ABSTRACT: Glucose-mediated regulation of glycerol uptake in Rhodosporidium toruloides: Insights through transcriptomic analysis on dual substrate fermentation
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.
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.
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.
Project description:Our study is the first investigation of proteome of oleaginous yeast Rhotodorula toruloides during conversion of xylose into lipids. We performed comparative proteomics of R. toruloides during cultivation of two carbon sources: glucose and xylose. Proteome analysis revealed significantly lower levels of ribosomal proteins and translation associated factors in xylose-grown cells as compared to glucose-grown cells. We showed that proteins involved in sugar transport, phospholipid and leucine biosynthesis as well as peroxisomal beta-oxidation and oxidative stress response were differentially regulated in response to carbon source.
