Academic Journal
Bi-directionalized promoter systems allow methanol-free production of hard-to-express peroxygenases with Komagataella Phaffii
| العنوان: | Bi-directionalized promoter systems allow methanol-free production of hard-to-express peroxygenases with Komagataella Phaffii |
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| المؤلفون: | Mihail Besleaga, Christian Zimmermann, Katharina Ebner, Robert L. Mach, Astrid R. Mach-Aigner, Martina Geier, Anton Glieder, Oliver Spadiut, Julian Kopp |
| المصدر: | Microbial Cell Factories, Vol 23, Iss 1, Pp 1-14 (2024) |
| بيانات النشر: | BMC |
| سنة النشر: | 2024 |
| المجموعة: | Directory of Open Access Journals: DOAJ Articles |
| مصطلحات موضوعية: | Komagataella phaffii, Bi-directionalized promoter, Unspecific peroxygenase, Recombinant protein production, Methanol-free, Derepressed feeding, Microbiology, QR1-502 |
| الوصف: | Background Heme-incorporating peroxygenases are responsible for electron transport in a multitude of organisms. Yet their application in biocatalysis is hindered due to their challenging recombinant production. Previous studies suggest Komagataella phaffi to be a suitable production host for heme-containing enzymes. In addition, co-expression of helper proteins has been shown to aid protein folding in yeast. In order to facilitate recombinant protein expression for an unspecific peroxygenase (AnoUPO), we aimed to apply a bi-directionalized expression strategy with Komagataella phaffii. Results In initial screenings, co-expression of protein disulfide isomerase was found to aid the correct folding of the expressed unspecific peroxygenase in K. phaffi. A multitude of different bi-directionalized promoter combinations was screened. The clone with the most promising promoter combination was scaled up to bioreactor cultivations and compared to a mono-directional construct (expressing only the peroxygenase). The strains were screened for the target enzyme productivity in a dynamic matter, investigating both derepression and mixed feeding (methanol-glycerol) for induction. Set-points from bioreactor screenings, resulting in the highest peroxygenase productivity, for derepressed and methanol-based induction were chosen to conduct dedicated peroxygenase production runs and were analyzed with RT-qPCR. Results demonstrated that methanol-free cultivation is superior over mixed feeding in regard to cell-specific enzyme productivity. RT-qPCR analysis confirmed that mixed feeding resulted in high stress for the host cells, impeding high productivity. Moreover, the bi-directionalized construct resulted in a much higher specific enzymatic activity over the mono-directional expression system. Conclusions In this study, we demonstrate a methanol-free bioreactor production strategy for an unspecific peroxygenase, yet not shown in literature. Hence, bi-directionalized assisted protein expression in K. phaffii, cultivated ... |
| نوع الوثيقة: | article in journal/newspaper |
| اللغة: | English |
| تدمد: | 1475-2859 |
| Relation: | https://doi.org/10.1186/s12934-024-02451-9; https://doaj.org/toc/1475-2859; https://doaj.org/article/59820955ea6f4b18a7aabf9052b1dd80 |
| DOI: | 10.1186/s12934-024-02451-9 |
| الاتاحة: | https://doi.org/10.1186/s12934-024-02451-9 https://doaj.org/article/59820955ea6f4b18a7aabf9052b1dd80 |
| رقم الانضمام: | edsbas.D9A2131 |
| قاعدة البيانات: | BASE |
Full Text Finder | تدمد: | 14752859 |
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| DOI: | 10.1186/s12934-024-02451-9 |