Abstract
Background
The methylotrophic yeast Pichia pastoris is frequently used for the production of recombinant proteins. However, expression levels can vary depending on the target protein. Allowing for simultaneous regulation of many genes, which may elicit a desired phenotype like increased protein production, overexpression of transcription factors can be used to overcome expression bottlenecks. Here, we present a novel P. pastoris transcription factor currently annotated as Aft1, activator of ferrous transport.
Results
The promoter regions of key secretory P. pastoris genes were screened for fungal transcription factor binding sites, revealing Aft1 as an interesting candidate for improving secretion. Genome wide analysis of transcription factor binding sites suggested Aft1 to be involved in the regulation of many secretory genes, but also indicated possible novel functions in carbohydrate metabolism. No Aft binding sites were found in promoters of characteristic iron homeostasis genes in P. pastoris. Microarrays were used to study the Aft1 regulon in detail, confirming Aft1 involvement in the regulation of carbon-responsive genes, and showing that iron regulation is dependent on FEP1, but not AFT1 expression levels. The positive effect of AFT1 overexpression on recombinant protein secretion was demonstrated for a carboxylesterase from Sphingopyxis sp. MTA144, for which secretion was improved 2.5-fold in fed batch bioreactor cultivations.
Conclusion
This study demonstrates that the transcription factor Aft1 can be used to improve recombinant protein secretion in P. pastoris. Furthermore, we discovered possible novel functions of Aft1 in carbohydrate metabolism and provide evidence arguing against a direct role of Aft1 in P. pastoris iron regulation.
The methylotrophic yeast Pichia pastoris is frequently used for the production of recombinant proteins. However, expression levels can vary depending on the target protein. Allowing for simultaneous regulation of many genes, which may elicit a desired phenotype like increased protein production, overexpression of transcription factors can be used to overcome expression bottlenecks. Here, we present a novel P. pastoris transcription factor currently annotated as Aft1, activator of ferrous transport.
Results
The promoter regions of key secretory P. pastoris genes were screened for fungal transcription factor binding sites, revealing Aft1 as an interesting candidate for improving secretion. Genome wide analysis of transcription factor binding sites suggested Aft1 to be involved in the regulation of many secretory genes, but also indicated possible novel functions in carbohydrate metabolism. No Aft binding sites were found in promoters of characteristic iron homeostasis genes in P. pastoris. Microarrays were used to study the Aft1 regulon in detail, confirming Aft1 involvement in the regulation of carbon-responsive genes, and showing that iron regulation is dependent on FEP1, but not AFT1 expression levels. The positive effect of AFT1 overexpression on recombinant protein secretion was demonstrated for a carboxylesterase from Sphingopyxis sp. MTA144, for which secretion was improved 2.5-fold in fed batch bioreactor cultivations.
Conclusion
This study demonstrates that the transcription factor Aft1 can be used to improve recombinant protein secretion in P. pastoris. Furthermore, we discovered possible novel functions of Aft1 in carbohydrate metabolism and provide evidence arguing against a direct role of Aft1 in P. pastoris iron regulation.
| Original language | English |
|---|---|
| Pages (from-to) | 120-120 |
| Journal | Microbial Cell Factories |
| Volume | 13 |
| DOIs | |
| Publication status | Published - 2014 |
Fields of Expertise
- Human- & Biotechnology
Treatment code (Nähere Zuordnung)
- Basic - Fundamental (Grundlagenforschung)
- Application
- Experimental