Cloning and analysis of Pycnoporus cinnabarinus cellobiose dehydrogenase

We have cloned and sequenced a gene encoding cellobiose dehydrogenase (CDH) from Pycnoporus cinnabarinus ( Pci). PCR primers that may recognize a homologous cdh were designed using regions of complete conservation of amino acid sequence within the known sequences of Trametes versicolor ( Tv) and Phanerochaete chrysosporium ( Pc) CDH. Upstream primers hybridized to regions encoding the heme domain, whereas downstream primers recognized highly conserved regions within the flavin domain. Eight different primer pairs yielded three PCR products close in size to the control amplification, which used cloned Tv cdh as template. The PCR products that were close to the control size were cloned, and one of these, a 1.8-kb product, was completely sequenced. The PCR product was highly homologous to both Tv and Pch cdh, and contained eight putative introns. The cloned product was used to isolate a full-length clone encoding CDH from a Pci genomic library. Pci cdh encoded a protein with 83% identity with Tv CDH and 74% identity with Pch CDH. Northern blot analysis revealed that Pci cdh was transcribed as a single mRNA species and was expressed in the presence of cellulose as the carbon source.