(447o) Secretion of Recombinant Lignin Peroxidase in the Yeast Kluyveromyces Lactis

The metalloprotein lignin peroxidase (LiP) is able to degrade the recalcitrant biopolymer lignin as well as a large number of environmental pollutants such as TNT, PCBs, azo dyes, etc. Production of the protein in the native white rot fungus Phanerochaete chrysosporium requires nutrient limited media and secretion is seen after 4 - 6 days. Heterologous production of this protein from numerous prokaryotic and eukaryotic hosts has been met with limited success.

Saccharomyces cerevisiae and the dairy yeast Kluyveromyces lactis have been successfully employed for large scale production of proteins. In this project, we screened numerous yeast host, vector backbones and promoters for production of lignin peroxidase. Culture conditions such as media composition, inducer concentration, induction time, were further optimized for maximum secretion of protein. In order to alleviate the plasmid segregational instability, the LiP expression cassette was integrated into the K. lactis host chromosome. Current work is aimed at using directed evolution techniques for modifying the protein so that it is easier to secrete by the cell.