Modeling meets metabolic engineering: The<i> i</i>Pichia consensus model as basis for metabolic studies in<i> Komagataella</i><i> phaffii</i>

Komagataella phaffii (syn. Pichia pastoris) has become one of the most commonly employed hosts for recombinant protein expression, with methanol-utilizing (Mut+) strains being used to produce more than 400 different proteins. The availability of fully sequenced and functionally annotated genomes has greatly facilitated systems biology studies and enabled the generation of genome-scale metabolic models (GEMs) for this species. In this study, two previously published GEMs (Kp.1.0 and iAUKM) were systematically merged in order to build a unified consensus model, referred to as iPichia, that offers a more complete description of K. phaffii metabolism for the first time. iPichia GEM was then extended by introducing enzyme capacity limitations through the GECKO 3.0 framework, yielding an enzyme-constrained genome-scale model (ecPichia GEM) for Komagataella phaffii. The resulting ecPichia GEM is the first enzyme constraint genome scale metabolic model for Komagataella phaffii. The predictive performance of ecPichia GEM for growth was evaluated using data from the literature. Gene essentiality analyses were carried with iPichia compared to protein-constrained ecPichia GEM, Kp.1.0 and iAUKM GEMs. Thereafter, the model was applied to uncover promising targets for metabolic engineering aimed at producing bisabolene - an industrially relevant sesquiterpene used in both biofuel and fragrance applications. Overall, the results underline the effectiveness of enzyme-constrained metabolic modeling as a tool to support rational strain development in the field of industrial biotechnology.