Exploring the potential of the glycerol-3-phosphate dehydrogenase 2 (GPD2) promoter for recombinant gene expression in Saccharomyces cerevisiae
A control point for keeping redox homeostasis in Saccharomyces cerevisiae during fermentative growth is the dynamic regulation of transcription for the glycerol-3-phosphate dehydrogenase 2 (GPD2) gene. In this study, the possibility to steer the activity of the GPD2 promoter was investigated by plac...
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| Format: | Article |
| Language: | English |
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Elsevier
2015-09-01
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| Series: | Biotechnology Reports |
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| Online Access: | http://www.sciencedirect.com/science/article/pii/S2215017X15000363 |
| _version_ | 1818386956443189248 |
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| author | Jan Dines Knudsen Ted Johanson Anna Eliasson Lantz Magnus Carlquist |
| author_facet | Jan Dines Knudsen Ted Johanson Anna Eliasson Lantz Magnus Carlquist |
| author_sort | Jan Dines Knudsen |
| collection | DOAJ |
| description | A control point for keeping redox homeostasis in Saccharomyces cerevisiae during fermentative growth is the dynamic regulation of transcription for the glycerol-3-phosphate dehydrogenase 2 (GPD2) gene. In this study, the possibility to steer the activity of the GPD2 promoter was investigated by placing it in strains with different ability to reoxidise NADH, and applying different environmental conditions. Flow cytometric analysis of reporter strains expressing green fluorescent protein (GFP) under the control of the GPD2 promoter was used to determine the promoter activity at the single-cell level. When placed in a gpd1Δgpd2Δ strain background, the GPD2 promoter displayed a 2-fold higher activity as compared to the strong constitutive glyceraldehyde-3-phosphate dehydrogenase (TDH3). In contrast, the GPD2 promoter was found to be inactive when cells were cultivated in continuous mode at a growth rate of 0.3 h−1 and in conditions with excess oxygen (i.e. with an aeration of 2.5 vvm, and a stirring of 800 rpm). In addition, a clear window of operation where the gpd1Δgpd2Δ strain can be grown with the same efficiency as wild type yeast was identified. In conclusion, the flow cytometry mapping revealed conditions where the GPD2 promoter was either completely inactive or hyperactive, which has implications for its implementation in future biotechnological applications such as for process control of heterologous gene expression. |
| first_indexed | 2024-12-14T04:02:17Z |
| format | Article |
| id | doaj.art-6d09766fc52e4ba8b3ee3a7a0e652b5b |
| institution | Directory Open Access Journal |
| issn | 2215-017X |
| language | English |
| last_indexed | 2024-12-14T04:02:17Z |
| publishDate | 2015-09-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Biotechnology Reports |
| spelling | doaj.art-6d09766fc52e4ba8b3ee3a7a0e652b5b2022-12-21T23:17:55ZengElsevierBiotechnology Reports2215-017X2015-09-017C10711910.1016/j.btre.2015.06.001Exploring the potential of the glycerol-3-phosphate dehydrogenase 2 (GPD2) promoter for recombinant gene expression in Saccharomyces cerevisiaeJan Dines Knudsen0Ted Johanson1Anna Eliasson Lantz2Magnus Carlquist3Division of Applied Microbiology, Department of Chemistry, Faculty of Engineering, Lund University, Getingevägen 60, SE-22100 Lund, SwedenDepartment of Systems Biology, Technical University of Denmark, Soltofts Plads, Building 223, DK-2800 Kgs. Lyngby, DenmarkDepartment of Chemical and Biochemical Engineering, Technical University of Denmark, Soltofts Plads, Building 228, DK-2800 Kgs. Lyngby, DenmarkDivision of Applied Microbiology, Department of Chemistry, Faculty of Engineering, Lund University, Getingevägen 60, SE-22100 Lund, SwedenA control point for keeping redox homeostasis in Saccharomyces cerevisiae during fermentative growth is the dynamic regulation of transcription for the glycerol-3-phosphate dehydrogenase 2 (GPD2) gene. In this study, the possibility to steer the activity of the GPD2 promoter was investigated by placing it in strains with different ability to reoxidise NADH, and applying different environmental conditions. Flow cytometric analysis of reporter strains expressing green fluorescent protein (GFP) under the control of the GPD2 promoter was used to determine the promoter activity at the single-cell level. When placed in a gpd1Δgpd2Δ strain background, the GPD2 promoter displayed a 2-fold higher activity as compared to the strong constitutive glyceraldehyde-3-phosphate dehydrogenase (TDH3). In contrast, the GPD2 promoter was found to be inactive when cells were cultivated in continuous mode at a growth rate of 0.3 h−1 and in conditions with excess oxygen (i.e. with an aeration of 2.5 vvm, and a stirring of 800 rpm). In addition, a clear window of operation where the gpd1Δgpd2Δ strain can be grown with the same efficiency as wild type yeast was identified. In conclusion, the flow cytometry mapping revealed conditions where the GPD2 promoter was either completely inactive or hyperactive, which has implications for its implementation in future biotechnological applications such as for process control of heterologous gene expression.http://www.sciencedirect.com/science/article/pii/S2215017X15000363GPD2 promoterInducible promoterGlycerol-deficient yeastGPD deletionFlow cytometryRedox reporterNADH/NAD+Population heterogeneityRecombinant protein production |
| spellingShingle | Jan Dines Knudsen Ted Johanson Anna Eliasson Lantz Magnus Carlquist Exploring the potential of the glycerol-3-phosphate dehydrogenase 2 (GPD2) promoter for recombinant gene expression in Saccharomyces cerevisiae Biotechnology Reports GPD2 promoter Inducible promoter Glycerol-deficient yeast GPD deletion Flow cytometry Redox reporter NADH/NAD+ Population heterogeneity Recombinant protein production |
| title | Exploring the potential of the glycerol-3-phosphate dehydrogenase 2 (GPD2) promoter for recombinant gene expression in Saccharomyces cerevisiae |
| title_full | Exploring the potential of the glycerol-3-phosphate dehydrogenase 2 (GPD2) promoter for recombinant gene expression in Saccharomyces cerevisiae |
| title_fullStr | Exploring the potential of the glycerol-3-phosphate dehydrogenase 2 (GPD2) promoter for recombinant gene expression in Saccharomyces cerevisiae |
| title_full_unstemmed | Exploring the potential of the glycerol-3-phosphate dehydrogenase 2 (GPD2) promoter for recombinant gene expression in Saccharomyces cerevisiae |
| title_short | Exploring the potential of the glycerol-3-phosphate dehydrogenase 2 (GPD2) promoter for recombinant gene expression in Saccharomyces cerevisiae |
| title_sort | exploring the potential of the glycerol 3 phosphate dehydrogenase 2 gpd2 promoter for recombinant gene expression in saccharomyces cerevisiae |
| topic | GPD2 promoter Inducible promoter Glycerol-deficient yeast GPD deletion Flow cytometry Redox reporter NADH/NAD+ Population heterogeneity Recombinant protein production |
| url | http://www.sciencedirect.com/science/article/pii/S2215017X15000363 |
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