Establishment of a biotechnological production process of a KlenTaq DNA polymerase with Pichia pastoris from cultivation to purification

Abstract

Recombinant production of thermostable DNA polymerases in Picha pastoris offers a promising alternative to classical E. coli based systems yet require reliable upstream and downstream integration. A previous thesis successfully expressed KlenTaq DNA polymerase in Erlenmeyer flasks. The objective of this work was therefore to establish a scalable bioprocess for KlenTaq production in a bioreactor, including cultivation, purification and evaluating enzymatic activity. Initial experiments confirmed KlenTaq expression in Erlenmeyer flasks by SDS-PAGE, and PCR assay demonstrated clear enzymatic activity of the produced polymerase. When transferring the process to the bioreactor, KlenTaq was expressed but consistently showed no PCR activity, despite ultrafiltration and diafiltration. Systematic cultivation parameter adjustments were therefore implemented to replicate Erlenmeyer flask conditions, including medium composition, process management, pH value during cultivation and production temperature. None of these modifications restored the activity. Only after increasing the concentration factor during ultrafiltration and performing a more extensive buffer exchange during ultrafiltration could KlenTaq activity be recovered. This indicates that insufficient purification stringency led to inhibitory components or destabilizing buffer environments that masked activity. Overall, this study establishes the first bioreactor-based process for KlenTaq DNA polymerase production in P. pastoris. The findings highlight the critical role of downstream processing, particularly concentration and buffer exchange, in retaining enzymatic activity and provide a foundation for further process optimization and scale up.