Cloning of the cbhI and cbhII genes involved in cellulose utilisation by the straw mushroom Volvariella volvacea

The straw mushroom Volvariella volvacea is cultivated on substrates rich in cellulose and has been shown to produce a family of cellulolytic enzymes. A PCR-based strategy was adopted to clone genes involved in cellulose utili sation, using degenerate primers designed to amplify conserved catalytic do main sequences of cellobiohydrolases (CBHs). PCR with these primers produce d two DNA fragments with sequence similarity to the cbhI and cbhII gene fam ilies detected in Trichoderma, Phanerochaete and Agaricus species. Full-len gth clones of these genes were obtained from an EMBL3 genomic library, and RACE-PCR was used to verify the presence of introns. The cbhI homologue has a coding region of 1722 bp, containing two introns, generating a 536 amino acid polypeptide product. The cbhII gene has a coding region of 1693 bp, c ontaining five introns, and gives rise to a 470-amino acid polypeptide prod uct. Northern and PCR analyses were used to study the expression of the gen es. These revealed that transcripts of both genes were induced on medium co ntaining cellulose - with cbhI being expressed more strongly than cbhII - b ut were repressed on medium containing glucose.