Redox Engineering by Ectopic Overexpression of NADH Kinase in Recombinant Pichia pastoris ( Komagataella phaffii ): Impact on Cell Physiology and Recombinant Production of Secreted Proteins

Abstract

Recombinant protein overproduction often results in oxidative stress, causing deviations from the optimal redox cofactor regeneration balance. This becomes one of the limiting factors in obtaining high levels of heterologous protein production. Overexpression of redox-affecting enzymes has been explored in other organisms, such as Saccharomyces cerevisiae , as a means to fine tune the cofactor regeneration balance in order to obtain higher protein titers. In the present work, this strategy is explored in P. pastoris . In particular, one NADH kinase enzyme from S. cerevisiae (Pos5) is used, either in the cytosol or in mitochondria of P. pastoris , and its impact on the production of a model protein (antibody fragment) is evaluated. A significant improvement in the production of the model protein is observed when the kinase is directed to the cytosol. These results are significant in the field of heterologous protein production in general and in particular in the development of improved metabolic engineering strategies for P. pastoris .