Orthologous promoters from related methylotrophic yeasts surpass expression of endogenous promoters of Pichia pastoris
Abstract Methylotrophic yeasts such as Komagataella phaffii (syn. Pichia pastoris, Pp), Hansenula polymorpha (Hp), Candida boidinii (Cb) and Pichia methanolica (Pm) are widely used protein production platforms. Typically, strong, tightly regulated promoters of genes coding for their methanol utiliza...
| Main Authors: | , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
SpringerOpen
2020-02-01
|
| Series: | AMB Express |
| Subjects: | |
| Online Access: | http://link.springer.com/article/10.1186/s13568-020-00972-1 |
| _version_ | 1798045931733516288 |
|---|---|
| author | Thomas Vogl Jasmin Elgin Fischer Patrick Hyden Richard Wasmayer Lukas Sturmberger Anton Glieder |
| author_facet | Thomas Vogl Jasmin Elgin Fischer Patrick Hyden Richard Wasmayer Lukas Sturmberger Anton Glieder |
| author_sort | Thomas Vogl |
| collection | DOAJ |
| description | Abstract Methylotrophic yeasts such as Komagataella phaffii (syn. Pichia pastoris, Pp), Hansenula polymorpha (Hp), Candida boidinii (Cb) and Pichia methanolica (Pm) are widely used protein production platforms. Typically, strong, tightly regulated promoters of genes coding for their methanol utilization (MUT) pathways are used to drive heterologous gene expression. Despite highly similar open reading frames in the MUT pathways of the four yeasts, the regulation of the respective promoters varies strongly between species. While most endogenous Pp MUT promoters remain tightly repressed after depletion of a repressing carbon, Hp, Cb and Pm MUT promoters are derepressed to up to 70% of methanol induced levels, enabling methanol free production processes in their respective host background. Here, we have tested a series of orthologous promoters from Hp, Cb and Pm in Pp. Unexpectedly, when induced with methanol, the promoter of the HpMOX gene reached very similar expression levels as the strong methanol, inducible, and most frequently used promoter of the Pp alcohol oxidase 1 gene (P PpAOX1 ). The HpFMD promoter even surpassed P PpAOX1 up to three-fold, when induced with methanol, and reached under methanol-free/derepressed conditions similar expression as the methanol induced P PpAOX1 . These results demonstrate that orthologous promoters from related yeast species can give access to otherwise unobtainable regulatory profiles and may even considerably surpass endogenous promoters in P. pastoris. |
| first_indexed | 2024-04-11T23:30:06Z |
| format | Article |
| id | doaj.art-97d5a3610e3a47d0ab8e28a36fad2898 |
| institution | Directory Open Access Journal |
| issn | 2191-0855 |
| language | English |
| last_indexed | 2024-04-11T23:30:06Z |
| publishDate | 2020-02-01 |
| publisher | SpringerOpen |
| record_format | Article |
| series | AMB Express |
| spelling | doaj.art-97d5a3610e3a47d0ab8e28a36fad28982022-12-22T03:57:11ZengSpringerOpenAMB Express2191-08552020-02-011011910.1186/s13568-020-00972-1Orthologous promoters from related methylotrophic yeasts surpass expression of endogenous promoters of Pichia pastorisThomas Vogl0Jasmin Elgin Fischer1Patrick Hyden2Richard Wasmayer3Lukas Sturmberger4Anton Glieder5Institute of Molecular Biotechnology, NAWI Graz, Graz University of TechnologyInstitute of Molecular Biotechnology, NAWI Graz, Graz University of TechnologyInstitute of Molecular Biotechnology, NAWI Graz, Graz University of TechnologyInstitute of Molecular Biotechnology, NAWI Graz, Graz University of TechnologyInstitute of Molecular Biotechnology, NAWI Graz, Graz University of TechnologyInstitute of Molecular Biotechnology, NAWI Graz, Graz University of TechnologyAbstract Methylotrophic yeasts such as Komagataella phaffii (syn. Pichia pastoris, Pp), Hansenula polymorpha (Hp), Candida boidinii (Cb) and Pichia methanolica (Pm) are widely used protein production platforms. Typically, strong, tightly regulated promoters of genes coding for their methanol utilization (MUT) pathways are used to drive heterologous gene expression. Despite highly similar open reading frames in the MUT pathways of the four yeasts, the regulation of the respective promoters varies strongly between species. While most endogenous Pp MUT promoters remain tightly repressed after depletion of a repressing carbon, Hp, Cb and Pm MUT promoters are derepressed to up to 70% of methanol induced levels, enabling methanol free production processes in their respective host background. Here, we have tested a series of orthologous promoters from Hp, Cb and Pm in Pp. Unexpectedly, when induced with methanol, the promoter of the HpMOX gene reached very similar expression levels as the strong methanol, inducible, and most frequently used promoter of the Pp alcohol oxidase 1 gene (P PpAOX1 ). The HpFMD promoter even surpassed P PpAOX1 up to three-fold, when induced with methanol, and reached under methanol-free/derepressed conditions similar expression as the methanol induced P PpAOX1 . These results demonstrate that orthologous promoters from related yeast species can give access to otherwise unobtainable regulatory profiles and may even considerably surpass endogenous promoters in P. pastoris.http://link.springer.com/article/10.1186/s13568-020-00972-1Pichia pastorisKomagataella phaffiiOrthologous promotersMethanol-freeDerepression |
| spellingShingle | Thomas Vogl Jasmin Elgin Fischer Patrick Hyden Richard Wasmayer Lukas Sturmberger Anton Glieder Orthologous promoters from related methylotrophic yeasts surpass expression of endogenous promoters of Pichia pastoris AMB Express Pichia pastoris Komagataella phaffii Orthologous promoters Methanol-free Derepression |
| title | Orthologous promoters from related methylotrophic yeasts surpass expression of endogenous promoters of Pichia pastoris |
| title_full | Orthologous promoters from related methylotrophic yeasts surpass expression of endogenous promoters of Pichia pastoris |
| title_fullStr | Orthologous promoters from related methylotrophic yeasts surpass expression of endogenous promoters of Pichia pastoris |
| title_full_unstemmed | Orthologous promoters from related methylotrophic yeasts surpass expression of endogenous promoters of Pichia pastoris |
| title_short | Orthologous promoters from related methylotrophic yeasts surpass expression of endogenous promoters of Pichia pastoris |
| title_sort | orthologous promoters from related methylotrophic yeasts surpass expression of endogenous promoters of pichia pastoris |
| topic | Pichia pastoris Komagataella phaffii Orthologous promoters Methanol-free Derepression |
| url | http://link.springer.com/article/10.1186/s13568-020-00972-1 |
| work_keys_str_mv | AT thomasvogl orthologouspromotersfromrelatedmethylotrophicyeastssurpassexpressionofendogenouspromotersofpichiapastoris AT jasminelginfischer orthologouspromotersfromrelatedmethylotrophicyeastssurpassexpressionofendogenouspromotersofpichiapastoris AT patrickhyden orthologouspromotersfromrelatedmethylotrophicyeastssurpassexpressionofendogenouspromotersofpichiapastoris AT richardwasmayer orthologouspromotersfromrelatedmethylotrophicyeastssurpassexpressionofendogenouspromotersofpichiapastoris AT lukassturmberger orthologouspromotersfromrelatedmethylotrophicyeastssurpassexpressionofendogenouspromotersofpichiapastoris AT antonglieder orthologouspromotersfromrelatedmethylotrophicyeastssurpassexpressionofendogenouspromotersofpichiapastoris |