A toolset of constitutive promoters for engineering Rhodosporidium toruloides
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ABSTRACT:
PROVIDER: PRJNA527258 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR8733520_1.fastq.gz | Fastqsanger.gz | |||
| SRR8733520_2.fastq.gz | Fastqsanger.gz | |||
| SRR8733521_1.fastq.gz | Fastqsanger.gz | |||
| SRR8733521_2.fastq.gz | Fastqsanger.gz | |||
| SRR8733512_1.fastq.gz | Fastqsanger.gz |
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Project description:Background: A key prerequisite for pathway engineering is the development of genetic tools and resources. Rhodosporidium toruloides is emerging as a promising host for the production of bioproducts from lignocellulosic biomass. However, there is a lack of characterized promoters to drive expression of heterologous genes for strain engineering in R. toruloides. Results: The resulting data describes a set of native R. toruloides promoters, characterized over time in four media commonly used for this yeast. The promoter sequences were sorted using transcriptional analysis and several of them were found to drive expression bidirectionally. We measured promoter expression by flow cytometry using a dual fluorescent reporter system. From these analyses, we found a total of 20 constitutive promoters (12 monodirectional and 8 bidirectional), that are strong, stable, and can reliably be used for genetic manipulation of this emergent host. Conclusions: We are presenting a list of robust constitutive promoters that are native to the emergent bioconversion host R. toruloides which helps to fulfill the lack of existing tools for this yeast and that can be applied in future metabolic engineering studies.
2020-03-02 | GSE128360 | GEO
Project description:Oleaginous yeasts are capable of accumulating high levels of intracellular storage lipids from excess carbon during times when other key nutrients are limited. The basidiomycete yeast Rhodosporidium toruloides is an emerging host for microbial cell factory applications thanks to its naturally high lipid and carotenoid production. However, the engineering toolbox in this non-model host is limited and is currently a bottleneck for implementation of metabolic engineering strategies. In this study, we performed differential gene expression analysis with the goal to identify promoters that are strongly induced or repressed by nitrogen-limitation, a condition that is commonly used to induce lipid accumulation in oleaginous yeasts. The genome-wide transcriptional response of R. toruloides BOT-A2 was analysed using RNAseq during exponential growth and nitrogen-starvation, with either glucose or xylose as the sole carbon source. To validate the differential gene expression findings, reporter genes were constructed by placing the candidate promoters in control of a fluorescent protein, integrated in BOT-A2 and evaluated in vivo.
2023-08-23 | GSE217097 | GEO
Project description:BackgroundRhodosporidium toruloides is a promising host for the production of bioproducts from lignocellulosic biomass. A key prerequisite for efficient pathway engineering is the availability of robust genetic tools and resources. However, there is a lack of characterized promoters to drive expression of heterologous genes for strain engineering in R. toruloides.ResultsThis data describes a set of native R. toruloides promoters, characterized over time in four different media commonly used for cultivation of this yeast. The promoter sequences were selected using transcriptional analysis and several of them were found to drive expression bidirectionally. Promoter expression strength was determined by measurement of EGFP and mRuby2 reporters by flow cytometry. A total of 20 constitutive promoters (12 monodirectional and 8 bidirectional) were found, and are expected to be of potential value for genetic engineering of R. toruloides.ConclusionsA set of robust and constitutive promoters to facilitate genetic engineering of R. toruloides is presented here, ranging from a promoter previously used for this purpose (P7, glyceraldehyde 3-phosphate dehydrogenase, GAPDH) to stronger monodirectional (e.g., P15, mitochondrial adenine nucleotide translocator, ANT) and bidirectional (e.g., P9 and P9R, histones H3 and H4, respectively) promoters. We also identified promoters that may be useful for specific applications such as late-stage expression (e.g., P3, voltage-dependent anion channel protein 2, VDAC2). This set of characterized promoters significantly expands the range of engineering tools available for this yeast and can be applied in future metabolic engineering studies.
| S-EPMC6599526 | biostudies-literature
Project description:Global proteomics datasets to study lignocellulosic carbon utilization in Rhodosporidium Toruloides
2021-09-09 | PXD022377 | Pride
Project description:Rhodosporidium toruloides is an oleaginous yeast under development with promising industrial applications. Since promoters of different strengths have been demonstrated as an efficient strategy to fine-tune gene expression in synthetic biology, a set of constitutive promoters with strengths varying over 2 orders of magnitude were identified in R. toruloides through transcriptome analysis under different growth conditions. Promoter candidates were first cloned and characterized using an enhanced green fluorescent protein (EGFP) as a reporter under eight conditions, and 31 promoters were identified with strength varied from 0.1 to 19.0 folds of the commonly used strong promoter of the glyceraldehyde-3-phosphate dehydrogenase gene (PGPD1). The resultant promoters were then used to optimize the linoleic acid biosynthetic pathway in R. toruloides in different media, including the use of lignocellulosic hydrolysate as the fermentation substrate, and improved the production of linoleic acid by up to 214.2% in minimal medium, with the highest production of 350.3 mg/L in Yeast Peptone Dextrose medium. This work has enriched the promoter library of R. toruloides, and helped develop R. toruloides as a platform organism for applications in biomanufacturing and synthetic biology.
| S-EPMC9908808 | biostudies-literature
Project description:The goal of the study is to use Next generation sequencing (RNA-seq) to study the underlying regulation of glycerol metabolism in mixed culture fermentation (glucose and glycerol) of Rhodosporidium toruloides. We sequenced the RNA from 4 different samples in the mixed culture (glucose and glycerol) with 2 replicates each. Transcriptional profiles showed that glycerol might be produced intracellularly and glycerol kinase (GUT1) and glycerol 3–phosphate dehydrogenase (GUT2) enzymes were not down-regulated in the presence of glucose at the transcriptional level. It also showed that this yeast has a different regulation compared to S.cerevisiae. Certain insights into lipid biosynthesis on these mixed cultures are provided at systems level. This analysis provides interesting targets for metabolic engineering in this organism growing on glucose and glycerol.
2016-05-12 | GSE74307 | GEO
Project description:Biorefining of renewable feedstocks is one of the most promising routes to replace fossil-based products. Since many common fermentation hosts, such as Saccharomyces cerevisiae, are naturally unable to convert many component plant cell wall polysaccharides, the identification of organisms with broad catabolism capabilities represents an opportunity to expand the range of substrates used in fermentation biorefinery approaches. The red basidiomycete yeast Rhodosporidium toruloides is a promising and robust host for lipid and terpene derived chemicals. Previous studies demonstrated assimilation of a range of substrates, from C5/C6-sugars to aromatic molecules similar to lignin monomers. In the current study, we analyzed R. toruloides potential to assimilate Dgalacturonic acid, a major sugar in many pectin-rich agricultural waste streams, including sugar beet pulp and citrus peels. D-galacturonic acid is not a preferred substrate for many fungi, but its metabolism was found to be on par with D-glucose and D-xylose in R. toruloides. A genome-wide analysis by combined RNAseq/RB-TDNAseq revealed those genes with high relevance for fitness on D-galacturonic acid. While R. toruloides was found to utilize the same non-phosphorylative catabolic pathway known from ascomycetes, the maximal velocities of several enzymes exceeded those previously reported. In addition, an efficient downstream glycerol catabolism and a novel transcription factor were found to be important for D-galacturonic acid utilization. These results set the basis for use of R. toruloides as a potential host for pectin-rich waste conversions and demonstrate its suitability as a model for metabolic studies in basidiomycetes.
2019-11-13 | GSE127536 | GEO
Project description:Nitrogen limitation is a major regulator to initiate lipid overproduction in oleaginous fungi. To examine the influence of nitrogen starvation, chemiostat cultures of R. toruloides in defined media with abundant ammonium (MM) or minute ammonium (MM-N) were performed to obtain steady-state samples. Then Illumina's digital gene expression (DGE) technology was used for high-throughput transcriptome profiling of these samples. Two samples cultured in minimum media with abundant ammonium (MM) or minute ammonium (MM-N)
2012-06-29 | E-GEOD-39023 | biostudies-arrayexpress
Project description:BackgroundLimonene is a widely used monoterpene in the production of food, pharmaceuticals, biofuels, etc. The objective of this work was to engineer Rhodosporidium toruloides as a cell factory for the production of limonene.ResultsBy overexpressing the limonene synthase (LS), neryl pyrophosphate synthase (NPPS)/geranyl pyrophosphate synthase and the native hydroxy-methyl-glutaryl-CoA reductase (HMGR), we established a baseline for limonene production based on the mevalonate route in Rhodosporidium toruloides. To further enhance the limonene titer, the acetoacetyl-CoA thiolase/HMGR (EfMvaE) and mevalonate synthase (EfMvaS) from Enterococcus faecalis, the mevalonate kinase from Methanosarcina mazei (MmMK) and the chimeric enzyme NPPS-LS were introduced in the carotenogenesis-deficient strain. The resulting strains produced a maximum limonene titer of 393.5 mg/L.ConclusionIn this study, we successfully engineered the carotenogenesis yeast R. toruloides to produce limonene. This is the first report on engineering R. toruloides toward limonene production based on NPP and the fusion protein SltNPPS-CltLS. The results demonstrated that R. toruloides is viable for limonene production, which would provide insights into microbial production of valuable monoterpenes.
| S-EPMC8697501 | biostudies-literature
Project description:Nitrogen limitation is a major regulator to initiate lipid overproduction in oleaginous fungi. To examine the influence of nitrogen starvation, chemiostat cultures of R. toruloides in defined media with abundant ammonium (MM) or minute ammonium (MM-N) were performed to obtain steady-state samples. Then Illumina's digital gene expression (DGE) technology was used for high-throughput transcriptome profiling of these samples.
2012-06-30 | GSE39023 | GEO