Development of an homologous transformation system for Acremonium chrysogenum based on the beta-tubulin gene.

Abstract

The beta-tubulin gene was isolated from the filamentous fungus Acremonium chrysogenum using a heterologous gene probe to screen an A. chrysogenum lambda library. Sequencing of the A. chrysogenum gene revealed a mosaic gene which contains five exons and four intervening sequences. The exons encode for a polypeptide of 447 amino-acid residues which showed a high degree of similarity when compared with amino-acid sequences from beta-tubulins of other eukaryotes. The introns are characterized by typical consensus sequences found in intervening sequences from other filamentous fungi. In-vitro mutagenesis of codon 167 of the beta-tubulin gene resulted in the substitution of a phenylalanine by a tyrosine in the corresponding polypeptide sequence. The mutated gene was used successfully in the transformation and co-transformation of A. chrysogenum to benomyl resistance. The molecular analysis of transformants provided evidence that they contain the mutated beta-tubulin gene in addition to the wild-type gene, as was proved by Southern-hybridization analysis and direct sequencing of PCR amplification products.