A toolset of constitutive promoters for engineering Rhodosporidium toruloides
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PROVIDER: PRJNA527258 | ENA |
REPOSITORIES: ENA
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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: Not available
| S-EPMC6599526 | biostudies-literature
Project description:Global proteomics datasets to study lignocellulosic carbon utilization in Rhodosporidium Toruloides
2021-09-09 | PXD022377 | Pride
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: Not available
| 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
Project description:Lipid accumulation by oleaginous microorganisms is of great scientific interest and biotechnological potential. While nitrogen limitation has been routinely employed, low-cost raw materials usually contain rich nitrogenous components, thus preventing from efficient lipid production. Inorganic phosphate (Pi) limitation has been found sufficient to promote conversion of sugars into lipids, yet the molecular basis of cellular response to Pi-limitation and concurrent lipid accumulation remains elusive. Here we performed multi-omic analyses of the oleaginous yeast Rhodosporidium toruloides to shield lights on Pi-limitation induced lipid accumulation. Samples were prepared under Pi-limited as well as Pi-replete chemostat conditions, and subjected to analysis at the transcriptomic, proteomic and metabolomic level. In total, 7970 genes, 4212 proteins and 123 metabolites were identified. Results showed that Pi-limitation facilitates up-regulation of Pi-associated metabolism, RNA degradation and triacylglycerol biosynthesis, while down-regulation of ribosome biosynthesis and tricarboxylic acid cycle. Pi-limitation leads to de-phosphorylation of adenosine monophosphate, the allosteric activator of isocitrate dehydrogenase key to lipid biosynthesis. It was found that NADPH, the key cofactor for fatty acid biosynthesis, is limited due to reduced flux through the pentose phosphate pathway and transhydrogenation cycle, and that this can be overcomed by overexpression of an endogenous malic enzyme. These phenomena are found distinctive from those under nitrogen-limitation. The information greatly enriches our understanding on microbial oleaginicity and Pi-related metabolism. Importantly, systems data may facilitate designing advanced cell factories for production of lipids and related oleochemicals.
2018-05-31 | GSE113097 | GEO