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.
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.
