of the gdhA p romoter for homologous and heterologous gene expression in fungi and Escher ichia coli was analyzed. Heterologous gene expression was ascertained by th e construction of gene fusions with the lacZ gene from E. coli and the bleo mycin-resistance determinant (ble(R)) from Streptoalloteichus hindustanus. Homologous gene expression was shown through the use of the penicillin-bios ynthetic genes pcbC and penDE from P. chrysogenum and the cephalosporin bio synthetic genes cefEF and cefG from Acremonium chrysogenum.