Expanding the genetic toolbox of Rhodotorula toruloides by identification and validation of six novel promoters induced or repressed under nitrogen starvation

Microbial Cell Factories · 5 authors, 4 centres

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TL;DR

This study identified and validated six novel promoters in the yeast Rhodotorula toruloides that are either induced or repressed during nitrogen starvation.

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The study used RNA sequencing to analyze gene expression in Rhodotorula toruloides strain BOT-A2 during exponential growth and nitrogen starvation on glucose or xylose. Genes with a |log2 fold change| ≥ 2 were identified, and promoter regions from 16 of these genes were tested in a fluorescent reporter assay. Three promoters were found to be upregulated and three downregulated under nitrogen starvation. One promoter (from gene RTBOTA2_003877) showed an on-off switch behavior, being active only under full nitrogen depletion. The study is limited by random integration of reporter constructs, which does not allow conclusions about absolute promoter strength. The findings provide new tools for engineering R. toruloides for specific regulation of genes in relation to nitrogen availability.

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other

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preclinical research

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ORIGINAL PAPER

PMC10440040

Expanding the genetic toolbox of Rhodotorula toruloides by identification and validation of six novel promoters induced or repressed under nitrogen starvation

Microbial Cell Factories

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ABSTRACT

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 |log 2 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. Supplementary Information The online version contains supplementary material available at 10.1186/s12934-023-02175-2.

CITATION EXPORT

Daniel P. Brink, Friederike Mierke, Joakim Norbeck et al.. Expanding the genetic toolbox of Rhodotorula toruloides by identification and validation of six novel promoters induced or repressed under nitrogen starvation. Microbial Cell Factories. (2023). https://doi.org/10.1186/s12934-023-02175-2

AUTHORS

5 · 4 CENTRES

Daniel P. Brink, Friederike Mierke, Joakim Norbeck, Verena Siewers, Thomas Andlid

1https://ror.org/040wg7k59grid.5371.00000 0001 0775 6028Systems and Synthetic Biology, Department of Life Sciences, Chalmers University of Technology, Gothenburg, Sweden
2https://ror.org/012a77v79grid.4514.40000 0001 0930 2361Applied Microbiology, Department of Chemistry, Lund University, Lund, Sweden
3https://ror.org/040wg7k59grid.5371.00000 0001 0775 6028Food and Nutrition Science, Department of Life Sciences, Chalmers University of Technology, Gothenburg, Sweden
4grid.5170.30000 0001 2181 8870 The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Lyngby, Denmark