[Molecular cloning and expression of alcohol dehydrogenase gene of Phanerochaete chrysosporium]

When Phanerochaete chrysosporium is grown under oxygen-limited condition, ethanol is one of the important metabolites. In order to understand the metabolic mechanism of P. chrysosporium grown under oxygen-limited condition, a cDNA sequence (1 071 bp) designated "PCAdh1" encoding an alcohol dehydrogenase (ADH) was cloned from the filamentous white-rot fungus P. chrysosporium. PCAdh1 gene encodes a protein of 356 amino acid residues. Although the catalytic domain and coenzyme-binding domain were highly conserved, the protein sequence of PCAdh1 showed a low level of similarity to other known ADH. The recombinant PCAdh1 protein was expressed in Escherichia coli and its enzyme activity was detected. The protein was purified and used to prepare antibody. Semi-quantitative RT-PCR and Western blot demonstrated that the expression level of PCAdh1 in P. chrysosporium remained stable despite the lowered oxygen content, indicating that the gene expression is constitutive. But with the reduction of oxygen content, the overall activity of ADH from the crude mycelia proteins was increased during the growing periods, implying that the expression of other Adh genes in P. chrysosporium is inductive.