Combinatorial Mutagenesis and Selection to Understand and Improve Yeast Promoters
Microbial promoters are important targets both for understanding the global gene expression and developing genetic tools for heterologous expression of proteins and complex biosynthetic pathways. Previously, we have developed and used combinatorial mutagenesis methods to analyse and improve bacteria...
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Online Access: | http://dx.doi.org/10.1155/2013/926985 |
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doaj-80c6d77e06a74442b842ca9c160b41012020-11-24T21:03:59ZengHindawi LimitedBioMed Research International2314-61332314-61412013-01-01201310.1155/2013/926985926985Combinatorial Mutagenesis and Selection to Understand and Improve Yeast PromotersLaila Berg0Trine Aakvik Strand1Svein Valla2Trygve Brautaset3Department of Biotechnology, Norwegian University of Science and Technology, Sem Sælands vei 6, 7491 Trondheim, NorwayDepartment of Molecular Biology, SINTEF Materials and Chemistry, Sem Sælands vei 2, 7465 Trondheim, NorwayDepartment of Biotechnology, Norwegian University of Science and Technology, Sem Sælands vei 6, 7491 Trondheim, NorwayDepartment of Molecular Biology, SINTEF Materials and Chemistry, Sem Sælands vei 2, 7465 Trondheim, NorwayMicrobial promoters are important targets both for understanding the global gene expression and developing genetic tools for heterologous expression of proteins and complex biosynthetic pathways. Previously, we have developed and used combinatorial mutagenesis methods to analyse and improve bacterial expression systems. Here, we present for the first time an analogous strategy for yeast. Our model promoter is the strong and inducible promoter in methylotrophic Pichia pastoris. The Zeocin resistance gene was applied as a valuable reporter for mutant promoter activity, and we used an episomal plasmid vector to ensure a constant reporter gene dosage in the yeast host cells. This novel design enabled direct selection for colonies of recombinant cells with altered Zeocin tolerance levels originating solely from randomly introduced point mutations in the promoter DNA sequence. We demonstrate that this approach can be used to select for promoter variants with abolished glucose repression in large mutant libraries. We also selected promoter variants with elevated expression level under induced conditions. The properties of the selected promoter variants were confirmed by expressing luciferase as an alternative reporter gene. The tools developed here should be useful for effective screening, characterization, and improvement of any yeast promoters.http://dx.doi.org/10.1155/2013/926985 |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Laila Berg Trine Aakvik Strand Svein Valla Trygve Brautaset |
spellingShingle |
Laila Berg Trine Aakvik Strand Svein Valla Trygve Brautaset Combinatorial Mutagenesis and Selection to Understand and Improve Yeast Promoters BioMed Research International |
author_facet |
Laila Berg Trine Aakvik Strand Svein Valla Trygve Brautaset |
author_sort |
Laila Berg |
title |
Combinatorial Mutagenesis and Selection to Understand and Improve Yeast Promoters |
title_short |
Combinatorial Mutagenesis and Selection to Understand and Improve Yeast Promoters |
title_full |
Combinatorial Mutagenesis and Selection to Understand and Improve Yeast Promoters |
title_fullStr |
Combinatorial Mutagenesis and Selection to Understand and Improve Yeast Promoters |
title_full_unstemmed |
Combinatorial Mutagenesis and Selection to Understand and Improve Yeast Promoters |
title_sort |
combinatorial mutagenesis and selection to understand and improve yeast promoters |
publisher |
Hindawi Limited |
series |
BioMed Research International |
issn |
2314-6133 2314-6141 |
publishDate |
2013-01-01 |
description |
Microbial promoters are important targets both for understanding the global gene expression and developing genetic tools for heterologous expression of proteins and complex biosynthetic pathways. Previously, we have developed and used combinatorial mutagenesis methods to analyse and improve bacterial expression systems. Here, we present for the first time an analogous strategy for yeast. Our model promoter is the strong and inducible promoter in methylotrophic Pichia pastoris. The Zeocin resistance gene was applied as a valuable reporter for mutant promoter activity, and we used an episomal plasmid vector to ensure a constant reporter gene dosage in the yeast host cells. This novel design enabled direct selection for colonies of recombinant cells with altered Zeocin tolerance levels originating solely from randomly introduced point mutations in the promoter DNA sequence. We demonstrate that this approach can be used to select for promoter variants with abolished glucose repression in large mutant libraries. We also selected promoter variants with elevated expression level under induced conditions. The properties of the selected promoter variants were confirmed by expressing luciferase as an alternative reporter gene. The tools developed here should be useful for effective screening, characterization, and improvement of any yeast promoters. |
url |
http://dx.doi.org/10.1155/2013/926985 |
work_keys_str_mv |
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