An integrated modelling and experimental approach to improving single-chain antibody fragment production in Pichia pastoris

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Kate Royle, David Leak, Cleo Kontoravdi

Imperial College London, United Kingdom

Pichia pastoris is a commonly used expression host for heterologous protein production, predominantly because it is amenable to genetic manipulation and can grow to high cell densities in cheap culture media. While considerable yields can be achieved in this way, the specific productivity is relatively low. Consequently, the full impact of this host on industrial biotechnology has not yet been realised. Previous studies to increase productivity have focused on bioengineering, such as targeting gene and strain characteristics, and fermentation conditions. Whereas the latter has been subject to systematic multivariate optimisations, the former has not. The majority of bioengineering studies have targeted one factor in isolation, and despite comparable strategies have variable outcomes. Here, an integrated experimental and computational modelling approach has been taken to understand how the factors interact and develop a global optimisation strategy. A deterministic single cell model of protein production in P. pastoris has been devised including the essential aspects of transcription, translation and folding in the endoplasmic reticulum (ER). Additionally, as heterologous protein production can induce stress responses, the unfolded protein response (UPR) and ER associated degradation pathway (ERAD) have been accounted for. Results from preliminary simulations highlighted key regulators of production capacity. To understand how yield arises from a balance of these, the regulators were characterised in strains which express a heterologous protein to different degrees. Consequently, two single chain antibody fragments were cloned into P. pastoris and a high and a low secretor of each identified. These strains were characterised with RT Q-PCR and LC-MS/MS and the results integrated into the model. Knowledge of the upper and lower bounds for parameters from the low and high secreting strains allowed for the optimal value to be predicted for the highest yield. This constitutes an integrated modelling and experimental approach to improving productivity in P. pastoris.