Novel transcriptional regulation of the GAP promoter in Pichia pastoris towards high expression of heterologous proteins.
P. pastoris
Constitutive expression
Heterologous protein
Transcription factor
pGAP
Journal
Microbial cell factories
ISSN: 1475-2859
Titre abrégé: Microb Cell Fact
Pays: England
ID NLM: 101139812
Informations de publication
Date de publication:
24 Jul 2024
24 Jul 2024
Historique:
received:
08
11
2023
accepted:
21
05
2024
medline:
24
7
2024
pubmed:
24
7
2024
entrez:
23
7
2024
Statut:
epublish
Résumé
Pichia pastoris (Komagataella phaffii) is a promising production host, but the usage of methanol limits its application in the medicine and food industries. To improve the constitutive expression of heterologous proteins in P. pastoris, four new potential transcription regulators (Loc1p, Msn2p, Gsm1p, Hot1p) of the glyceraldehyde triphosphate dehydrogenase promoter (pGAP) were revealed in this study by using cellulase E4 as reporter gene. On this basis, a series of P. pastoris strains with knockout or overexpression of transcription factors were constructed and the deletion of transcription factor binding sites on pGAP was confirmed. The results showed that Loc1p and Msn2p can inhibit the activity of pGAP, while Gsm1p and Hot1p can enhance the activity of pGAP; Loc1p, Gsm1p and Hot1p can bind directly to pGAP, while Msn2p must be treated to expose the C-terminal domain to bind to pGAP. Moreover, manipulating a single transcription factor led to a 0.96-fold to 2.43-fold increase in xylanase expression. In another model protein, aflatoxin oxidase, knocking out Loc1 based on AFO-∆Msn2 strain resulted in a 0.63-fold to 1.4-fold increase in expression. It can be demonstrated that the combined use of transcription factors can further improve the expression of exogenous proteins in P. pastoris. These findings will contribute to the construction of pGAP-based P. pastoris systems towards high expression of heterologous proteins, hence improving the application potential of yeast.
Sections du résumé
BACKGROUND
BACKGROUND
Pichia pastoris (Komagataella phaffii) is a promising production host, but the usage of methanol limits its application in the medicine and food industries.
RESULTS
RESULTS
To improve the constitutive expression of heterologous proteins in P. pastoris, four new potential transcription regulators (Loc1p, Msn2p, Gsm1p, Hot1p) of the glyceraldehyde triphosphate dehydrogenase promoter (pGAP) were revealed in this study by using cellulase E4 as reporter gene. On this basis, a series of P. pastoris strains with knockout or overexpression of transcription factors were constructed and the deletion of transcription factor binding sites on pGAP was confirmed. The results showed that Loc1p and Msn2p can inhibit the activity of pGAP, while Gsm1p and Hot1p can enhance the activity of pGAP; Loc1p, Gsm1p and Hot1p can bind directly to pGAP, while Msn2p must be treated to expose the C-terminal domain to bind to pGAP. Moreover, manipulating a single transcription factor led to a 0.96-fold to 2.43-fold increase in xylanase expression. In another model protein, aflatoxin oxidase, knocking out Loc1 based on AFO-∆Msn2 strain resulted in a 0.63-fold to 1.4-fold increase in expression. It can be demonstrated that the combined use of transcription factors can further improve the expression of exogenous proteins in P. pastoris.
CONCLUSION
CONCLUSIONS
These findings will contribute to the construction of pGAP-based P. pastoris systems towards high expression of heterologous proteins, hence improving the application potential of yeast.
Identifiants
pubmed: 39044288
doi: 10.1186/s12934-024-02435-9
pii: 10.1186/s12934-024-02435-9
doi:
Substances chimiques
Transcription Factors
0
Fungal Proteins
0
Recombinant Proteins
0
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
206Subventions
Organisme : National Key Research and Development Program of China
ID : No. 2021YFC2103002
Organisme : National Key Research and Development Program of China
ID : No. 2021YFC2103002
Organisme : National Key Research and Development Program of China
ID : No. 2021YFC2103002
Organisme : National Key Research and Development Program of China
ID : No. 2021YFC2103002
Organisme : National Key Research and Development Program of China
ID : No. 2021YFC2103002
Organisme : National Key Research and Development Program of China
ID : No. 2021YFC2103002
Organisme : National Key Research and Development Program of China
ID : No. 2021YFC2103002
Organisme : National Key Research and Development Program of China
ID : No. 2021YFC2103002
Informations de copyright
© 2024. The Author(s).
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