Effect of transcription promoters on the optimal production of secreted protein in fed-batch reactors.

Abstract

Production of heterologous proteins by yeast secretion imposes additional factors that need to be considered, which do not appear with production by direct expression. These include additional intracellular polypeptide processing dynamics through the secretory organelles and the protein concentration in the culture medium, which is the usual final destination of the product. Optimal control theory is applied to optimize fed-batch production of secreted protein. We maximize an objective function that includes both total production rate and product concentration. A mutant invertase is chosen as the model heterologous secretory protein. Optimal control control strategies have been obtained for the use of two different promoters for the gene transcription, a dere-pressible SUC2 promoter and a strong glycolytic GPD promoter. With the use of the strong GPD promoter, achieving maximum production occurs on the singular arc of maximum specific growth rate. As the object switches to maximum product concentration, operation occurs for longer periods of time at a slow glucose singular arc condition. The optimal control for maximizing protein production with the weak SUC2 promoter requires transitions between high and low glucose concentrations associated with multiple distinct singular arc conditions. For maximum product concentration, the high concentration branches of the singular arc supporting maximum growth rate and maximum secretion rate disappear. Operation stays essentially on the low glucose concentration branch of the singular arc, which maximizes the protein production rate and minimizes the dilution of the broth product concentration.