toruloides cultivated on several different carbon sources was used to identify constitutive promoters driving high and medium expression levels of native genes (predicted expression levels are classified as described in “ Methods” section). Of the set of promoters tested, several appear to be good candidates for driving heterologous expression in this host, thus expanding the toolset available for genome editing.Ĭonstruction and characterization of promoter library toruloides promoters by monitoring expression of two fluorescent reporters in four different media over a cultivation period of 7 days. The aim of this study was to characterize native R. Several of these promoters were predicted to be bidirectional. toruloides that are likely to result in high- and medium-level constitutive expression. RNA sequencing analysis was performed to select promoters from R. For this work, a high-efficiency chemical transformation method was adopted from a protocol developed for Saccharomyces cerevisiae to streamline the engineering workflow for R. toruloides is Agrobacterium tumefaciens-mediated transformation (ATMT), a useful strategy for genomic integration of heterologous genes but is a laborious and time-consuming process. The most common method used for transformation of R. In this context, functional bidirectional promoters would be a valuable addition to the toolset, since they can be convenient for optimization of multi-gene pathways. Solid v dotted line in map snapgene viewer full#While promoters that are responsive to certain metabolites are valuable tools, a toolset of well characterized constitutive promoters remains necessary to explore the full potential of strain engineering. toruloides, the majority of them being metabolite-responsive promoters rather than constitutive. A small selection of promoters has already been characterized to modulate expression of heterologous genes in R. A growing branch of synthetic biology involves the pursuit and improvement of standard parts that are reliable, orthogonal and robust for non-conventional hosts. A major need on the tools front is for efficient and stable promoters to express genes of interest and selection markers. To improve prospects for more advanced metabolic engineering in this fungal host, the development of robust genetic tools and resources is an immediate priority. However, the high lipid content indicates a suitability for applications to any acetyl-CoA-derived product, from fatty alcohols, terpenes and methyl ketones to TCA-cycle derived products, with many applications such as fuels, chemicals, and pharmaceuticals. toruloides has focused on lipid accumulation. So far, much of the work on metabolic engineering or bioproduct formation in R. The oleaginous, carotenogenic yeast Rhodosporidium toruloides (also known as Rhodotorula toruloides) is a promising host for sustainable bioproduction due to its innate capacity to efficiently utilize both hexoses and pentoses present in lignocellulosic biomass, as well as its capacity to grow on lignin derived intermediates and aromatics, such as p-coumaric acid and ferulic acid. With the aim of replacing many petrochemical-derived fuels and chemicals with renewable alternatives, the field of metabolic engineering is moving towards the development of microbial hosts that use lignocellulosic biomass as feedstocks for bioproduction. 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. 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). 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. ConclusionsĪ set of robust and constitutive promoters to facilitate genetic engineering of R. 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. Promoter expression strength was determined by measurement of EGFP and mRuby2 reporters by flow cytometry. The promoter sequences were selected using transcriptional analysis and several of them were found to drive expression bidirectionally. toruloides promoters, characterized over time in four different media commonly used for cultivation of this yeast. However, there is a lack of characterized promoters to drive expression of heterologous genes for strain engineering in R. A key prerequisite for efficient pathway engineering is the availability of robust genetic tools and resources. Rhodosporidium toruloides is a promising host for the production of bioproducts from lignocellulosic biomass.
0 Comments
Leave a Reply. |
AuthorWrite something about yourself. No need to be fancy, just an overview. ArchivesCategories |