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Expanding the genet...
Expanding the genetic toolbox of Rhodotorula toruloides by identification and validation of six novel promoters induced or repressed under nitrogen starvation
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- Brink, Daniel P. (författare)
- Chalmers University of Technology,Lund University,Lunds universitet,Teknisk mikrobiologi,Centrum för tillämpade biovetenskaper,Kemiska institutionen,Institutioner vid LTH,Lunds Tekniska Högskola,Applied Microbiology,Center for Applied Life Sciences,Department of Chemistry,Departments at LTH,Faculty of Engineering, LTH
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- Mierke, Friederike, 1992 (författare)
- Chalmers University of Technology,Chalmers tekniska högskola
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- Norbeck, Joakim, 1965 (författare)
- Chalmers University of Technology,Chalmers tekniska högskola
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- Siewers, Verena, 1976 (författare)
- Chalmers University of Technology,Chalmers tekniska högskola,Novo Nordisk Fonden,Novo Nordisk Foundation
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- Andlid, Thomas, 1963 (författare)
- Chalmers University of Technology,Chalmers tekniska högskola
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(creator_code:org_t)
- 2023
- 2023
- Engelska.
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Ingår i: Microbial Cell Factories. - 1475-2859. ; 22:1
- Relaterad länk:
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http://dx.doi.org/10... (free)
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https://doi.org/10.1...
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https://research.cha...
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https://lup.lub.lu.s...
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Abstract
Ämnesord
Stäng
- BACKGROUND: The non-conventional yeast Rhodotorula toruloides is an emerging host organism in biotechnology by merit of its natural capacity to accumulate high levels of carotenoids and intracellular storage lipids from a variety of carbon sources. While the number of genetic engineering strategies that employ R. toruloides is increasing, the lack of genetic tools available for modification of this yeast is still limiting strain development. For instance, several strong, constitutive R. toruloides promoters have been characterized, but to date, only five inducible promoters have been identified. Although nitrogen-limited cultivation conditions are commonly used to induce lipid accumulation in this yeast, no promoters regulated by nitrogen starvation have been described for R. toruloides. RESULTS: In this study, we used a combination of genomics and transcriptomics methods to identify novel R. toruloides promoter sequences that are either inducible or repressible by nitrogen starvation. RNA sequencing was used to assess gene expression in the recently isolated strain R. toruloides BOT-A2 during exponential growth and during nitrogen starvation, when cultivated with either glucose or xylose as the carbon source. The genome of BOT-A2 was sequenced using a combination of long- and short-read sequencing and annotated with support of the RNAseq data. Differential expression analysis was used to identify genes with a |log2 fold change|≥ 2 when comparing their expression during nitrogen depletion to that during exponential growth. The promoter regions from 16 of these genes were evaluated for their ability to drive the expression of a fluorescent reporter gene. Three promoters that were clearly upregulated under nitrogen starvation and three that were downregulated were selected and further characterized. One promoter, derived from gene RTBOTA2_003877, was found to function like an on-off switch, as it was only upregulated under full nitrogen depletion and downregulated in the presence of the nitrogen source. CONCLUSIONS: Six new R. toruloides promoters that were either upregulated or downregulated under nitrogen-starvation were identified. These substantially contribute to the available promoters when engineering this organism and are foreseen to be particularly useful for future engineering strategies requiring specific regulation of target genes in accordance with nitrogen availability.
Ämnesord
- NATURVETENSKAP -- Biologi -- Biokemi och molekylärbiologi (hsv//swe)
- NATURAL SCIENCES -- Biological Sciences -- Biochemistry and Molecular Biology (hsv//eng)
- NATURVETENSKAP -- Biologi -- Mikrobiologi (hsv//swe)
- NATURAL SCIENCES -- Biological Sciences -- Microbiology (hsv//eng)
- NATURVETENSKAP -- Biologi -- Genetik (hsv//swe)
- NATURAL SCIENCES -- Biological Sciences -- Genetics (hsv//eng)
- NATURVETENSKAP -- Biologi -- Bioinformatik och systembiologi (hsv//swe)
- NATURAL SCIENCES -- Biological Sciences -- Bioinformatics and Systems Biology (hsv//eng)
Nyckelord
- Nitrogen starvation
- Xylose
- Rhodotorula toruloides
- Rhodosporidium
- Promoters
- RNAseq
- MinION
- Differential gene expression
- Glucose
- Differential gene expression
- Glucose
- MinION
- Nitrogen starvation
- Promoters
- Rhodosporidium
- Rhodotorula toruloides
- RNAseq
- Xylose
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- ref (ämneskategori)
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