Rhodotorula Toruloides Research Articles

Induction of resistance mechanisms in Rhodotorula toruloides for growth in sugarcane hydrolysate with high inhibitor content.

The oleaginous yeast Rhodotorula toruloides is a potential lipid producer for biodiesel production. However, this yeast shows growth inhibition due to harmful compounds when cultivated in hemicellulose hydrolysate. Here, we present a comparative analysis of colony selection and heterologous adaptive laboratory enhancement (ALE) strategies for obtaining robust strains. We implemented these ALE strategies for R. toruloides in a culture medium containing sugarcane hemicellulose hydrolysate. Our comparison study showed that the strain obtained with heterogeneous ALE strategy (Rth) reached a µmax of 55% higher than the parental strain. It also exhibited higher biomass production (6.51g/l) and lipid content (60%). ALE with colony selection strategy (Rtc) had a fitness gain in terms of shortening of the lag phase (9h) when compared to Rth and parental strain (11.67, 12.33h, respectively). When cultivated in Eucalyptus urograndis hemicellulose hydrolysate, the Rth strain achieved a high lipid content, 64%. Kinetics studies showed a strong effect of acetic acid as a repressor of xylose consumption during R. toruloides cultivation.Key points• Distinct adaptive laboratory strategies resulted in strains with different physiologies.• Heterologous adaptive laboratory enhancement provided the best results (fitness gain of 55% in µmax).• The Rth strain achieved a lipid content of 64.3% during cultivation in eucalyptus hemicellulose hydrolysate.

Chromosome-level genome assembly and transcriptome-based annotation of the oleaginous yeast Rhodotorula toruloides CBS 14

Rhodotorula toruloides is an oleaginous yeast with high biotechnological potential. In order to understand the molecular physiology of lipid synthesis in R. toruloides and to advance metabolic engineering, a high-resolution genome is required. We constructed a genome draft of R. toruloides CBS 14, using a hybrid assembly approach, consisting of short and long reads generated by Illumina and Nanopore sequencing, respectively. The genome draft consists of 23 contigs and 3 scaffolds, with a N50 length of 1,529,952 bp, thus largely representing chromosomal organization. The total size of the genome is 20,534,857 bp and the overall GC content is 61.83%. Transcriptomic data from different growth conditions was used to aid species-specific gene annotation. We annotated 9464 genes and identified 11,691 transcripts. Furthermore, we demonstrated the presence of a potential plasmid, an extrachromosomal circular structure of about 11 kb with a copy number about three times as high as the other chromosomes.

From a genome assembly to full regulatory network prediction: the case study of Rhodotorula toruloides putative Haa1-regulon

Numerous genomes are sequenced and made available to the community through the NCBI portal. However, and, unlike what happens for gene function annotation, annotation of promoter sequences and the underlying prediction of regulatory associations is mostly unavailable, severely limiting the ability to interpret genome sequences in a functional genomics perspective. Here we present an approach where one can download a genome of interest from NCBI in the GenBank Flat File (.gbff) format and, with a minimum set of commands, have all the information parsed, organized and made available through the platform web interface. Also, the new genomes are compared with a given genome of reference in search of homologous genes, shared regulatory elements and predicted transcription associations. We present this approach within the context of Community YEASTRACT of the YEASTRACT + portal, thus benefiting from immediate access to all the comparative genomics queries offered in the YEASTRACT + portal. Besides the yeast community, other communities can install the platform independently, without any constraints. In this work, we exemplify the usefulness of the presented tool, within Community YEASTRACT, in constructing a dedicated database and analysing the genome of the highly promising oleaginous red yeast species Rhodotorula toruloides currently poorly studied at the genome and transcriptome levels and with limited genome editing tools. Regulatory prediction is based on the conservation of promoter sequences and available regulatory networks. The case-study examined is focused on the Haa1 transcription factor—a key regulator of yeast resistance to acetic acid, an important inhibitor of industrial bioconversion of lignocellulosic hydrolysates. The new tool described here led to the prediction of a RtHaa1 regulon with expected impact in the optimization of R. toruloides robustness for lignocellulosic and pectin-rich residue biorefinery processes.

Oleaginous yeasts respond differently to carbon sources present in lignocellulose hydrolysate

BackgroundMicrobial oils, generated from lignocellulosic material, have great potential as renewable and sustainable alternatives to fossil-based fuels and chemicals. By unravelling the diversity of lipid accumulation physiology in different oleaginous yeasts grown on the various carbon sources present in lignocellulose hydrolysate (LH), new targets for optimisation of lipid accumulation can be identified. Monitoring lipid formation over time is essential for understanding lipid accumulation physiology. This study investigated lipid accumulation in a variety of oleaginous ascomycetous and basidiomycetous strains grown in glucose and xylose and followed lipid formation kinetics of selected strains in wheat straw hydrolysate (WSH).ResultsTwenty-nine oleaginous yeast strains were tested for their ability to utilise glucose and xylose, the main sugars present in WSH. Evaluation of sugar consumption and lipid accumulation revealed marked differences in xylose utilisation capacity between the yeast strains, even between those belonging to the same species. Five different promising strains, belonging to the species Lipomyces starkeyi, Rhodotorula glutinis, Rhodotorula babjevae and Rhodotorula toruloides, were grown on undiluted wheat straw hydrolysate and lipid accumulation was followed over time, using Fourier transform-infrared (FTIR) spectroscopy. All five strains were able to grow on undiluted WSH and to accumulate lipids, but to different extents and with different productivities. R. babjevae DVBPG 8058 was the best-performing strain, accumulating 64.8% of cell dry weight (CDW) as lipids. It reached a culture density of 28 g/L CDW in batch cultivation, resulting in a lipid content of 18.1 g/L and yield of 0.24 g lipids per g carbon source. This strain formed lipids from the major carbon sources in hydrolysate, glucose, acetate and xylose. R. glutinis CBS 2367 also consumed these carbon sources, but when assimilating xylose it consumed intracellular lipids simultaneously. Rhodotorula strains contained a higher proportion of polyunsaturated fatty acids than the two tested Lipomyces starkeyi strains.ConclusionsThere is considerable metabolic diversity among oleaginous yeasts, even between closely related species and strains, especially when converting xylose to biomass and lipids. Monitoring the kinetics of lipid accumulation and identifying the molecular basis of this diversity are keys to selecting suitable strains for high lipid production from lignocellulose.

Engineering oleaginous yeast Rhodotorula toruloides for overproduction of fatty acid ethyl esters

BackgroundProduction of biofuels and green chemicals by microbes is currently of great interest due to the increasingly limited reserves of fossil fuels. Biodiesel, especially fatty acid ethyl esters (FAEEs), is considered as an attractive alternative because of its similarity with petrodiesel and compatibility with existing infrastructures. Cost-efficient bio-production of FAEEs requires a highly lipogenic production host that is suitable for large-scale fermentation. As a non-model oleaginous yeast that can be cultured to an extremely high cell density and accumulate over 70% cell mass as lipids, Rhodotorula toruloides represents an attractive host for FAEEs production.ResultsWe first constructed the FAEE biosynthetic pathways in R. toruloides by introducing various wax ester synthase genes from different sources, and the bifunctional wax ester synthase/acyl-CoA-diacyglycerol acyltransferase (WS/DGAT) gene from Acinetobacter baylyi was successfully expressed, leading to a production of 826 mg/L FAEEs through shake-flask cultivation. We then mutated this bifunctional enzyme to abolish the DGAT activity, and further improved the titer to 1.02 g/L. Finally, to elevate the performance of Δku70-AbWS* in a bioreactor, both batch and fed-batch cultivation strategies were performed. The FAEEs titer, productivity and yield were 4.03 g/L, 69.5 mg/L/h and 57.9 mg/g (mg FAEEs/g glucose) under batch cultivation, and 9.97 g/L, 90.6 mg/L/h, and 86.1 mg/g under fed-batch cultivation. It is worth mentioning that most of the produced FAEEs were secreted out of the cell, which should greatly reduce the cost of downstream processing.ConclusionWe achieved the highest FAEEs production in yeast with a final titer of 9.97 g/L and demonstrated that the engineered R. toruloides has the potential to serve as a platform strain for efficient production of fatty acid-derived molecules.

Screening and Growth Characterization of Non-conventional Yeasts in a Hemicellulosic Hydrolysate.

Lignocellulosic biomass is an attractive raw material for the sustainable production of chemicals and materials using microbial cell factories. Most of the existing bioprocesses focus on second-generation ethanol production using genetically modified Saccharomyces cerevisiae, however, this microorganism is naturally unable to consume xylose. Moreover, extensive metabolic engineering has to be carried out to achieve high production levels of industrially relevant building blocks. Hence, the use of non-Saccharomyces species, or non-conventional yeasts, bearing native metabolic routes, allows conversion of a wide range of substrates into different products, and higher tolerance to inhibitors improves the efficiency of biorefineries. In this study, nine non-conventional yeast strains were selected and screened on a diluted hemicellulosic hydrolysate from Birch. Kluyveromyces marxianus CBS 6556, Scheffersomyces stipitis CBS 5773, Lipomyces starkeyi DSM 70295, and Rhodotorula toruloides CCT 7815 were selected for further characterization, where their growth and substrate consumption patterns were analyzed under industrially relevant substrate concentrations and controlled environmental conditions in bioreactors. K. marxianus CBS 6556 performed poorly under higher hydrolysate concentrations, although this yeast was determined among the fastest-growing yeasts on diluted hydrolysate. S. stipitis CBS 5773 demonstrated a low growth and biomass production while consuming glucose, while during the xylose-phase, the specific growth and sugar co-consumption rates were among the highest of this study (0.17 h–1 and 0.37 g/gdw*h, respectively). L. starkeyi DSM 70295 and R. toruloides CCT 7815 were the fastest to consume the provided sugars at high hydrolysate conditions, finishing them within 54 and 30 h, respectively. R. toruloides CCT 7815 performed the best of all four studied strains and tested conditions, showing the highest specific growth (0.23 h–1), substrate co-consumption (0.73 ± 0.02 g/gdw*h), and xylose consumption (0.22 g/gdw*h) rates. Furthermore, R. toruloides CCT 7815 was able to produce 10.95 ± 1.37 gL–1 and 1.72 ± 0.04 mgL–1 of lipids and carotenoids, respectively, under non-optimized cultivation conditions. The study provides novel information on selecting suitable host strains for biorefinery processes, provides detailed information on substrate consumption patterns, and pinpoints to bottlenecks possible to address using metabolic engineering or adaptive evolution experiments.

Functional characterization and overexpression of Δ12-desaturase in the oleaginous yeast Rhodotorula toruloides for production of linoleic acid-rich lipids

Linoleic acid (LA) has garnered much attention due to its potential applications in the oleochemical and nutraceutical industries. The oleaginous yeast Rhodotorula toruloides has outstanding lipogenecity, and is considered a potential alternative to the current plant-based platforms for LA production. Δ12-fatty acid desaturases (Δ12-Fads) are involved in LA synthesis in various fungi and yeasts, but their functions in R.toruloides remain poorly understood. To achieve the production of LA-rich lipids in R.toruloides, we investigated the function of the native Δ12-FAD (RtFAD2). First, the overexpression of RtFAD2 and its co-overexpression with RtFAD1 (encoding R.toruloides Δ9-Fad) and their effects on LA production in R.toruloides were investigated. The function of RtFad2 was confirmed by heterologous expression in Saccharomyces cerevisiae. Overexpression of RtFAD2 significantly elevated the LA contents and titers in the wild-type strain R.toruloides DMKU3-TK16 (TK16) and in a thermotolerant derivative of TK16 (L1-1). Additionally, overexpression of RtFAD2 in R.toruloides strains also increased the lipid titer and content. Overexpression of RtFAD1 was down-regulated in the RtFAD1 and RtFAD2 co-overexpressing strains, suggesting that the elevated LA content may function as a key regulator of RtFAD1 expression to control C18 fatty-acid synthesis in R.toruloides. We characterized the function of RtFAD2 and showed that its overexpression in R.toruloides increased the lipid and LA production. These findings may assist in the rational design of metabolic engineering related to LA or polyunsaturated fatty acid production in R.toruloides.

Complete Utilization of the Major Carbon Sources Present in Sugar Beet Pulp Hydrolysates by the Oleaginous Red Yeasts Rhodotorula toruloides and R. mucilaginosa.

Agro-industrial residues are low-cost carbon sources (C-sources) for microbial growth and production of value-added bioproducts. Among the agro-industrial residues available, those rich in pectin are generated in high amounts worldwide from the sugar industry or the industrial processing of fruits and vegetables. Sugar beet pulp (SBP) hydrolysates contain predominantly the neutral sugars d-glucose, l-arabinose and d-galactose, and the acidic sugar d-galacturonic acid. Acetic acid is also present at significant concentrations since the d-galacturonic acid residues are acetylated. In this study, we have examined and optimized the performance of a Rhodotorula mucilaginosa strain, isolated from SBP and identified at the molecular level during this work. This study was extended to another oleaginous red yeast species, R. toruloides, envisaging the full utilization of the C-sources from SBP hydrolysate (at pH 5.0). The dual role of acetic acid as a carbon and energy source and as a growth and metabolism inhibitor was examined. Acetic acid prevented the catabolism of d-galacturonic acid and l-arabinose after the complete use of the other C-sources. However, d-glucose and acetic acid were simultaneously and efficiently metabolized, followed by d-galactose. SBP hydrolysate supplementation with amino acids was crucial to allow d-galacturonic acid and l-arabinose catabolism. SBP valorization through the production of lipids and carotenoids by Rhodotorula strains, supported by complete catabolism of the major C-sources present, looks promising for industrial implementation.

Lipidomic Profile of Rhodotorula toruloides by GC/MS and Antioxidant Capacity of the Oil by DPPH and TLC-Plate Methods

ABSTRACT This work was undertaken to evaluate the antioxidant capacity of Rhodotorula toruloides lipid extract in TLC plate, using the (DPPH) (1,1-diphenyl-2-picril-hydrazine) method as an innovative way to visualise lipid groups that comprise this activity. Similarly, carotenoids and crude oil were analysed for antioxidant capacity by the DPPH and β-carotene/linoleic acid methods. The lipidomic profile extract analysis was performed by GC/MS and HPLC/DAD. The sample preparation for the GC/MS analysis was made by ultrasound-assisted transesterification. Free compounds were silylated with BSTFA (N,O-Bis (trimethylsilyl) trifluoracetamide) + 1% TMCS (Trimethylchlorosilane). The analysis of the lipid extract showed that in the saponifiable fraction saturated fatty acids (SFA) and monounsaturated fatty acids (MUFA) were present; and in the unsaponifiable fraction were steroids and carotenoids. The antioxidant capacity was expressed as IC50 reaching 6.4 mg/L that means relative efficiency. The oil profile, using TLC, shows the chemical groups: carotenoids, acylglycerols, free fatty acids and steroids. Similarly, the GC/ MS analysis shows the fatty acids and steroids. The HPLC analysis describes the carotenoids profile, highlighting b-carotene as the majority and the presence of ß-carotene-5,8-epoxide, zeaxanthin and b-cryptoxanthin, characterising the lipidomic study of this yeast. Keywords: DPPH-TLC, lipid metabolite classes, TLC-antioxidant system.

Chemical‐ vs sonochemical‐assisted synthesis of ZnO nanoparticles from a new zinc complex for improvement of carotene biosynthesis fromRhodotorula toruloidesMH023518

The complex [ZnL2] {HL = 2‐(((3,4‐dimethylphenyl)imino)methyl)phenol} was introduced by two routes (chemical vs. sonochemical) and acted as a source of different size zinc‐oxide (ZnO) nanoparticles (NPs) (nonsonicated ZnO NPs of Brunauer–Emmett–Teller (BET) surface area of 19.208 m2g−1, total pore volume of 0.03493 cc g−1, and particle diameter of 20.49–114.89 nm vs. sonicated ZnO NPs of BET surface area of 38.383 m2g−1, total pore volume of 0.08723 cc g−1and particle diameter of 15.09–55.31 nm). The NPs generate cell stress that leads to the formation of reactive oxygen species (ROS). Consequently, carotene biosynthesis can be enhanced to overcome the ROS.Rhodotorula toruloidesMH023518, genetically identified with 18S rRNA, produced carotenoids (124 ± 4.5 mg L−1) and lipids (0.897 ± 0.01 mg L−1) under no NPs' stress, but these values increased to total carotenoids of 264 ± 0.6 and 297.78 ± 0.8 mg L−1and total lipids of 0.97 ± 0.007 and 1.096 ± 0.015 mg L−1under stress conditions of 50 ppm of the nonsonicated and sonicated ZnO NPs, respectively. These results may represent potential industrial utilization of the as‐formed ZnO NPs (particularly the sonicated sample) as carotene and lipid stimulator. Additionally in this article, the soluble proteins, total antioxidants, and catalase and superoxide dismutase specific activities of the yeast in the presence of various concentrations of the as‐formed ZnO NPs were determined.

Xylose Metabolism and the Effect of Oxidative Stress on Lipid and Carotenoid Production in Rhodotorula toruloides: Insights for Future Biorefinery.

The use of cell factories to convert sugars from lignocellulosic biomass into chemicals in which oleochemicals and food additives, such as carotenoids, is essential for the shift toward sustainable processes. Rhodotorula toruloides is a yeast that naturally metabolises a wide range of substrates, including lignocellulosic hydrolysates, and converts them into lipids and carotenoids. In this study, xylose, the main component of hemicellulose, was used as the sole substrate for R. toruloides, and a detailed physiology characterisation combined with absolute proteomics and genome-scale metabolic models was carried out to understand the regulation of lipid and carotenoid production. To improve these productions, oxidative stress was induced by hydrogen peroxide and light irradiation and further enhanced by adaptive laboratory evolution. Based on the online measurements of growth and CO2 excretion, three distinct growth phases were identified during batch cultivations. Majority of the intracellular flux estimations showed similar trends with the measured protein levels and demonstrated improved NADPH regeneration, phosphoketolase activity and reduced β-oxidation, correlating with increasing lipid yields. Light irradiation resulted in 70% higher carotenoid and 40% higher lipid content compared to the optimal growth conditions. The presence of hydrogen peroxide did not affect the carotenoid production but culminated in the highest lipid content of 0.65 g/gDCW. The adapted strain showed improved fitness and 2.3-fold higher carotenoid content than the parental strain. This work presents a holistic view of xylose conversion into microbial oil and carotenoids by R. toruloides, in a process toward renewable and cost-effective production of these molecules.

The complete mitochondrial genome of the lipid-producing yeast Rhodotorula toruloides

Mitochondria are semi-autonomous organelles with their own genome and crucial to cellular material and energy metabolism. Here, we report the complete mitochondrial genome of a lipid-producing basidiomycetous yeast Rhodotorula toruloides NP11. The mitochondrial genome of R. toruloides NP11 was assembled into a circular DNA molecule of 125937bp, encoding 15 proteins, 28 transfer RNAs, 2 ribosomal RNA subunits and 10 open reading frames with unknown function. The G + C content (41%) of the mitochondrial genome is substantially lower than that of the nuclear genome (62%) of R. toruloides NP11. Further reanalysis of the transcriptome data confirmed the transcription of four mitochondrial genes. The comparison of the mitochondrial genomes of R. toruloides NP11 and NBRC0880 revealed a significant genetic divergence. These data can complement our understanding of the genetic background of R. toruloides and provide fundamental information for further genetic engineering of this strain.

Oleic acid formation from Stearoyl-CoA and NADH in vitro with a Δ-9-desaturase KRICT Rt9 recombinant protein

As a strategy to produce oleic acid using an enzyme biocatalyst in vitro, the Δ-9-desaturase gene from Rhodotorula toruloides NP11 was synthesized and codon optimized for expression in E. coli. Thus, 21.8% of the DNA sequence of the new gene was changed from the native gene of XP_016270987Rt9FAD. When the total amount of fatty acids were compared in the E. coli control (BL21) and the transformant (Rt9), it was revealed that the amount of unsaturated fatty acid (oleic acid, C18: 1, n-9) was increased by greater than 3.25-folds after induction. At the same time, the existing intracellular saturated-fatty acid (Stearic acid, C18: 0) in the standard E. coli had disappeared. Finally, we efficiently purified a new Δ-9-desaturase KRICT Rt9 protein from the transgenic E. coli. The purified enzyme Δ-9-desaturase KRICT Rt9 was reacted with Stearoyl-CoA and NADH to produce oleic acid in vitro.

Whole genome sequencing and comparative genomic analysis of oleaginous red yeast Sporobolomyces pararoseus NGR identifies candidate genes for biotechnological potential and ballistospores-shooting

BackgroundSporobolomyces pararoseus is regarded as an oleaginous red yeast, which synthesizes numerous valuable compounds with wide industrial usages. This species hold biotechnological interests in biodiesel, food and cosmetics industries. Moreover, the ballistospores-shooting promotes the colonizing of S. pararoseus in most terrestrial and marine ecosystems. However, very little is known about the basic genomic features of S. pararoseus. To assess the biotechnological potential and ballistospores-shooting mechanism of S. pararoseus on genome-scale, the whole genome sequencing was performed by next-generation sequencing technology.ResultsHere, we used Illumina Hiseq platform to firstly assemble S. pararoseus genome into 20.9 Mb containing 54 scaffolds and 5963 predicted genes with a N50 length of 2,038,020 bp and GC content of 47.59%. Genome completeness (BUSCO alignment: 95.4%) and RNA-seq analysis (expressed genes: 98.68%) indicated the high-quality features of the current genome. Through the annotation information of the genome, we screened many key genes involved in carotenoids, lipids, carbohydrate metabolism and signal transduction pathways. A phylogenetic assessment suggested that the evolutionary trajectory of the order Sporidiobolales species was evolved from genus Sporobolomyces to Rhodotorula through the mediator Rhodosporidiobolus. Compared to the lacking ballistospores Rhodotorula toruloides and Saccharomyces cerevisiae, we found genes enriched for spore germination and sugar metabolism. These genes might be responsible for the ballistospores-shooting in S. pararoseus NGR.ConclusionThese results greatly advance our understanding of S. pararoseus NGR in biotechnological potential and ballistospores-shooting, which help further research of genetic manipulation, metabolic engineering as well as its evolutionary direction.

Rhodotorula toruloides Single Cell Oil Production Using Eucalyptus urograndis Hemicellulose Hydrolysate as a Carbon Source

Microbial oil is a potential substitute for vegetable oils in the biodiesel industry. Efforts to obtain cheap carbon sources for the cultivation of lipid-producing microorganisms comprise an active research area. This work aimed to extract the hemicellulose fraction from Eucalyptus uograndis and to use its hydrolysate as a carbon source for Rhodotorula toruloides (an oleaginous yeast) cultivation for microbial oil production. Hemicellulose hydrothermal extractions were performed at different temperatures, times, and ratios of solid to liquid (S/L). Temperature and time showed a stronger effect on the solubilization of hemicellulose. Hemicellulose extraction at 155 °C, 195 min, and an S/L ratio of 1/2 resulted in a hydrolysate with a xylose content of 37.0 g/l. R. toruloides cultivation in this hydrolysate showed that initial pH had a strong influence on cell growth. At an initial pH of 6.2, cells grew to 6.0 g/l of biomass with a lipid content of 50%. Therefore, we believe that E. urograndis hemicellulose hydrolysate could be a potential substrate for R. toruloides for lipid production based on the biorefinery concept.

C/N ratio and carbon source-dependent lipid production profiling in Rhodotorula toruloides

Microbial oils are lipids produced by oleaginous microorganisms, which can be used as a potential feedstock for oleochemical production. The oleaginous yeast Rhodotorula toruloides can co-produce microbial oils and high-value compounds from low-cost substrates, such as xylose and acetic acid (from hemicellulosic hydrolysates) and raw glycerol (a byproduct of biodiesel production). One step towards economic viability is identifying the best conditions for lipid production, primarily the most suitable carbon-to-nitrogen ratio (C/N). Here, we aimed to identify the best conditions and cultivation mode for lipid production by R. toruloides using various low-cost substrates and a range of C/N ratios (60, 80, 100, and 120). Turbidostat mode was used to achieve a steady state at the maximal specific growth rate and to avoid continuously changing environmental conditions (i.e., C/N ratio) that inherently occur in batch mode. Regardless of the carbon source, higher C/N ratios increased lipid yields (up to 60% on xylose at a C/N of 120) but decreased the specific growth rate. Growth on glycerol resulted in the highest specific growth and lipid production (0.085 g lipids/gDW*h) rates at C/Ns between 60 and 100. We went on to study lipid production using glycerol in both batch and fed-batch modes, which resulted in lower specific lipid production rates compared with turbisdostat, however, fed batch is superior in terms of biomass production and lipid titers. By combining the data we obtained in these experiments with a genome-scale metabolic model of R. toruloides, we identified targets for improvements in lipid production that could be carried out either by metabolic engineering or process optimization.

Molecular identification and antifungal susceptibility testing of Pucciniomycotina red yeast clinical isolates from Rio de Janeiro, Brazil.

Infections caused by Rhodotorula spp. are increasing worldwide. This study identified, through the light of the new taxonomic advances on the subphylum Pucciniomycotina, 16 isolates from blood cultures and compared their antifungal susceptibility on microdilution and gradient diffusion methods. Internal transcriber spacer sequencing identified Rhodotorula mucilaginosa (n = 12), Rhodotorula toruloides (n = 2), Rhodotorula dairenensis (n = 1), and Cystobasidium minutum (n = 1). Amphotericin B was the most effective drug. A good essential agreement was observed on MIC values of amphotericin B and voriconazole determined by the two methods. Therefore, the gradient method is useful for susceptibility tests of R. mucilaginosa against these drugs.

Genome-scale model of Rhodotorula toruloides metabolism.

The basidiomycete red yeast Rhodotorula toruloides is a promising platform organism for production of biooils. We present rhto-GEM, the first genome-scale model (GEM) of R. toruloides metabolism, that was largely reconstructed using RAVEN toolbox. The model includes 852 genes, 2,731 reactions, and 2,277 metabolites, while lipid metabolism is described using the SLIMEr formalism allowing direct integration of lipid class and acyl chain experimental distribution data. The simulation results confirmed that the R. toruloides model provides valid growth predictions on glucose, xylose, and glycerol, while prediction of genetic engineering targets to increase production of linolenic acid, triacylglycerols, and carotenoids identified genes-some of which have previously been engineered to successfully increase production. This renders rtho-GEM valuable for future studies to improve the production of other oleochemicals of industrial relevance including value-added fatty acids and carotenoids, in addition to facilitate system-wide omics-data analysis in R. toruloides. Expanding the portfolio of GEMs for lipid-accumulating fungi contributes to both understanding of metabolic mechanisms of the oleaginous phenotype but also uncover particularities of the lipid production machinery in R. toruloides.

Proteome analysis of xylose metabolism in Rhodotorula toruloides during lipid production

BackgroundRhodotorula toruloides is a promising platform organism for production of lipids from lignocellulosic substrates. Little is known about the metabolic aspects of lipid production from the lignocellolosic sugar xylose by oleaginous yeasts in general and R. toruloides in particular. This study presents the first proteome analysis of the metabolism of R. toruloides during conversion of xylose to lipids.ResultsRhodotorula toruloides cultivated on either glucose or xylose was subjected to comparative analysis of its growth dynamics, lipid composition, fatty acid profiles and proteome. The maximum growth and sugar uptake rate of glucose-grown R. toruloides cells were almost twice that of xylose-grown cells. Cultivation on xylose medium resulted in a lower final biomass yield although final cellular lipid content was similar between glucose- and xylose-grown cells. Analysis of lipid classes revealed the presence of monoacylglycerol in the early exponential growth phase as well as a high proportion of free fatty acids. Carbon source-specific changes in lipid profiles were only observed at early exponential growth phase, where C18 fatty acids were more saturated in xylose-grown cells. Proteins involved in sugar transport, initial steps of xylose assimilation and NADPH regeneration were among the proteins whose levels increased the most in xylose-grown cells across all time points. The levels of enzymes involved in the mevalonate pathway, phospholipid biosynthesis and amino acids biosynthesis differed in response to carbon source. In addition, xylose-grown cells contained higher levels of enzymes involved in peroxisomal beta-oxidation and oxidative stress response compared to cells cultivated on glucose.ConclusionsThe results obtained in the present study suggest that sugar import is the limiting step during xylose conversion by R. toruloides into lipids. NADPH appeared to be regenerated primarily through pentose phosphate pathway although it may also involve malic enzyme as well as alcohol and aldehyde dehydrogenases. Increases in enzyme levels of both fatty acid biosynthesis and beta-oxidation in xylose-grown cells was predicted to result in a futile cycle. The results presented here are valuable for the development of lipid production processes employing R. toruloides on xylose-containing substrates.

Dichloromethane as an alternative to chloroform in the extraction of single cell oil from Rhodotorula toruloides

Dichloromethane (methylene chloride) was evaluated as an alternative to chloroform in the extraction of single cell oils from the yeast Rhodotorula toruloides. Dichloromethane was proposed as a potentially more environmentally friendly substitute since it is nearly 15% less expensive and has a toxicity threshold to human health that is approximately 20 times higher if compared to chloroform.A three-factor factorial experiment was designed in order to test the efficacy of the proposed solvent in the extraction process, as well as the volume of solvent and time of extraction, the response variable being the mass fraction of extracted lipids. The results show that all the three variables had a significant and positive effect on the response variable since all p-values were less than 0.005 (99% confidence). Therefore, for this study, the best experimental condition for the extraction was determined to be one with 80 mL of dichloromethane and 24 h of extraction. In general, the results showed that dichloromethane is an alternative to chloroform in lipid extraction from Rhodotorula toruloides, without compromise on extraction efficiency.