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Background  A tannic acid-inducible and mycoviral-regulated laccase3 ( lac 3) from the chestnut blight fungus  Cryphonectria parasitica  has recently been identified, but further characterization was hampered because of the precipitation of protein products by tannic acid supplementation. The present study investigated the heterologous expression of the functional laccase3 using a yeast  Saccharomyces cerevisiae .    Results  Laccase activity in the culture broth of transformants measured using a laccase-specific substrate suggested that the  lac 3 gene was successfully expressed and the corresponding protein product secreted into the culture media. In addition, activity staining and Western blot analysis of a native gel revealed that the enzyme activity co-existed with the protein product specific to anti-laccase3 antibody, confirming that the cloned  lac 3 gene is responsible for the laccase activity. When transformants were grown on plates containing tannic acid-supplemented media, brown coloration was observed around transformed cells, indicating the oxidation of tannic acid. However, the enzymatic activity was measurable only in the selective ura -  media and was negligible in nonselective nutrient-rich culture conditions. This was in part because of the increased plasmid instability in the nonselective media. Moreover, the protein product of  lac 3 appears to be sensitive to the cultured nonselective nutrient-rich broth, because a rapid decline in enzymatic activity was observed when the cultured broth of ura -  media was mixed with that of nonselective nutrient-rich broth. In addition, constitutive expression of the  lac 3 gene resulted in a reduced cell number of the  lac 3 transformants compared to that of vector-only transformed control. However, the presence of recombinant vector without  lac 3 induction did not affect the growth of transformants.    Conclusions  The results suggest that expression of the  lac 3 gene has an inhibitory effect on the growth of transformed  S. cerevisiae  and that the controlled expression of  lac 3 is appropriate for the possible application of recombinant yeast to the treatment of phenolic compounds.

Heterologous expression of a tannic acid-inducible laccase3 of Cryphonectria parasitica in Saccharomyces cerevisiae