A Genetic Screen for the Isolation of Mutants with Increased Flux in the Isoprenoid Pathway of Yeast.
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| Názov: | A Genetic Screen for the Isolation of Mutants with Increased Flux in the Isoprenoid Pathway of Yeast. |
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| Autori: | Wadhwa M; Department of Biological Sciences, Indian Institute of Science Education and Research, Mohali, S.A.S Nagar, Punjab, India., Bachhawat AK; Department of Biological Sciences, Indian Institute of Science Education and Research, Mohali, S.A.S Nagar, Punjab, India. anand@iisermohali.ac.in. |
| Zdroj: | Methods in molecular biology (Clifton, N.J.) [Methods Mol Biol] 2019; Vol. 1927, pp. 231-246. |
| Spôsob vydávania: | Journal Article; Research Support, Non-U.S. Gov't |
| Jazyk: | English |
| Informácie o časopise: | Publisher: Humana Press Country of Publication: United States NLM ID: 9214969 Publication Model: Print Cited Medium: Internet ISSN: 1940-6029 (Electronic) Linking ISSN: 10643745 NLM ISO Abbreviation: Methods Mol Biol Subsets: MEDLINE |
| Imprint Name(s): | Publication: Totowa, NJ : Humana Press Original Publication: Clifton, N.J. : Humana Press, |
| Výrazy zo slovníka MeSH: | Genes, Fungal* , Genetic Testing* , Mutation*, Terpenes/*metabolism , Yeasts/*genetics , Yeasts/*metabolism, Carotenoids/metabolism ; Cloning, Molecular/methods ; Escherichia coli/genetics ; Escherichia coli/metabolism ; Metabolic Networks and Pathways ; Mutagenesis ; Plasmids/genetics ; Reproducibility of Results ; Saccharomyces cerevisiae/genetics ; Saccharomyces cerevisiae/metabolism ; Transformation, Genetic |
| Abstrakt: | The yeast Saccharomyces cerevisiae is one of the preferred hosts for the production of terpenoids through metabolic engineering. A genetic screen to identify novel mutants that can increase the flux in the isoprenoid pathway has been lacking. We present here the method that has led to the development of a carotenoid based visual screen by exploiting the carotenogenic genes from the red yeast Rhodosporidium toruloides, an organism known to have high levels of carotenoids. We also discuss the methods to use this screen for the identification of mutants that can lead to higher isoprenoid flux. The carotenoid based screen was developed in S. cerevisiae using phytoene synthase RtPSY1 and a hyperactive mutant of the enzyme phytoene dehydrogenase, RtCRTI(A393T) from Rhodosporidium toruloides. As validation of the genetic screen is critical at all stages, we describe the method to validate the screen using a known flux increasing gene, a truncated HMG1 (tHMG1). To demonstrate how this screen can be exploited to isolate mutants, we described how targeted mutagenesis of candidate gene, SPT15 a TATA binding protein involved in the global transcription machinery can be carried out to yield novel mutants with increased metabolic flux. Since it is also important to ensure that the isolated mutants are enhancing general isoprenoid flux, we describe how this can be established using an alternate isoprenoid, α-farnesene. |
| Contributed Indexing: | Keywords: Carotenoids; Mutagenesis; Phytoene dehydrogenase; Phytoene synthase; R. toruloides; S. cerevisiae; SPT15 |
| Substance Nomenclature: | 0 (Terpenes) 36-88-4 (Carotenoids) |
| Entry Date(s): | Date Created: 20190222 Date Completed: 20190621 Latest Revision: 20190621 |
| Update Code: | 20250114 |
| DOI: | 10.1007/978-1-4939-9142-6_16 |
| PMID: | 30788796 |
| Databáza: | MEDLINE |
Nájsť tento článok vo Web of Science | Abstrakt: | The yeast Saccharomyces cerevisiae is one of the preferred hosts for the production of terpenoids through metabolic engineering. A genetic screen to identify novel mutants that can increase the flux in the isoprenoid pathway has been lacking. We present here the method that has led to the development of a carotenoid based visual screen by exploiting the carotenogenic genes from the red yeast Rhodosporidium toruloides, an organism known to have high levels of carotenoids. We also discuss the methods to use this screen for the identification of mutants that can lead to higher isoprenoid flux. The carotenoid based screen was developed in S. cerevisiae using phytoene synthase RtPSY1 and a hyperactive mutant of the enzyme phytoene dehydrogenase, RtCRTI(A393T) from Rhodosporidium toruloides. As validation of the genetic screen is critical at all stages, we describe the method to validate the screen using a known flux increasing gene, a truncated HMG1 (tHMG1). To demonstrate how this screen can be exploited to isolate mutants, we described how targeted mutagenesis of candidate gene, SPT15 a TATA binding protein involved in the global transcription machinery can be carried out to yield novel mutants with increased metabolic flux. Since it is also important to ensure that the isolated mutants are enhancing general isoprenoid flux, we describe how this can be established using an alternate isoprenoid, α-farnesene. |
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| ISSN: | 1940-6029 |
| DOI: | 10.1007/978-1-4939-9142-6_16 |