Analysis of the light regulatory mechanism in carotenoid production in Rhodosporidium toruloides NBRC 10032.

Light stimulates carotenoid production in an oleaginous yeast Rhodosporidium toruloides NBRC 10032 by promoting carotenoid biosynthesis genes. These genes undergo two-step transcriptional activation. The potential light regulator, Cryptochrome DASH (CRY1), has been suggested to contribute to this mechanism. In this study, based on KU70 (a component of nonhomologous end joining (NHEJ)) disrupting background, CRY1 disruptant was constructed to clarify CRY1 function. From analysis of CRY1 disruptant, it was suggested that CRY1 has the activation role of the carotenogenic gene expression. To obtain further insights into the light response, mutants varying carotenoid production were generated. Through analysis of mutants, the existence of the control two-step gene activation was proposed. In addition, our data analysis showed the strong possibility that R. toruloides NBRC 10032 is a homo-diploid strain.

Effect of light on carotenoid and lipid production in the oleaginous yeast Rhodosporidium toruloides.

The oleaginous yeast Rhodosporodium toruloides is receiving widespread attention as an alternative energy source for biofuels due to its unicellular nature, high growth rate and because it can be fermented on a large-scale. In this study, R. toruloides was cultured under both light and dark conditions in order to understand the light response involved in lipid and carotenoid biosynthesis. Our results from phenotype and gene expression analysis showed that R. toruloides responded to light by producing darker pigmentation with an associated increase in carotenoid production. Whilst there was no observable difference in lipid production, slight changes in the fatty acid composition were recorded. Furthermore, a two-step response was found in three genes (GGPSI, CAR1, and CAR2) under light conditions and the expression of the gene encoding the photoreceptor CRY1 was similarly affected.

Multi-omics metabolism analysis on irradiation-induced oxidative stress to Rhodotorula glutinis.

Oxidative stress is induced in many organisms by various natural abiotic factors including irradiation. It has been demonstrated that it significantly improves growth rate and lipid production of Rhodotorula glutinis. However, the specific mechanism of how irradiation influences the metabolism of R. glutinis remains still unavailable. To investigate and better understand the mechanisms involved in irradiation-induced stress resistance in R. glutinis, a multi-omics metabolism analysis was implemented. The results confirmed that irradiation indeed not only improved cell biomass but also accelerated the production of carotenoids and lipids, especially neutral lipid. Compared with the control, metabolome profiling in the group exposed to irradiation exhibited an obvious difference in the activation of the tricarboxylic acid cycle and triglyceride (TAG) production. The results of proteome analysis (data are available via ProteomeXchange with identifier PXD009678) showed that 423 proteins were changed significantly, and proteins associated with protein folding and transport, the Hsp40 and Sec12, were obviously upregulated, indicating that cells responded to irradiation by accelerating the protein folding and transport of correctly folded proteins as well as enhanced the degradation of misfolded proteins. A significant upregulation of the carotenoid biosynthetic pathway was observed which revealed that increased carotenoid content is a cellular defense mechanism against oxidative stress generated by irradiation. Therefore, the results of comprehensive omics analysis provide intensive insights on the response mechanism of R. glutinis to irradiation-induced oxidative stress which could be helpful for using irradiation as an effective strategy to enhance the joint production of the neutral lipid and carotene.

The ability of a cold-adapted Rhodotorula mucilaginosa strain from Tibet to control blue mold in pear fruit.

Cold-adapted yeasts were isolated from soil samples collected in Tibet and evaluated as potential biocontrol agents against blue mold (Penicillium expansum) of pear fruit in cold storage. YC1, an isolate identified as Rhodotorula mucilaginosa, was found to exhibit the greatest biocontrol activity among the different isolates that were screened. A washed cell suspension of YC1 exhibited the best biocontrol activity among three different preparations that were used in the current study. A concentration of 10(8) cells/ml reduced the incidence of decay to 35 %, compared to the control where decay incidence was 100 %. A higher intracellular level of trehalose and a higher proportion of polyunsaturated acids present in YC1, was associated with increased the tolerance of this strain to low temperatures, relative to the other strains that were evaluated. The increased tolerance to low temperature allowed the YC1 strain of yeast to more effectively compete for nutrients and space in wounded pear fruit that had been inoculated with spores of P. expansum and placed in cold storage. The present study demonstrated the ability to select cold-adapted yeasts from cold climates and use them as biocontrol agents of postharvest diseases of fruit placed in cold storage.

The radiation resistance and cobalt biosorption activity of yeast strains isolated from the Lanyu low-level radioactive waste repository in Taiwan.

The ubiquitous nature of microbes has made them the pioneers in radionuclides adsorption and transport. In this study, the radiation resistance and nuclide biosorption capacity of microbes isolated from the Lanyu low-level radioactive waste (LLRW) repository in Taiwan was assessed, the evaluation of the possibility of using the isolated strain as biosorbents for (60)Co and Co (II) from contaminated aqueous solution and the potential impact on radionuclides release. The microbial content of solidified waste and broken fragments of containers at the Lanyu LLRW repository reached 10(5) CFU/g. Two yeast strains, Candida guilliermondii (CT1) and Rhodotorula calyptogenae (RT1) were isolated. The radiation dose necessary to reduce the microbial count by one log cycle of CT1 and RT1 was 2.1 and 0.8 kGy, respectively. Both CT1 and RT1 can grow under a radiation field with dose rate of 6.8 Gy/h, about 100 times higher than that on the surface of the LLRW container in Lanyu repository. CT1 and RT1 had the maximum (60)Co biosorption efficiency of 99.7 ± 0.1% and 98.3 ± 0.2%, respectively in (60)Co aqueous solution (700 Bq/mL), and the (60)Co could stably retained for more than 30 days in CT 1. Nearly all of the Co was absorbed and reached equilibrium within 1 h by CT1 and RT1 in the 10 µg/g Co (II) aqueous solution. Biosorption efficiency test showed almost all of the Co (II) was adsorbed by CT1 in 20 µg/g Co (II) aqueous solution, the efficiency of biosorption by RT1 in 10 µg/g of Co (II) was lower. The maximum Co (II) sorption capacity of CT1 and RT1 was 5324.0 ± 349.0 µg/g (dry wt) and 3737.6 ± 86.5 µg/g (dry wt), respectively, in the 20 µg/g Co (II) aqueous solution. Experimental results show that microbial activity was high in the Lanyu LLRW repository in Taiwan. Two isolated yeast strains, CT1 and RT1 have high potential for use as biosorbents for (60)Co and Co (II) from contaminated aqueous solution, on the other hand, but may have the impact on radionuclides release from LLRW repository.

Lipid and carotenoid production by Rhodotorula glutinis under irradiation/high-temperature and dark/low-temperature cultivation.

The capacity of lipid and carotenoid production by Rhodotorula glutinis was investigated under different irradiation conditions, temperatures and C/N ratios. The results showed that dark/low-temperature could enhance lipid content, while irradiation/high-temperature increased the yields of biomass and carotenoid. The optimum C/N ratio for production was between 80 and 100. A two-stage cultivation strategy was used for lipid and carotenoid production in a 5L fermenter. In the first stage, the maximum biomass reached 28.1g/L under irradiation/high-temperature. Then, the cultivation condition was changed to dark/low-temperature, and C/N ratio was adjusted to 90. After the second stage, the biomass, lipid content and carotenoid reached 86.2g/L, 26.7% and 4.2mg/L, respectively. More significantly, the yields of biomass and lipid were 43.1% and 11.5%, respectively. Lipids contained 79.7% 18C and 16.8% 16C fatty acids by GC analysis. HPLC quantified the main carotenoids were ß-carotene (68.4%), torularhodin (21.5%) and torulene (10.1%).

From crude glycerol to carotenoids by using a Rhodotorula glutinis mutant.

In this work eighteen red yeasts were screened for carotenoids production on glycerol containing medium. Strain C2.5t1 of Rhodotorula glutinis, that showed the highest productivity, was UV mutagenized. Mutant 400A15, that exhibited a 280 % increase in ß-carotene production in respect to the parental strain, was selected. A central composite design was applied to 400A15 to optimize carotenoids and biomass productions. Regression analyses of the quadratic polynomial equations obtained (R(2) = 0.87 and 0.94, for carotenoids and biomass, respectively) suggest that the models are reliable and significant (P < 0.0001) in the prediction of carotenoids and biomass productions on the basis of the concentrations of crude glycerol, yeast extract and peptone. Accordingly, total carotenoids production achieved (14.07 ± 1.45 mg l(-1)) under optimized growth conditions was not statistically different from the maximal predicted (14.64 ± 1.57 mg l(-1)) (P < 0.05), and it was about 100 % higher than that obtained under un-optimized conditions. Therefore mutant 400A15 may represent a biocatalyst of choice for the bioconversion of crude glycerol into value-added metabolites, and a tool for the valorization of this by-product of the biodiesel industry.

Determination of the effect of Nd:YAG laser irradiation through dentinal tubules on several oral pathogens.

The effects of Nd:YAG laser irradiations at different power settings on several oral pathogens were evaluated. A total of 252 dentin samples were divided into seven groups consisting of 36 dentin specimens each. In each group, 9 of the 36 specimens were used as controls, thereby including a control in every group. The remaining 27 specimens were divided into three subgroups consisting of nine specimens according to different Nd:YAG laser settings (1.5, 1.8, and 2 W). Each group was inoculated on the nonpulpal side with one of the following microorganisms: Candida glabrata, Candida tropicalis, Candida krusei, Candida sake, Candida lusitaniae, Candida kefyr, and Rhodotorula mucilaginosa. The following irradiation procedure was used: the specimens were irradiated on the bacteria-free side (the side consisting of the pulpal wall) using contact mode under the constant scanning movement of the optical fiber at an angle of 10°. One lasing cycle consisted of four irradiation cycles of 10 s each, with 15-s intervals in between each irradiation cycle. The remainder of the controls and the lased specimens of each group were prepared for the microbiological investigation. After incubation for 24 h at 37 °C, the colonies were counted, and the total number of surviving microorganisms was statistically assessed. Microorganisms irradiated with Nd:YAG laser at power settings 2 W, 15 pps did not survive. Although there was a significant reduction of microorganisms at 1.5 and 1.8 W, when comparing Nd:YAG laser irradiation with the control group, sterilization did not occur.

Enhancement of cell growth rate by light irradiation in the cultivation of Rhodotorula glutinis.

A yeast, Rhodotorula glutinis, is regarded as a potential microbial oil producer, due to its high lipid content. The flask results of this study indicated that irradiation could increase the growth of R. glutinis compared to that of a batch without irradiation. Further 5-l fermenter results confirmed that irradiation could greatly enhance the cells' growth rate and total lipid productivity. The maximum lipid productivity obtained in the fed-batch operation with 3 LED (light emitting diode) lamps was 0.39 g/l h as compared to 0.34 g/l h in the batch with 3 LED lamps and 0.19 g/l h in the batch without irradiation. Conclusively, the irradiation could significantly increase the cells' growth rate, which, in turn, could be applied to the commercialized production of biodiesel from single cell oils.

Photoprotection by carotenoid pigments in the yeast Rhodotorula mucilaginosa: the role of torularhodin.

In this paper we report the relationship between carotenoids and ergosterol and cell UV-B resistance in different strains of the yeast Rhodotorula mucilaginosa. Cell survival was studied using a set of 13 strains; additionally, two mutants (a hyper-producing one and a colourless one) in combination with diphenylamine (DPA), a carotenogenesis inhibitor, were used. A positive correlation between total carotenoids and survival to UV-B radiation was found. However, when individual carotenoid concentrations were tested, only torularhodin was found to be significantly related to UV-B survival. On the contrary, ergosterol did not affect survival. The hyper-pigmented strain showed an enhanced survival (up to 250%) compared to the parental strain, while the survival of the albino mutant was similar to that experienced by the parental strain; however, observed changes in survival were dose dependent. The cyclobutane pyrimidine dimers (CPDs), one of the major forms of DNA damage caused by UV exposure, appears as unrelated to the accumulation of carotenoids and cell survival. These results indicate that bearing higher torularhodin concentrations enhances UV-B survival in yeasts and, thus, the accumulation of this pigment constitutes an important mechanism that improves the resistance of yeasts to UV-B.

[Biosorption ability of mutants of Rhodotorula mucilaginosa UCM Y-1776].

Twenty stable mutants with various coloration intensity have been allocated in carotene-synthesizing natural strain Rhodotorula mucilaginosa UCM Y-1776 (wild type) after nitrosoguanidine action. Two brightly orange mutants 4L and 11 and one non-pigmented mutant 2 were chosen for the further researches. The ultraviolet was inefficient as a mutagen. Resistance to high concentration of copper ions (up to 200 mg/g), high sorption ability (Qmax = 9.1 mmol/g) was characteristic of R. mucilaginosa UCM Y-1776. Concentration of copper ions 50 mg/l was toxic for mutants 4L, 11 and 2, which sorption ability was lower in comparison with carotene pigmented R. mucilaginosa UCM Y-1776. It was shown, for the first time that there was a direct dependence between the presence of carotenoid pigments, resistance to high concentration of copper ions and sorption ability for yeast R. mucilaginosa UCM Y-1776.

Mycosporines from freshwater yeasts: a trophic cul-de-sac?

Mycosporine-like amino-acids (MAAs) are found in aquatic bacteria, algae, and animals. A related compound, the mycosporine-glutaminol-glucoside (myc-glu-glu), has recently been reported in freshwater yeasts. Although animals depend on other organisms as their source of MAAs, they can efficiently accumulate them in their tissues. In this work we assessed the potential transfer of the yeast mycosporine myc-glu-glu from the diet into the copepod Boeckella antiqua and the ciliate Paramecium bursaria. For this purpose, we performed experiments to study the feeding of B. antiqua and P. bursaria on the yeast Rhodotorula minuta and their ability to bioaccumulate myc-glu-glu. Bioaccumulation of myc-glu-glu in B. antiqua was assessed through long-term factorial experiments manipulating the diet (Chlamydomonas reinhardii and C. reinhardii + yeasts) and radiation exposure (PAR and PAR + UVR). Shorter term experiments were designed in the case of P. bursaria. The composition and concentration of MAAs in the diet and in the consumers were determined by HPLC analyses. Our results showed that even though both consumers ingested yeast cells, they were unable to accumulate myc-glu-glu. Moreover, when exposed to conditions that stimulated the accumulation of photoprotective compounds (i.e. UVR exposure), an increase in MAAs concentration occurred in copepods fed C. reinhardii plus yeasts as well as in those fed only C. reinhardii. This suggests that the copepods were able to modify their tissue concentrations of MAAs in response to environmental clues but also that the contribution of yeast mycosporines to total MAAs concentration was negligible.

Constitutive and UV-inducible synthesis of photoprotective compounds (carotenoids and mycosporines) by freshwater yeasts.

Twelve yeasts isolated from lakes of Northwestern Patagonia, Argentina, belonging to eight genera (Sporobolomyces, Sporidiobolus, Rhodotorula, Rhodosporidium, Cystofilobasidium, Cryptococcus, Torulaspora, and Candida) were analysed for their ability to produce photoprotective compounds. For this purpose, three laboratory experiments were performed to study the effect of photosynthetically active radiation (PAR) and PAR in combination with UV radiation (PAR + UVR) on the production of carotenoids and mycosporines. The synthesis of carotenoid compounds was clearly stimulated in six out of nine red yeast strains tested upon exposure to PAR or PAR + UVR; however, the latter conditions produced a stronger response than PAR alone. The increase in carotenoids in the red strains under PAR + UVR irradiation showed a negative exponential relationship with their basal carotenoid content, suggesting that cells with higher constitutive levels of carotenoids are less responsive to induction by PAR + UVR. Three red yeasts, Rhodotorula minuta, Rh. pinicola, and Rhodotorula sp., and the colourless Cryptococcus laurentii produced a UV-absorbing compound when exposed to PAR or PAR + UVR. This compound showed an absorption maximum at 309-310 nm and was identified as mycosporine-glutaminol-glucoside (myc-glu-glu). In these strains, exposure to PAR or PAR + UVR resulted in elevated concentrations of both carotenoids and myc-glu-glu. This is the first report on the production of mycosporines by yeasts. All strains that developed under PAR + UVR were able to synthesise carotenoids either constitutively or in response to PAR exposure, and a few of them also produced myc-glu-glu when exposed to PAR. Collectively, our results suggest that the presence of carotenoids, either alone or in combination with mycosporines, are required for sustaining growth under exposure to PAR + UVR in the freshwater yeast strains studied.

Manipulation of temperature and illumination conditions for enhanced beta-carotene production by mutant 32 of Rhodotorula glutinis.

AIMS: Enhancement in the production of beta-carotene by the hyper producer mutant 32 of Rhodotorula glutinis by manipulation of temperature and illumination. METHODS AND RESULTS: Growth and beta-carotene production was investigated in a 1 litre fermenter at different temperature and illumination conditions. The optimum temperature for growth and beta-carotene production was 30 and 20 degrees C, respectively. At 30 degrees C, beta-carotene production was 125 +/- 2 mg l-1 and accounted for 66% of the total carotenoids in 72 h; at 20 degrees C, it was 250 +/- 7 mg l-1 and accounted for 92% of total carotenoid content. Continuous illumination of the fermenter by 1000 lx white light hampered growth as well as carotenoid synthesis. At 30 degrees C, illuminating the fermenter in late logarithmic phase resulted in a 58% increase in beta-carotene production with a concurrent decrease in torulene; at 20 degrees C, however, it showed no appreciable increase. SIGNIFICANCE AND IMPACT OF THE STUDY: Proper manipulation of culture conditions enhanced beta-carotene production by R. glutinis which makes it a significant source of beta-carotene.

Production of beta-carotene by a mutant of Rhodotorula glutinis.

Wild strains of Rhodotorula glutinis and R. rubra were investigated concerning their carotenoid production, proportion of beta-carotene and cell mass yield. R. glutinis NCIM 3353 produced 2.2 mg carotenoid/l in 72 h; and the amount of beta-carotene was 14% (w/w) of the total carotenoid content (17 microg/g cell dry weight). It was subjected to mutagenesis using UV radiation for strain improvement. Out of 2,051 isolates screened, the yellow coloured mutant 32 produced 120-fold more beta-carotene (2,048 microg/g cell dry weight) than the parent culture in 36 h, which was 82% (w/w) of the total carotenoid content. Mutant 32 was grown on different carbon and nitrogen sources. The best yield of beta-carotene (33+/-3 mg/l) was obtained when glucose and yeast extract were supplied as carbon and nitrogen sources, respectively. Divalent cation salts further increased the total carotenoid content (66+/-2 mg/l) with beta-carotene as the major component (55+/-2%, w/w).

Phenylalanine ammonia lyase production by gamma irradiated and analog-resistant mutants of Rhodotorula glutinis.

Mutants resistant to phenylalanine analogs (L-tyrosine, p-fluoro-D, L-phenylalanine (PFP) and trans-cinnamic acid) were isolated from a wild type strain of Rhodotorula glutinis A-97 by mutagenic treatment with gamma radiation and screened for phenylalanine ammonia lyase (PAL) production. One such mutant, gammaT11 (resistant to L-tyrosine), exhibited four times the PAL activity of the parent wild strain A-97. Mutant isolate gammaTFP5.6 which was selected as L-tyrosine and PFP resistant isolate, produced inducible PAL activity at levels 5.94-fold higher than the wild-type A-97 and 2.66-fold higher than its parent mutant isolate gammaT5 which was resistant to L-tyrosine. The mutant isolate gammaTC5d which was resistant to L-tyrosine and trans-cinnamic acid, exhibited 3.48 and 1.56-fold increase in PAL activity compared to the parent wild strain A-97 and its parent mutant isolate gammaT5, respectively. Different media have been examined for the induction of PAL.

[Effect of stress on the composition of yeast lipids]. / Vliianie stressovykh vozdeistvii na sostav lipidov drozhzhei.

Pigmented (Rhodotorula glutinis) and nonpigmented (Lipomyces starkeyi) yeasts were studied. Exogenous stressors (UV irradiation and methylene blue) were shown to change the composition of yeast lipids (especially the ratio of unsaturated fatty acids) and to increase the content of lipid peroxidation products formed (particularly in nonpigmented yeasts). In carotene-synthesizing yeasts, these stressors decreased the amount of carotenoids produced and did not affect the ratio between carotenoid pigments (beta-carotene, torulene, and torularhodin).

Induction of colony morphology variation in Rhodotorula gracilis by UV irradiation.

Nonlethal UV irradiation induced an unusually high frequency of colony morphology variation in Rhodotorula gracilis. The variation was not fixed but indicated further variability in subsequent platings. Microscopic examination of the cultures indicated that UV-irradiated variants had grossly varying types of shapes and arrangements of cells in contrast to the uniformly shaped and budding cells of the nonirradiated culture. Flow-cytometric analysis of a colony variant suggested a slightly higher proportion of cells with variable DNA content than the nonirradiated culture. Extensive biochemical characterization revealed only one difference in that the nonirradiated culture had a partial requirement for pantothenate while the colony variant was completely independent of this requirement. We speculate that UV triggers a yet unstudied means of variability in R. gracilis with possible accompanying recombinational events.

Pseudohyphal variations of yeasts exposed to specific space flight parameters.

Phenotypes of Saccharomyces cerevisiae and Rhodotorula rubra exposed to specific parameters of space flight, which were measured both quantitatively and qualitatively, produced variations in pseudohyphal formation. Both the length of the parent and branch psuedohyphal filaments varied according to specific wavelengths and energy levels of UV light exposures when phenotypic isolates were compared with the parent or ground control isolate of each yeast species.

Free radical production in photodynamically inactivated cells of Rhodotorula glutinis.

In an investigation of die-off of micro-organisms irradiated with high-intensity light, as might be expected in a space environment, the yeast Rhodotorula glutinis was used as a prototype. Previous results have shown that the yeast Rhodotorula glutinis can be photodynamically inactivated by laser radiation and by other intense light sources such as a xenon arc lamp. Experiments were conducted to determine if free radicals were involved in the inactivation of Rhodotorula glutinis when irradiated with light of wavelengths 300 nm and longer. Presumptive evidence that free radicals were involved in the photodynamic inactivation of the cells was found when it was shown that compounds capable of trapping free radicals were able to provide some protection to the cells. Further presumptive evidence that free radicals are involved was provided when it was shown that lipid peroxidation, which can be mediated by free radicals, is caused when the cells are irradiated. The actual production of free radicals was demonstrated by the detection of the presence of unpaired electron paramagnetic resonance spectra. These studies were conducted at both -30 degrees C and -160 degrees C. It was found that free radicals were produced at both temperatures and that cysteine could decrease the free radical population at -30 degrees C but not at -160 degrees C.