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Phase contrast microscopy photos of Rhodosporidium toruloides BOT-A2 (left photo) and Pseudozyma hubeinensis BOT-O (right photo). Storage lipids are seen as bright/yellow lipid bodies inside the cells, indicated by arrows
Source publication
Background
Sustainable production of oil for food, feed, fuels and other lipid-based chemicals is essential to meet the demand of the increasing human population. Consequently, novel and sustainable resources such as lignocellulosic hydrolysates and processes involving these must be explored. In this paper we screened for naturally-occurring xylose...
Citations
... Several R. toruloides transcriptome datasets are available from a variety of different culture conditions, including nitrogen limitation [14,39], phosphate limitation [5], stress response during growth on hydrolysates [11,40,41] and growth on different carbon sources [10,30,33,39,42], but no study has yet compared the effect of hexose and pentose sugars during nitrogen starvation. We here generated a highly contiguous, functionally annotated genome assembly from our recently isolated Rhodotorula toruloides strain BOT-A2 (previously classified as Rhodosporidium toruloides) [43] and performed RNA sequencing (RNAseq) on samples cultivated in nitrogenlimited media with either glucose or xylose as the sole carbon source. Differential expression analysis of samples taken during the exponential growth phase and at nitrogen depletion was used to identify putative promoter candidate sequences, which were further evaluated using reporter gene expression. ...
... The natural yeast isolate Rhodotorula toruloides BOT-A2 [43] and its derived strains were used in all experiments and cultivated at 30 °C. A list of all strains can be found in Table 3. ...
... The KOH/ethanol extraction method [60] was used to extract fatty acids from the cultivations for RNA sequencing, with slight changes as previously described [43]. In short, after freeze-drying of the samples, biomass was determined using a micro scale. ...
Background
The non-conventional yeast Rhodotorula toruloides is an emerging host organism in biotechnology by merit of its natural capacity to accumulate high levels of carotenoids and intracellular storage lipids from a variety of carbon sources. While the number of genetic engineering strategies that employ R. toruloides is increasing, the lack of genetic tools available for modification of this yeast is still limiting strain development. For instance, several strong, constitutive R. toruloides promoters have been characterized, but to date, only five inducible promoters have been identified. Although nitrogen-limited cultivation conditions are commonly used to induce lipid accumulation in this yeast, no promoters regulated by nitrogen starvation have been described for R. toruloides.
Results
In this study, we used a combination of genomics and transcriptomics methods to identify novel R. toruloides promoter sequences that are either inducible or repressible by nitrogen starvation. RNA sequencing was used to assess gene expression in the recently isolated strain R. toruloides BOT-A2 during exponential growth and during nitrogen starvation, when cultivated with either glucose or xylose as the carbon source. The genome of BOT-A2 was sequenced using a combination of long- and short-read sequencing and annotated with support of the RNAseq data. Differential expression analysis was used to identify genes with a |log2 fold change|≥ 2 when comparing their expression during nitrogen depletion to that during exponential growth. The promoter regions from 16 of these genes were evaluated for their ability to drive the expression of a fluorescent reporter gene. Three promoters that were clearly upregulated under nitrogen starvation and three that were downregulated were selected and further characterized. One promoter, derived from gene RTBOTA2_003877, was found to function like an on–off switch, as it was only upregulated under full nitrogen depletion and downregulated in the presence of the nitrogen source.
Conclusions
Six new R. toruloides promoters that were either upregulated or downregulated under nitrogen-starvation were identified. These substantially contribute to the available promoters when engineering this organism and are foreseen to be particularly useful for future engineering strategies requiring specific regulation of target genes in accordance with nitrogen availability.
... In addition, glycerol was also tested as an alternative substrate for lipid production using yeast (Fabricio et al. 2019, Bansal et al. 2020. Recently, natural yeast isolates which were identified as Pseudozyma hubeiensis and Rhodotorula toruloides strains were found to accumulate high levels of lipids using xylose and glycerol (Qvirist et al. 2022). Exploring yeast diversity to produce lipids using various substrates efficiently is receiving increasing attention for sustainable production (Mota et al. 2022). ...
Oleaginous yeasts utilize renewable resources to produce lipids, which benefits sustainable development, and it is of great interest to screen robust lipid producers. Curvibasidium sp. belongs to nonconventional yeast that are very limitedly studied. Here, two cold-adaptive strains of Curvibasidium sp., namely, Y230 and Y231, isolated from the medicinal lichen Usnea diffracta were investigated for their potential in lipid production. Genome mining of Curvibasidium sp. Y231 was performed, and the special features related to fatty acid biosynthesis were revealed. Glucose, xylose and glycerol were tested as sole carbon sources for yeast cell growth and lipid production. The total lipid contents of Curvibasidium sp. Y230 and Y231 range from 38.43% to 54.62% of the cell dry cell weight at 20°C, and glucose is the optimal carbon source. These results indicate that the Curvibasidium sp. strains are promising for sustainable lipid production. Our study provides basis for exploration of lichen-derived strains for biotechnological applications, and also benefits utilization of other nonconventional yeasts for sustainable production based on genome-based studies.
... In this study, we investigated the P. hubeiensis strain BOT-O, which we previously isolated from plant leaves and twigs in a greenhouse in the botanical garden in Gothenburg, Sweden. The strain was initially screened for growth and lipid production on xylose and showed higher lipid production than any other strain isolated from the same environment (Qvirist et al., 2022). The aim of the study was to further characterise the oleaginous nature of P. hubeiensis and its ability to naturally co-utilise glucose and xylose, with a focus on physiology and gene expression. ...
... The oleaginous yeast strain P. hubeiensis BOT-O was previously isolated from the Gothenburg Botanical Garden, Sweden (Qvirist et al., 2022) and was cultivated at 30 • C. The strain was maintained on YPD (20 g/L yeast extract, 10 g/L peptone, 20 g/L glucose) plates with 20 g/L agar. The BOT-O cryostock solution was stored in 25 % (w/w) glycerol at − 80 • C. Growth on xylan was assessed after incubation at 30 • C for three days on YP-plates (YPD without glucose) supplemented with 10 g/ L of different xylans: beech wood xylan, birch wood xylan and wheat arabinoxylan. ...
... The fatty acids were extracted using the KOH/ethanol extraction method described by Andlid et al. (1995), using the slight alterations described in Qvirist et al. (2022). Samples were freeze-dried, and weighed with a micro scale (Precisa Gravimetrics AG, Dietikon, Switzerland). ...
Pseudozyma hubeiensis is a basidiomycete yeast that has the highly desirable traits for lignocellulose valorisation of being equally efficient at utilization of glucose and xylose, and capable of their co-utilization. The species has previously mainly been studied for its capacity to produce secreted biosurfactants in the form of mannosylerythritol lipids, but it is also an oleaginous species capable of accumulating high levels of triacylglycerol storage lipids during nutrient starvation. In this study, we aimed to further characterize the oleaginous nature of P. hubeiensis by evaluating metabolism and gene expression responses during storage lipid formation conditions with glucose or xylose as a carbon source. The genome of the recently isolated P. hubeiensis BOT-O strain was sequenced using MinION long-read sequencing and resulted in the most contiguous P. hubeiensis assembly to date with 18.95 Mb in 31 contigs. Using transcriptome data as experimental support, we generated the first mRNA-supported P. hubeiensis genome annotation and identified 6540 genes. 80% of the predicted genes were assigned functional annotations based on protein homology to other yeasts. Based on the annotation, key metabolic pathways in BOT-O were reconstructed, including pathways for storage lipids, mannosylerythritol lipids and xylose assimilation. BOT-O was confirmed to consume glucose and xylose at equal rates, but during mixed glucose-xylose cultivation glucose was found to be taken up faster. Differential expression analysis revealed that only a total of 122 genes were significantly differentially expressed at a cut-off of |log2 fold change| ≥ 2 when comparing cultivation on xylose with glucose, during exponential growth and during nitrogen-starvation. Of these 122 genes, a core-set of 24 genes was identified that were differentially expressed at all time points. Nitrogen-starvation resulted in a larger transcriptional effect, with a total of 1179 genes with significant expression changes at the designated fold change cut-off compared with exponential growth on either glucose or xylose.
Biosurfactants generated from lactic acid bacteria (LAB) offer an advantage over standard microbial surfactants due to their antifungal, antibacterial and antiviral capabilities. Many LAB strains have been related to the manufacture of biosurfactant, an essential chemical with uses in the treatment of a number of illnesses. Furthermore, their effectiveness as anti-adhesive agents against a diverse variety of pathogens proves their utility as anti-adhesive coating agents for medical insertional materials, reducing hospital infections without the need of synthetic drugs and chemicals. LAB produces both low and high molecular weight biosurfactants. Biosurfactants from L. pentosus, L. gasseri and L. jensenii have been reported to produce glycolipopeptides that comprise carbohydrates, proteins and lipids in the ratio of 1:3:6 with palmitic, stearic acid, and linoelaidic acid as the major fatty acid component, whereas L. plantarum has been reported to make surlactin due to the presence of non- ribosomal peptide synthetase genes (NRPS) genes. Antimicrobial activity of sophorolipids and rhamnolipids generated from LAB against B. subtilis, P. aeruginosa, S. epidermidis, Propionibacterium acnes and E. coli has been demonstrated. The safety of biosurfactants is being evaluated in compliance with a number of regulatory standards that emphasize the importance of safety in the pharmaceutical industry. This review attempts, for the first time, to provide a comprehensive evaluation of several approaches for the synthesis of biosurfactant-mediated molecular modulation in terms of their biological value. Future biosurfactant directions, as well as regulatory considerations that are crucial for the synthesis of biosurfactants from novel LAB, have also been explored.
Oleaginous yeasts-derived microbial lipids provide a promising alternative feedstock for the biodiesel industry. However, hyperosmotic stress caused by high sugar concentration during fermentation significantly prevents high cell density and productivity. Isolation of new robust osmophilic oleaginous species from specific environment possibly resolves this issue to some extent. In this study, the cultivable yeast composition of honeycombs was investigated. Totally, 11 species of honeycomb-associated cultivable yeast were identified and characterized. Among them, an osmophilic yeast strain, designated as Rhodotorula toruloides C23 was featured with excellent lipogenic and carotenogenic capacity and remarkable cell growth using glucose, xylose or glycerol as feedstock, with simultaneous production of 24.41 g/L of lipids and 15.50 mg/L of carotenoids from 120 g/L glucose in 6.7-L fermentation. Comparative transcriptomic analysis showed that C23 had evolved a dedicated molecular regulation mechanism to maintain their high simultaneous accumulation of intracellular lipids and carotenoids and cell growth under high sugar concentration.
