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Ultrasonics Sonochemistry Mar 2020The extraction of Rhodotorula glutinis carotenoids by ultrasound under pressure (manosonication) in an aqueous medium has been demonstrated. The influence of treatment...
The extraction of Rhodotorula glutinis carotenoids by ultrasound under pressure (manosonication) in an aqueous medium has been demonstrated. The influence of treatment time, pressure, and ultrasound amplitude on R. glutinis inactivation and on the extraction of carotenoids was evaluated, and the obtained data were described mathematically. The extraction yields were lineal functions of those three parameters, whereas inactivation responded to a more complex equation. Under optimum treatment conditions, 82% of carotenoid content was recovered. Extraction of carotenoids in an aqueous medium was attributed to the capacity of ultrasound for cell disruption and emulsification. Cavitation caused the rupture of cell envelopes and the subsequent formation of small droplets of carotenoids surrounded by the phospholipids of the cytoplasmic membrane that would stabilize the emulsion. Analysis of the dispersed particle size of the extracts demonstrated that a fine, homogeneous emulsion was formed after treatment (average size: 230 nm; polydispersity <0.22). This research describes an innovative green process for extracting carotenoids from fresh biomass of R. glutinis in which only two unit operations are required: ultrasonic treatment, followed by a centrifugation step to discard cell debris. The extract obtained thanks to this procedure is rich in carotenoids (25 mg/L) and could be directly incorporated as a pigment in foods, beverages, and diet supplements; it can also be utilized as an ingredient in drugs or cosmetics.
Topics: Biomass; Carotenoids; Rhodotorula; Solvents; Ultrasonic Waves
PubMed: 31669840
DOI: 10.1016/j.ultsonch.2019.104833 -
World Journal of Microbiology &... Oct 2019In this study, we aimed to determine the effect of exogenous stress factors (sodium chloride as osmotic stressor, hydrogen peroxide as an inducer of oxidative stress,...
In this study, we aimed to determine the effect of exogenous stress factors (sodium chloride as osmotic stressor, hydrogen peroxide as an inducer of oxidative stress, white light irradiation, and low temperature) on the biosynthesis of carotenoids and lipids by red yeast (Rhodotorula glutinis, R. mucilaginosa, and R. gracilis) during cultivation in media containing potato wastewater and glycerol. According to our results, the yeast were able to grow and biosynthesize lipids and carotenoids in the presence of the applied stress factors. Low temperature caused an increase in the biosynthesis of intracellular lipids and carotenoids. R. gracilis synthesized lipids (21.1 g/100 g) and carotenoids (360.4 µg/g) in greater quantities than that of other strains. Under these conditions, there was also an increase in the content of unsaturated fatty acids, especially linoleic and linolenic acids. The highest percentage of polyunsaturated fatty acid (PUFA) (30.4%) was synthesized by the R. gracilis yeast after cultivation at 20°C. Their quantity was 2.5-fold greater than that of the biomass grown in control conditions. The contribution of individual carotenoid fractions depended both on the yeast strain and the culture conditions. Induction of osmotic stress and low temperature intensified the biosynthesis of β-carotene (up to 73.9% of the total carotenoid content). In oxidative stress conditions, yeast synthesized torulene (up to 82.2%) more efficiently than under other conditions, whereas white light irradiation increased the production of torularhodin (up to 20.0%).
Topics: Carotenoids; Culture Media; Industrial Waste; Lipids; Rhodotorula
PubMed: 31576445
DOI: 10.1007/s11274-019-2732-8 -
Bioengineered Dec 2019Selenium-enriched yeast can transform toxic inorganic selenium into absorbable organic selenium, which is of great significance for human health and pharmaceutical...
Selenium-enriched yeast can transform toxic inorganic selenium into absorbable organic selenium, which is of great significance for human health and pharmaceutical industry. A yeast X-20 we obtained before has good selenium-enriched ability, but its selenium content is still low for industrial application. In this study, strategies of process optimization and transport regulation of selenium were thus employed to further improve the cell growth and selenium enrichment. Through engineering phosphate transporters from into X-20, the selenium content was increased by 21.1%. Through using mixed carbon culture (20 g L, glycerol: glucose 3:7), both biomass and selenium content were finally increased to 5.3 g L and 5349.6 µg g (cell dry weight, DWC), which were 1.14 folds and 6.77 folds compared to their original values, respectively. Our results indicate that high selenium-enrichment ability and biomass production can be achieved through combining process optimization and regulation of selenium transport.
Topics: Biological Transport; Biomass; Culture Media; Fermentation; Gene Expression; Glucose; Glycerol; Metabolic Engineering; Phosphate Transport Proteins; Phosphates; Plasmids; Proton-Phosphate Symporters; Rhodotorula; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Selenium; Sodium-Phosphate Cotransporter Proteins, Type III; Transgenes
PubMed: 31322471
DOI: 10.1080/21655979.2019.1644853 -
Biotechnology For Biofuels 2019Lipid extraction for quantification of fat content in oleaginous yeasts often requires strong acids and harmful organic solvents; it is laborious and time-consuming....
BACKGROUND
Lipid extraction for quantification of fat content in oleaginous yeasts often requires strong acids and harmful organic solvents; it is laborious and time-consuming. Therefore, in most cases just endpoint measurements of lipid accumulation are performed and kinetics of intracellular lipid accumulation is difficult to follow. To address this, we created a prediction model using Fourier-transform near-infrared (FT-NIR) spectroscopy. This method allows to measure lipid content in yeast.
METHODS
The FT-NIR calibration sets were constructed from spectra of freeze-dried cells of the oleaginous yeasts CBS 14, CBS 1807 and CBS 6114. The yeast cells were obtained from different cultivation conditions. Freeze-dried cell pellets were scanned using FT-NIR in the Multi Purpose Analyser (MPA) from Bruker. The obtained spectra were assigned corresponding to total fat content, obtained from lipid extraction using a modified Folch method. Quantification models using partial least squares (PLS) regression were built, and the calibration sets were validated on independently cultivated samples. The model was additionally tested on DBVPG 8058 and CBS 2387.
RESULTS
The of the FT-NIR model for was 98%, and the root mean square error of cross-validation (RMSECV) was 1.53. The model was validated using a separate set of samples with a root mean square error of prediction (RMSEP) of 3.21. The of the model was 96%, with RMSECV 2.4 and RMSEP 3.8. The of the mixed model, including all tested yeast strains, was 90.5%, with RMSECV 2.76 and RMSEP 3.22, respectively. The models were verified by predicting the total fat content in newly cultivated and freeze-dried samples. Additionally, the kinetics of lipid accumulation of a culture were followed and compared with standard lipid extraction methods.
CONCLUSIONS
Using FT-NIR spectroscopy, we have developed a faster, less laborious and non-destructive quantification of yeast intracellular lipid content compared to methods using lipid extraction.
PubMed: 31297157
DOI: 10.1186/s13068-019-1513-9 -
Journal of Biological Engineering 2019Coumarins play roles in many biological processes. is one of the major sources of coumarins in China. Due to increasing demand for coumarins in the marketplace,...
BACKGROUND
Coumarins play roles in many biological processes. is one of the major sources of coumarins in China. Due to increasing demand for coumarins in the marketplace, traditional extraction from plants is now considered economically insufficient and unsustainable. Microbial synthesis is a promising strategy for scalable production of coumarins. However, the biosynthetic pathway of coumarin remains poorly understood, and even more, the genes associated with this process have not been characterized in .
RESULTS
RNA-seq was employed to elucidate the umbelliferone biosynthetic pathway. The results indicated that three enzymes, phenylalanine ammonia-lyase (PAL), 4-Coumarate: Coenzyme A Ligase (4CL), and -coumaroyl CoA 2'-hydroxylase (C2'H) were involved in umbelliferone biosynthesis. Using the cloned genes, we generated a synthetic biology based microbial cell factory that produces coumarins from tyrosine utilizing tyrosine ammonia lyase (RgTAL) to bypass cinnamic acid 4-hydroxylase (C4H). With metabolic engineering strategies, we deleted and and overexpressed six related genes involved in tyrosine biosynthesis, to drive the carbon flux from tyrosine. To overcome the limitation of 4CL, a virtual screening and site-specific mutagenesis-based protein engineering approach was applied. In addition, induction/culture conditions and different ions were employed to further improve the yield of umbelliferone. Finally, a yield of 356.59 mg/L umbelliferone was obtained.
CONCLUSIONS
The current study elucidated the umbelliferone biosynthesis pathway in . The results also demonstrated the feasibility of integrating gene mining with synthetic biology techniques to produce natural compounds.
PubMed: 31139252
DOI: 10.1186/s13036-019-0174-3 -
Applied Biochemistry and Biotechnology Oct 2019AbstractThe objective of this study was to determine the possibility of simultaneous biosynthesis of lipids and carotenoids by the Rhodotorula yeast strains in media...
AbstractThe objective of this study was to determine the possibility of simultaneous biosynthesis of lipids and carotenoids by the Rhodotorula yeast strains in media with waste glycerol and deproteinized potato wastewater and to determine the level of pollution reduction by media. On the basis of results obtained during the yeast microcultures in the Bioscreen C system, it was found that potato wastewater and glycerol can be used as components of media for Rhodotorula glutinis, Rhodotorula mucilaginosa, and Rhodotorula gracilis yeast strains. The amount of glycerol added to media higher than 10% significantly decreased the growth rate of yeast. The results of yeast culture in the laboratory shaker flasks showed a possibility of simultaneous production of lipids and carotenoids by R. glutinis, R. mucilaginosa, and R. gracilis yeast strains during cultivation in media containing only waste glycerol and deproteinized potato wastewater. A higher intracellular lipid content (approximately 15 g/100 g) was obtained for R. mucilaginosa and R. gracilis yeast biomass after cultivation in experimental media with waste glycerol and potato wastewater. In conclusion, the yeast grown in media with potato wastewater supplemented with 3% or 5% glycerol synthesized carotenoids, and their content in biomass did not exceed 230 μg/g
Topics: Carotenoids; Glycerol; Lipids; Rhodotorula; Solanum tuberosum; Wastewater
PubMed: 31073981
DOI: 10.1007/s12010-019-03023-z -
RSC Advances May 2019With the continuous development of the livestock breeding industry, the amount of piggery wastewater discharged increases year by year, and the pressure of controlling...
With the continuous development of the livestock breeding industry, the amount of piggery wastewater discharged increases year by year, and the pressure of controlling environmental pollution continuously increases. A novel method using a co-culture of and in piggery wastewater was proposed in this study, which was aimed at treating piggery wastewater and producing useful products. The results showed that the optimal inoculum ratio of algae to yeast was 3 : 1 in the wastewater, which achieved the removal efficiencies of 58.53%, 36.07%, 33.20% and 56.25% for ammoniacal nitrogen (NH-N), total nitrogen (TN), total protein (TP) and chemical oxygen demand (COD), respectively, after 6 d. The synergistic relationship of and was preliminarily validated using the oxygen/carbon dioxide exchange balance and scanning electron microscopy images. The co-cultivation system gained 59.8% (w/w) protein within 5 d which can be used as a feed additive, and produces aquatic animals with better growth and quality. Thus, the 1000 litre pilot scale bioreactor was used indoors and removed 82.65% of TN, 53.51% of TP, 93.48% of NH-N and 85.44% of COD in 21 d which gave a better performance for TN ( < 0.05) than the bench scale results. This system improves the nutrition removal and protein production efficiencies, and is a promising method for piggery wastewater treatment and the pig breeding industry.
PubMed: 35519326
DOI: 10.1039/c9ra01884k -
Drug Delivery Dec 2019The potential advantages of recombinant microbes as oral drug carriers for curing diseases have attracted much attention. The use of recombinant oil microbes as living...
The potential advantages of recombinant microbes as oral drug carriers for curing diseases have attracted much attention. The use of recombinant oil microbes as living cell liposomes to carry polypeptide drugs may be an ideal polypeptide oral drug delivery system. GM4-ΔTS was constructed by LFH-PCR from Rhodotorula glutinis GM4, which was screened and preserved in our laboratory, and then transferred into choline-phosphate cytidylyltransferase (CCT), which is a rate-limiting enzyme for lecithin synthesis. The results showed that the CCT gene was highly expressed in the GM4-ΔTS strain and could significantly increase fatty acid and lecithin contents in GM4-ΔTS-PGK1-CCT. Moreover, insulin, H22-LP, and α-MSH were successfully introduced into cells in vitro, and the strain no longer proliferated in vivo, for safe and controllable polypeptide drug delivery. In vivo, normal mice were intragastrically administered with recombinant strains carrying insulin and α-MSH, and different levels of polypeptide drugs were detected in serum and tissue, respectively. Then, recombinant strains carrying insulin were administered to type II diabetes mellitus mice. The results showed that the strains could effectively reduce blood glucose levels in mice, which indicated that the recombinant strains could carry insulin into the body, and the drug effect was remarkable. Therefore, recombinant GM4-ΔTS-PGK1-CCT strains were successfully used as living cell liposomes to carry insulin, H22-LP, and α-MSH peptides into the body for the first time; additionally, these strains have enhanced safety, controllability, and efficacy.
Topics: Animals; Diabetes Mellitus, Experimental; Drug Carriers; Insulin; Liposomes; Mice; Mice, Inbred BALB C; Peptides; Random Allocation; Rhodotorula
PubMed: 30744426
DOI: 10.1080/10717544.2018.1551439 -
AMB Express Aug 2018The constant growing demand for vegetable oil for biodiesel and food is raising many environmental concerns about the sustainability of its production based on crops....
The constant growing demand for vegetable oil for biodiesel and food is raising many environmental concerns about the sustainability of its production based on crops. Oleaginous yeasts show great potential to end with those concerns due to their high lipid productivity in small areas. To evaluate their productivity in lipids, an efficient and reproducible extraction process should be used. As no standard extraction process is available for the extraction of yeast lipids, an optimized extraction process is presented. In this work, the lipids extraction process for the yeasts Rhodotorula glutinis and Lipomyces kononenkoae is optimized using bead beating for cell rupture and introducing adaptations of the two most used extraction methods (Bligh and Dyer and Folch). For Rhodotorula g. the optimum extraction conditions are obtained by the Bligh and Dyer method applying 4.8 cycles of 47 s with 0.7 g of glass beads. For Lipomyces k. the optimum extraction conditions make use of the Folch method applying seven cycles of 42 s with 0.54 g of glass beads. These results reinforce the idea that, for each yeast, different extraction processes may be needed to correctly determine the lipid yield. The extraction procedure was further evaluated with less harmful solvents. Toluene was tested as a possible substitute of chloroform, and ethanol as a possible substitute of methanol. With the optimized extraction process, better results for Lipomyces k. were obtained using toluene and ethanol, while for Rhodotorula g. toluene proved to be a valid substitute of chloroform but ethanol is far less effective than methanol.
PubMed: 30083943
DOI: 10.1186/s13568-018-0658-4 -
Scientific Reports Jul 2018Rhodotorula glutinis, an oleaginous red yeast, intrinsically produces several bio-products (i.e., lipids, carotenoids and enzymes) and is regarded as a potential host...
Rhodotorula glutinis, an oleaginous red yeast, intrinsically produces several bio-products (i.e., lipids, carotenoids and enzymes) and is regarded as a potential host for biorefinery. In view of the limited available genetic engineering tools for this yeast, we have developed a useful genetic transformation method and transformed the β-carotene biosynthesis genes (crtI, crtE, crtYB and tHMG1) and cellulase genes (CBHI, CBHII, EgI, EgIII, EglA and BGS) into R. glutinis genome. The transformant P4-10-9-63Y-14B produced significantly higher β-carotene (27.13 ± 0.66 mg/g) than the wild type and also exhibited cellulase activity. Furthermore, the lipid production and salt tolerance ability of the transformants were unaffected. This is the first study to engineer the R. glutinis for simultaneous β-carotene and cellulase production. As R. glutinis can grow in sea water and can be engineered to utilize the cheaper substrates (i.e. biomass) for the production of biofuels or valuable compounds, it is a promising host for biorefinery.
Topics: Biofuels; Cellulase; Fungal Proteins; Gene Expression Regulation, Fungal; Genetic Engineering; Genome, Fungal; Industrial Microbiology; Rhodotorula; beta Carotene
PubMed: 30022171
DOI: 10.1038/s41598-018-29194-z