Engineering and production of laccase from Trametes versicolor in the yeast Yarrowia lipolytica.

The lcc1 gene coding for the laccase from Trametes versicolor DSM11269 was cloned into the genome of Yarrowia lipolytica using either single or multiple integration sites. The levels of the recombinant laccase activity secreted in the culture media were 0.25 and 1 U ml(-1) for single and multiple integrations, respectively. The strain with a single integration was successfully used to express variant libraries which were screened on ABTS substrate. The strain encoding the double mutant L185P/Q214K (rM4A) showed a sixfold enhancement in secreted enzyme activity. The catalytic efficiency of the purified rM-4A laccase was respectively increased 2.4- and 2.8-fold towards ABTS and 2,6-dimethoxyphenol, compared to the rWT. Culture supernatants containing either rWT or rM-4A catalyzed the almost complete decolorization of an Amaranth solution (70 nMs(-1)). Taken together, our results open new perspectives for the use of Y. lipolytica as a molecular evolution platform to engineer laccases with improved properties.