| Title: | Bioreactor-scale cell performance and protein production can be substantially increased by using a secretion signal that drives co-translational translocation in Pichia pastoris |
| Author(s): | Barrero JJ; Pagazartaundua A; Glick BS; Valero F; Ferrer P; |
| Address: | "Department of Chemical, Biological and Environmental Engineering, Escola d'Enginyeria, Universitat Autonoma de Barcelona, Bellaterra (Cerdanyola del Valles), 08193, Catalonia, Spain; Department of Molecular Genetics and Cell Biology, University of Chicago, 920 East 58th Street, Chicago, IL 60637, USA. Electronic address: juanjose.barrero@uab.cat. Department of Chemical, Biological and Environmental Engineering, Escola d'Enginyeria, Universitat Autonoma de Barcelona, Bellaterra (Cerdanyola del Valles), 08193, Catalonia, Spain; Department of Molecular Genetics and Cell Biology, University of Chicago, 920 East 58th Street, Chicago, IL 60637, USA. Electronic address: alejandropagaza6@gmail.com. Department of Chemical, Biological and Environmental Engineering, Escola d'Enginyeria, Universitat Autonoma de Barcelona, Bellaterra (Cerdanyola del Valles), 08193, Catalonia, Spain; Department of Molecular Genetics and Cell Biology, University of Chicago, 920 East 58th Street, Chicago, IL 60637, USA. Electronic address: bsglick@uchicago.edu. Department of Chemical, Biological and Environmental Engineering, Escola d'Enginyeria, Universitat Autonoma de Barcelona, Bellaterra (Cerdanyola del Valles), 08193, Catalonia, Spain; Department of Molecular Genetics and Cell Biology, University of Chicago, 920 East 58th Street, Chicago, IL 60637, USA. Electronic address: francisco.valero@uab.cat. Department of Chemical, Biological and Environmental Engineering, Escola d'Enginyeria, Universitat Autonoma de Barcelona, Bellaterra (Cerdanyola del Valles), 08193, Catalonia, Spain; Department of Molecular Genetics and Cell Biology, University of Chicago, 920 East 58th Street, Chicago, IL 60637, USA. Electronic address: Pau.Ferrer@uab.cat" |
| DOI: | 10.1016/j.nbt.2020.09.001 |
| ISSN/ISBN: | 1876-4347 (Electronic) 1871-6784 (Print) 1871-6784 (Linking) |
| Abstract: | "Pichia pastoris (Komagataella spp.) has become one of the most important host organisms for production of heterologous proteins of biotechnological interest, many of them extracellular. The protein secretion pathway has been recognized as a limiting process in which many roadblocks have been pinpointed. Recently, we have identified a bottleneck at the ER translocation level. In earlier exploratory studies, this limitation could be largely overcome by using an improved chimeric secretion signal to drive proteins through the co-translational translocation pathway. Here, we have further tested at bioreactor scale the improved secretion signal consisting of the pre-Ost1 signal sequence, which drives proteins through co-translational translocation, followed by the pro region from the secretion signal of the Saccharomyces cerevisiae alpha-factor mating pheromone. For comparison, the commonly used full-length alpha-factor secretion signal, which drives proteins through post-translational translocation, was tested. These two secretion signals were fused to three different model proteins: the tetrameric red fluorescent protein E2-Crimson, which can be used to visualize roadblocks in the secretory pathway; the lipase 2 from Bacillus thermocatenulatus (BTL2); and the Rhizopus oryzae lipase (ROL). All strains were tested in batch cultivation to study the different growth parameters obtained. The strains carrying the improved secretion signal showed increased final production of the proteins of interest. Interestingly, they were able to grow at significantly higher maximum specific growth rates than their counterparts carrying the conventional secretion signal. These results were corroborated in a 5 L fed-batch cultivation, where the final product concentration and volumetric productivity were also shown to be improved" |
| Keywords: | *Bioreactors Fungal Proteins/*biosynthesis Pichia/cytology/*metabolism Protein Transport Saccharomyces cerevisiae/metabolism Alpha-factor Heterologous protein production Pichia pastoris Secretion Signal sequence Translocation; |
| Notes: | "MedlineBarrero, Juan J Pagazartaundua, Alejandro Glick, Benjamin S Valero, Francisco Ferrer, Pau eng R01 GM104010/GM/NIGMS NIH HHS/ Netherlands 2020/10/13 N Biotechnol. 2021 Jan 25; 60:85-95. doi: 10.1016/j.nbt.2020.09.001. Epub 2020 Oct 9" |
