Sort Lecture : Expanding the toolbox of the Pichia pastoris platform and Systems biology based identification of novel promoters, secretion leaders and metabolic engineering targets
Pichia pastoris is the most frequently used yeast system for heterologous protein production today, however, the toolbox of available genetic elements is rather limited. Instead of classical genetic approaches, we applied systems biology tools to improve several aspects of the P. pastoris production platform.
A set of novel regulated promoters, enabling induction without methanol, was successfully identified by using DNA microarrays and shown to be suitable for high level expression of recombinant proteins in glucose-based protein production processes.
Analysis of the secretome of P. pastoris revealed that the secretion leader of the most abundant secretory protein can be successfully used to target several recombinant human proteins for secretion, even exceeding the secretion levels obtained with the commonly used Saccharomyces cerevisiae MFα secretion leader and generating a correct N-terminus. Surprisingly, this leader is undergoing a unusual 3-step processing on its way to the cell exterior, making the novel secretion leader sequences not only a valuable tool for recombinant protein production, but also for basic research of intracellular transport.
The incorporation of heterologous protein production into the genome-scale metabolic model allows the investigation of interplay between protein production, energy demand and biomass formation, and the prediction of cell engineering targets. Thereby, enhancement of recombinant protein production by model based redirections of carbon fluxes and energy production, with the example of intracellular production of human copper/zinc superoxide dismutase in P. pastoris was achieved.
Taken together, these novel elements expand the toolbox of the P. pastoris platform and enable more robust and cost-effective production processes for biopharmaceutical proteins and for industrial enzymes.