Integration of these genes into the yeast chromosomes eliminates the segregational instability and copy number variation associated with plasmid-based systems. The goal of the work is to develop methods for the efficient, regulated integration of precise numbers of stable cloned genes. The relationship between expression and cloned gene number, the structural stability of the integrations, and the ability of each system to insert a series of different genes are also being investigated. These studies will provide an important tool for the introduction of metabolic pathways into yeast and for fine-tuning cloned gene copy number. The yeast Kluyveromyces lactis is a promising recombinant host for the production and secretion of complex proteins.
LiP acts by a free radical mechanism, and has been shown to degrade a wide range of pollutants.
The goal of this research is to enhance the production and secretion of LiP by employing alternate hosts, in particular the yeasts Saccharomyces cerevisiae and Kluyveromyces lactis . The work includes implementing strategies for the synthesis and secretion of this foreign protein in yeast, and evaluating the resulting expression level, secretion efficiency, and localization of active and inactive product. The results will be used to design immobilized yeast and cell-free enzyme systems for the biodegradation of toxic wastes.