The object of this project is to determine and quantify the environmental factors that influence the productivity of a cloned protein by a recombinant yeast population. The system used as a model is Saccharomyces cerevisiae transformed with a 2u hybrid plasmid. This is a host- vector system with significant practical applicability due to the potential of 2u-based plasmids for good stability and relatively high copy numbers. The effects of the bioculturing environment on host cell physiology and recombinant productivity will be studied for the system S. cerevisiae - 2u hybrid plasmid under the control of constitutive and inducible promoters. The identity and concentration of medium components, the nature of nutrient limitation in the chemostat, and the bioreactor operation strategy will be examined systematically in order to assess quantitatively the influence of these parameters on plasmid stability, copy number, growth rate, and cloned protein expression rate. Saccharomyces cerevisiae (baker's yeast) is an excellent microorganism for the production of recombinant proteins since it is non-pathogenic to humans and has been grown at an industrial scale for centuries. Large-scale fermentation of recombinant yeast is currently used for the production of several valuable products, including therapeutic proteins. The proposed research program will provide a better understanding of the factors that influence the efficiency of recombinant protein production in yeast using a system of commercial significance, the 2u hybrid plasmid. This project will also suggest appropriate manipulations of the various fermentation parameters that can result in more economic production of biomedically important proteins through this biotechnological route. The generation of new knowledge in this area and the potential development of industrial operational strategies will increase the competitive edge of the United States in biotechnology.
