Identification of improved signal peptides for heterologous expression in Saccharomyces using a screen that exploits Gaussia luciferase
Abstract A high-throughput and sensitive screen for the improved expression of gene targets in Saccharomyces cerevisiae is described that is based upon the activity of the luciferase from Gaussia princeps. Using the Unspecific Peroxygenase (UPO) from Agrocybe aegerita (AaeUPO) as a model protein, im...
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Nature Portfolio
2025-07-01
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| Series: | Scientific Reports |
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| Online Access: | https://doi.org/10.1038/s41598-025-09669-6 |
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| author | Ginevra Camboni Jared Cartwright Gideon Grogan |
| author_facet | Ginevra Camboni Jared Cartwright Gideon Grogan |
| author_sort | Ginevra Camboni |
| collection | DOAJ |
| description | Abstract A high-throughput and sensitive screen for the improved expression of gene targets in Saccharomyces cerevisiae is described that is based upon the activity of the luciferase from Gaussia princeps. Using the Unspecific Peroxygenase (UPO) from Agrocybe aegerita (AaeUPO) as a model protein, improvements in expression, effected through error-prone PCR-based mutation within the signal peptide (SP) domain, can be detected using fusion of the target to Gaussia luciferase encoded downstream of the first folded domain of the AaeUPO protein and luminescent assay of expression supernatants. In this way, previously undiscovered mutations within the SP of AaeUPO that improve expression were revealed, and then applied to the expression of full-length AaeUPO in S. cerevisiae. The system was validated against control expression constructs that were well or poorly expressed and indicated a 13.9-fold improvement in expression for the best mutant over the wild-type SP sequence. It is envisaged that this protocol may be applied generally to the high-throughput detection of improved expression in S. cerevisiae for constructs that are engineered using directed evolution techniques. |
| format | Article |
| id | doaj-art-21d2e12ed5d1400dabcbf8bec6b7a5fa |
| institution | DOAJ |
| issn | 2045-2322 |
| language | English |
| publishDate | 2025-07-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Scientific Reports |
| spelling | doaj-art-21d2e12ed5d1400dabcbf8bec6b7a5fa2025-08-20T03:03:36ZengNature PortfolioScientific Reports2045-23222025-07-0115111110.1038/s41598-025-09669-6Identification of improved signal peptides for heterologous expression in Saccharomyces using a screen that exploits Gaussia luciferaseGinevra Camboni0Jared Cartwright1Gideon Grogan2Department of Chemistry, University of YorkDepartment of Biology, University of YorkDepartment of Chemistry, University of YorkAbstract A high-throughput and sensitive screen for the improved expression of gene targets in Saccharomyces cerevisiae is described that is based upon the activity of the luciferase from Gaussia princeps. Using the Unspecific Peroxygenase (UPO) from Agrocybe aegerita (AaeUPO) as a model protein, improvements in expression, effected through error-prone PCR-based mutation within the signal peptide (SP) domain, can be detected using fusion of the target to Gaussia luciferase encoded downstream of the first folded domain of the AaeUPO protein and luminescent assay of expression supernatants. In this way, previously undiscovered mutations within the SP of AaeUPO that improve expression were revealed, and then applied to the expression of full-length AaeUPO in S. cerevisiae. The system was validated against control expression constructs that were well or poorly expressed and indicated a 13.9-fold improvement in expression for the best mutant over the wild-type SP sequence. It is envisaged that this protocol may be applied generally to the high-throughput detection of improved expression in S. cerevisiae for constructs that are engineered using directed evolution techniques.https://doi.org/10.1038/s41598-025-09669-6Komagataella phaffiiPichia pastorisGaussia luciferaseSignal peptideUnspecific peroxygenaseDirected evolution |
| spellingShingle | Ginevra Camboni Jared Cartwright Gideon Grogan Identification of improved signal peptides for heterologous expression in Saccharomyces using a screen that exploits Gaussia luciferase Scientific Reports Komagataella phaffii Pichia pastoris Gaussia luciferase Signal peptide Unspecific peroxygenase Directed evolution |
| title | Identification of improved signal peptides for heterologous expression in Saccharomyces using a screen that exploits Gaussia luciferase |
| title_full | Identification of improved signal peptides for heterologous expression in Saccharomyces using a screen that exploits Gaussia luciferase |
| title_fullStr | Identification of improved signal peptides for heterologous expression in Saccharomyces using a screen that exploits Gaussia luciferase |
| title_full_unstemmed | Identification of improved signal peptides for heterologous expression in Saccharomyces using a screen that exploits Gaussia luciferase |
| title_short | Identification of improved signal peptides for heterologous expression in Saccharomyces using a screen that exploits Gaussia luciferase |
| title_sort | identification of improved signal peptides for heterologous expression in saccharomyces using a screen that exploits gaussia luciferase |
| topic | Komagataella phaffii Pichia pastoris Gaussia luciferase Signal peptide Unspecific peroxygenase Directed evolution |
| url | https://doi.org/10.1038/s41598-025-09669-6 |
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