Bioprocess engineering and metabolic modeling as a roadmap to enhance recombinant protein production in Pichia pastoris

Abstract

Pichia pastoris is recognized as a biotechnological workhorse for recombinant protein expression. Based on past achievements and novel developments, systems biotechnology of P. pastoris has significantly progressed over the last two decades. In this doctoral thesis, a systematic analysis of operational conditions in conjunction with the development of computational toolboxes and optimal mechanical bioreactor configurations were developed as a roadmap to enhance the production of recombinant proteins through bioprocess engineering and metabolic modeling approaches. Although we used a P. pastoris strain expressing constitutively the sweet protein thaumatin, as case study, the developments achieved in this work are also applicable for the expression of other recombinant proteins of interest. First, we present an integrated framework for revealing the metabolic effects of two operational parameters – specific growth rate and dissolved oxygen concentration - in glucose-limited continuous cultures. More specifically, we employed a rational experimental design to calculate the significant statistical effects from multiple chemostat data, which was later contextualized using a curated genome-scale metabolic model. Our xxii xxii results revealed a negative effect of the oxygenation on the specific product formation rate, and a positive effect on the biomass yield. Notably, we identified a novel synergistic effect of both parameters on the specific product formation rate. Finally, model predictions indicated an opposite relationship between the oxygenation level and growth-associated ATP requirement, suggesting higher metabolic growth costs under low oxygenation. We then assembled a robust dynamic genome-scale metabolic model for glucose- limited, aerobic cultures of Pichia pastoris. The model was employed to analyze the metabolic flux distribution of a fed-batch culture and to unravel genetic and process engineering strategies to improve the production of the recombinant Human Serum Albumin (HSA). Simulations of single knock-outs indicated that carbon deviations towards cysteine and tryptophan formation could improve 63 fold HSA production. Moreover, the model suggested that implementation of a decreasing specific growth rate during the feed phase of a fed-batch culture results in a 25% increase of the volumetric productivity of the protein. Finally, optimization of Oxygen Transfer Rate (OTR) in 1-L reactors was carried out. For this purpose, we first formulated an automatic algorithm able to achieve reliable kLa estimations under different hydrodynamic conditions. Then, we presented a road map to optimize oxygen transfer rate in 1-L bioreactor, using different impeller-sparger configurations. The relative importance of aeration and agitation under several configurations and hydrodynamic conditions was assessed. Finally, we proposed a decision tree for the selection of the best configuration to improve oxygen transfer rates in bioreactors, according to viscosity and the range of operational parameters. Overall, this thesis attempted to analyze and understand metabolic effects using rational design of experiments and metabolic modeling. Moreover, this systematic analysis in conjunction with the optimization of the oxygen transfer rate in bioreactors will allow to improve recombinant protein production in Pichia pastoris.