Continous Bioprocessing - BISY
Most biopharmaceutical and technical enzymes are manufactured in classical batch or fed batch-based cultivation systems. This results in high batch to batch variations and renders difficulties for the downstream. Furthermore, reactor setup is expensive and sterilization and sanitization with base is not only energy consuming but also harmful to the environment. The establishment of continuous biomanufacturing is highly desired by industry as space-time yields can be improved at stable product quality. Unfortunately, microbial hosts suffer from instable productivity in common continuous cultivations, due to yet not understood reasons.
Figure 1: Schematic drawing of cascaded continuous bioprocessing
P. pastoris is a frequently used host in multiple biotechnological applications, as cheap cultivations can be conducted at feasible growth rates and glycosylated protein secretion into the supernatant is possible at high specific productivities. Cascaded continuous cultivation has shown to be superior to common continuous cultivation systems. Hence, bi-directional promotor systems which can separate production of helper proteins and target protein, should be ideal for cascaded continuous cultivation.
Figure 2: DIC micrographs of PpMuts adapted from 10.1038/srep03279, öffnet eine externe URL in einem neuen Fenster
Figure 3: PpMuts cultivation in 1.5L bioreactor cultivation
In previous studies, conducted in this working group, we established a cascaded continuous cultivation system to ensure constant productivity over a period of at least two weeks of induction with E. coli. The generated knowledge should be transferred to P. pastoris within the project BI(U)SY Pichia. We want to combine the benefits of the yeast host using bidirectional promotor systems supplied by BISY GmbH. with the benefits of the cascade culturing system. In using the bidirectional systems in the cascade, we will be able to spatially resolve the production of different genes of interest and enable a stable continuous production platform for P. pastoris. A variety of strains will thus be screened using different process parameters.
Kopp, J., Kittler, S., Slouka, C., Herwig, C., Spadiut, O., & Wurm, D. J. (2020) Repetitive Fed-Batch: A Promising Process Mode for Biomanufacturing With E. coli. Frontiers in Bioengineering and Biotechnology 8, 573607.
Kittler, S., Kopp, J., Veelenturf, P.G., Spadiut, O., Delvigne, F., Herwig, C., Slouka, C., (2020) The Lazarus Escherichia coli Effect: Recovery of Productivity on Glycerol/Lactose Mixed Feed in Continuous Biomanufacturing. Frontiers in Bioengineering and Biotechnology 8.
Kopp, J., Slouka C, Spadiut O and Herwig C (2019) The Rocky Road From Fed-Batch to Continuous Processing With E. coli. Front. Bioeng. Biotechnol. 7:328.
Kopp, J., Kolkmann, A.-M., Veleenturf, P.G., Spadiut, O., Herwig, C., and Slouka, C. (2019). Boosting Recombinant Inclusion Body Production—From Classical Fed-Batch Approach to Continuous Cultivation. Frontiers in Bioengineering and Biotechnology 7, 297.
Slouka, C., Kopp, J., Strohmer, D., Kager, J., Spadiut, O., and Herwig, C. (2019). Monitoring and control strategies for inclusion body production in E. coli based on glycerol consumption. J Biotechnol 296, 75-82.
Wurm, D.J., Spadiut, O., (2019) Efficient development of a mixed feed process for Pichia pastoris. Methods in Molecular Biology, pp. 323-333.
Vogl, T., Kickenweiz, T., Pitzer, J. et al. Engineered bidirectional promoters enable rapid multi-gene co-expression optimization. Nat Commun 9, 3589 (2018).
Pekarsky, A., Veiter, L., Rajamanickam, V., Herwig, C., Grünwald-Gruber, C., Altmann, F., Spadiut, O., (2018) Production of a recombinant peroxidase in different glyco-engineered Pichia pastoris strains: A morphological and physiological comparison. Microbial Cell Factories 17.
Rajamanickam, V., Metzger, K., Schmid, C., Spadiut, O., (2017) A novel bi-directional promoter system allows tunable recombinant protein production in Pichia pastoris. Microbial Cell Factories 16.
Capone, S., Ćorajević, L., Bonifert, G., Murth, P., Maresch, D., Altmann, F., Herwig, C., Spadiut, O., (2015) Combining protein and strain engineering for the production of Glyco-engineered horseradish peroxidase C1A in Pichia pastoris. International Journal of Molecular Sciences 16, 23127-23142.
Krainer, F.W., Capone, S., Jäger, M., Vogl, T., Gerstmann, M., Glieder, A., Herwig, C., Spadiut, O., (2015) Optimizing cofactor availability for the production of recombinant heme peroxidase in Pichia pastoris. Microbial Cell Factories 14.
Gmeiner, C., Spadiut, O., (2015) Effects of different media supplements on the production of an active recombinant plant peroxidase in a Pichia pastoris Δoch1 strain. Bioengineered 6, 175-178.
Further reading links