Isolation of five laccase gene sequences from the white-rot fungus Trametes sanguinea by PCR, and cloning, characterization and expression of the laccase cDNA in yeasts

To obtain laccase-gene-specific sequences from the white-rot fungus Trametes sanguinea M85-2, a PCR screening method was used. Degenerate primers were designed based on highly conserved copper-binding regions 1 and 4 of known laccases and used to amplify laccase sequences from T. sanguinea M85-2 genomic DNA. A single 1.6-kbp DNA band was amplified and cloned into a vector. Partial sequences of 21 clones were classified into five groups (lcc1-5) and the deduced amino acid sequences were all homologous to known laccase sequences. Based on the partial sequence of lcc1, the 5'-end of its cDNA was obtained by a PCR termed 5' rapid amplification of cDNA ends (5'-RACE), and RT-PCR was then carried out using the 5'-primer and the poly-dT primer to obtain the full-length lcc1 cDNA. The obtained cDNA encoded a protein consisting of 518 amino acid residues and its first 21 amino acid residues were predicted to be the signal peptide for secretion. The conserved characteristic structures of laccase, such as copper-binding ligands, N-glycosylation sites, and cysteine residues for disulfide bridges, were observed. The genomic DNA sequence of the led gene was also cloned by PCR method and the sequence revealed 10 introns. The lcc1 cDNA was inserted into yeast vectors for heterologous expression by Saccharomyces cerevisiae and Pichia pastoris. Phenol-oxidizing activity was detected from transformants of the yeasts, indicating that the obtained cDNA encodes a laccase. Previously, two laccase isozymes were biochemically characterized and purified from T. sanguinea M85-2. Using the sequential PCR method presented here, we have obtained partial sequences of at least five laccase genes and one cDNA clone encoding a protein with laccase activity but without any enzymatic information, suggesting that expressed enzymes under restricted conditions may not represent all the isozymes in target microorganisms. PCR cloning and heterologous expression of the cloned genes can be an alternative method of screening enzymes if these enzymes have conserved sequences.