P21: Development and scale up Pichia pastoris producing Rhizopus oryzae lipase through the use of microbioreactor and lab scale bioreactor systems for clone screening and evaluation of fed-batch strategies

In Pichia pastoris Bioprocess Engineering, clone selection and bioprocess optimization under PAOX1, using methanol as an inducing substrate, can result in low reproducibility which increases time and resource needs. An automated microfermentation platform, the RoboLector, was successfully tested to overcome the chronic problems of clone selection and optimization of fed-batch strategies with high cell density cultures. Clones from Mut⁺ P. pastoris phenotype strains expressing heterologous Rhizopus oryzae lipase (ROL) were tested to select the best performers for recombinant protein production. The RoboLector showed high performance for the selection and optimization of media with minimal cost and time. The RoboLector microbioreactor was also tested for fed-batch strategies with three clones producing lipase. The first strategy was the enzymatic release of glucose from a polymer and methanol addition every 24 hours. The second used glycerol as co-substrate with methanol and a nitrogen source at two different feeding rates. Although specific activity was higher using glycerol as co-substrate, specific activity with respect to substrate release of glucose was better. The implementation of these simple strategies increased the levels of lipolytic activity 80-fold compared with classical strategies for clone selection. Thus, these fed-batch strategies minimize the risk of error in the clone selection step. Finally, the performance of two fed-batch strategies and clone selection for lipase production were compared between the RoboLector and 5L bioreactor for three selected clones. Clone ranking was the same at both scales. When ROL production was compared with the mixed substrate fed-batch strategies the results were also quite similar.