Utilization of olive mill wastewater for selective production of lipids and carotenoids by Rhodotorula glutinis.

Olive mill wastewater (OMW) is a zero-cost substrate for numerous value-added compounds. Although several studies on the production of lipids and carotenoids by Rhodotorula glutinis in OMW exist, none of them has specifically focused on the conditions for a target lipid or carotenoid. This study presents cultivation conditions that selectively stimulate the cell biomass, individual carotenoids and lipids. It was found that supplemental carbon and nitrogen sources as well as illumination affected cell biomass the most. High temperature, low initial pH, illumination, lack of urea and presence of glycerol stimulated the lipid synthesis. The highest total lipid content obtained in undiluted OMW supplemented with urea was 11.08 ± 0.17% (w/w) whilst it was 41.40 ± 0.21% (w/w) when supplemented with glycerol. Moreover, the main fatty acid produced by R. glutinis in all media was oleic acid, whose fraction reached 63.94 ± 0.58%. Total carotenoid yield was significantly increased with low initial pH, high temperature, illumination, certain amounts of urea, glycerol and cultivation time. Up to 192.09 ± 0.16 µg/g cell carotenoid yield was achieved. Torularhodin could be selectively produced at high pH, low temperature and with urea and glycerol supplementation. To selectively induce torulene synthesis, cultivation conditions should have low pH, high temperature and illumination. In addition, low pH, high temperature and urea supplementation served high production of ß-carotene. Up to 85.40 ± 0.76, 80.67 ± 1.40 and 39.45 ± 0.69% of torulene, torularhodin and ß-carotene, respectively, were obtained under selected conditions. KEY POINTS: • Cultivation conditions selectively induced target carotenoids and lipids • 41.40 ± 0.21% (w/w) lipid content and 192.09 ± 0.16 µg/g cell carotenoid yield were achieved • Markedly high selectivity values for torularhodin and torulene were achieved.

Valorization of food wastes with a sequential two-step process for microbial ß-carotene production: A zero waste approach.

Here, two consecutive ß-carotene fermentation processes were carried out with Rhodotorula glutinis yeast in the growth media obtained from orange and grape wastes. Firstly, waste biomasses were subjected to hot water extraction. Effects of waste type, drying pretreatment, particle size and solid/liquid ratio on the total concentration and yield of sugars recovered were tested. The highest sugar concentration was obtained by the hot water extraction of fresh grape pomace as 61.2 g total reducing sugars (TRS)/L at a solid/liquid ratio of 100 g/L. In the first fermentation process, effect of solid/liquid ratio (initial TRS concentration) on ß-carotene production pattern of R. glutinis was investigated in the media obtained directly by hot water extraction of the wastes. Microorganism and ß-carotene concentrations increased with increasing solid/liquid ratio (range 10-100 g/L), and the microbial growth data fit the Monod model well for all cases. Maximum ß-carotene concentration in the growth medium obtained from hot water extraction of 100 g/L of grape pomace was determined as 5988.6 mg/L. In the second fermentation process, ß-carotene was produced in the acid hydrolysate of extraction residues. 10.1 g/L and 6.7 g/L of TRS was obtained after acid hydrolysis of orange and grape residues, respectively, and the highest ß-carotene concentration of 370.0 mg/L was found in the medium of hydrolyzed orange peel extraction residue. Total ß-carotene production increased to 1777.1 and 3279.6 mg/L (26% and 4.9% of increase) after the second fermentation step. 85.3% and 80.2% of reduction in orange and grape waste weights were observed at the end of the process, which was an indicator of efficient waste biomass disposal. Two sequential ß-carotene fermentation steps offered significant advantages in terms of both efficiency and a zero waste approach.

Novel flavonoid C-8 hydroxylase from Rhodotorula glutinis: identification, characterization and substrate scope.

BACKGROUND: The regioselective hydroxylation of phenolic compounds, especially flavonoids, is still a bottleneck of classical organic chemistry that could be solved using enzymes with high activity and specificity. Yeast Rhodotorula glutinis KCh735 in known to catalyze the C-8 hydroxylation of flavones and flavanones. The enzyme F8H (flavonoid C8-hydroxylase) is involved in the reaction, but the specific gene has not yet been identified. In this work, we present identification, heterologous expression and characterization of the first F8H ortho-hydroxylase from yeast. RESULTS: Differential transcriptome analysis and homology to bacterial monooxygenases, including also a FAD-dependent motif and a GD motif characteristic for flavin-dependent monooxygenases, provided a set of coding sequences among which RgF8H was identified. Phylogenetic analysis suggests that RgF8H is a member of the flavin monooxygenase group active on flavonoid substrates. Analysis of recombinant protein showed that the enzyme catalyzes the C8-hydroxylation of naringenin, hesperetin, eriodyctiol, pinocembrin, apigenin, luteolin, chrysin, diosmetin and 7,4'-dihydroxyflavone. The presence of the C7-OH group is necessary for enzymatic activity indicating ortho-hydroxylation mechanism. The enzyme requires the NADPH coenzyme for regeneration prosthetic group, displays very low hydroxyperoxyflavin decupling rate, and addition of FAD significantly increases its activity. CONCLUSIONS: This study presents identification of the first yeast hydroxylase responsible for regioselective C8-hydroxylation of flavonoids (F8H). The enzyme was biochemically characterized and applied in in vitro cascade with Bacillus megaterium glucose dehydrogenase reactions. High in vivo activity in Escherichia coli enable further synthetic biology application towards production of rare highly antioxidant compounds.

Farnesol and tyrosol: novel inducers for microbial production of carotenoids and prodigiosin.

This study was performed to elucidate the effects of two fungal quorum sensing molecules (tyrosol and farnesol) on carotenoid synthesis in the yeast Rhodotorula glutinis and prodigioin synthesis in the bacterium Serratia marcencens. Farnesol or tyrosol was directly added to the flask cultures at the beginning (immediately after inoculation with the preculture) of day 1 or the beginning (49th h) of day 3. The results demonstrated that tyrosol supplementation increased the synthesis of carotenoids but farnesol supplementation increased the synthesis of prodigiosin. It was found that adding farnesol or tyrosol into the culture on day 3 compared to day 1 caused more increments in pigment synthesis. The maximum increase (fivefold) in the synthesis of prodigiosin was achieved with 200 µL/L farnesol supplementation, whereas the maximum increase (2.13 fold) in the synthesis of carotenoids was achieved with 4 mg/L tyrosol supplementation. This is the first report about the effects of fungal quorum sensing molecules (farnesol and tyrosol) on the synthesis of carotenoids and prodigiosin in microorganisms. Due to non-human toxicity and low price and of farnesol and tyrosol, these molecules can be used as novel inducers for large-scale production of microbial pigments.

Genomics and lipidomics analysis of the biotechnologically important oleaginous red yeast Rhodotorula glutinis ZHK provides new insights into its lipid and carotenoid metabolism.

BACKGROUND: Rhodotorula glutinis is recognized as a biotechnologically important oleaginous red yeast, which synthesizes numerous meritorious compounds with wide industrial usages. One of the most notable properties of R. glutinis is the formation of intracellular lipid droplets full of carotenoids. However, the basic genomic features that underlie the biosynthesis of these valuable compounds in R. glutinis have not been fully documented. To reveal the biotechnological potential of R. glutinis, the genomics and lipidomics analysis was performed through the Next-Generation Sequencing and HPLC-MS-based metabolomics technologies. RESULTS: Here, we firstly assemble the genome of R. glutinis ZHK into 21.8 Mb, containing 30 scaffolds and 6774 predicted genes with a N50 length of 14, 66,672 bp and GC content of 67.8%. Genome completeness assessment (BUSCO alignment: 95.3%) indicated the genome assembly with a high-quality features. According to the functional annotation of the genome, we predicted several key genes involved in lipids and carotenoids metabolism as well as certain industrial enzymes biosynthesis. Comparative genomics results suggested that most of orthologous genes have underwent the strong purifying selection within the five Rhodotorula species, especially genes responsible for carotenoids biosynthesis. Furthermore, a total of 982 lipids were identified using the lipidomics approaches, mainly including triacylglycerols, diacylglyceryltrimethylhomo-ser and phosphatidylethanolamine. CONCLUSION: Using whole genome shotgun sequencing, we comprehensively analyzed the genome of R. glutinis and predicted several key genes involved in lipids and carotenoids metabolism. By performing comparative genomic analysis, we show that most of the ortholog genes have undergone strong purifying selection within the five Rhodotorula species. Furthermore, we identified 982 lipid species using lipidomic approaches. These results provided valuable resources to further advance biotechnological applications of R .glutinis.

Fatty acids profiles and estimation of the biodiesel quality parameters from Rhodotorula spp. from Antarctica.

OBJECTIVE: Oleaginous yeasts are a renewable and alternative source of oil for third-generation biodiesel. This work aimed to evaluate the effects of glucose concentration (30-100 g L-1) on growth, lipid synthesis, and fatty acids (FA) profile of three Rhodotorula spp. (R. glacialis R15, R. glutinis R4, and R. glutinis R48) isolated from Antarctica, and estimate the key quality parameters of the biodiesel produced by yeasts to confirm their potential as feedstocks for third-generation biodiesel synthesis. RESULTS: Yeasts accumulated 50-69.5% of lipids (w/w) under nitrogen-limitation and glucose-excess (C/N = 40-133). Glucose concentration increase influenced positively lipid accumulation (69.5% w/w) and FA profile of R. glacialis R15. Lipid accumulation (53% on average) of R. glutinis strains was not significantly affected by glucose concentration; content of saturated (~ 30%) and polyunsaturated FA (~ 29-30%) was slightly influenced. FA profiles of lipids synthesized by R15, R4, and R48 are similar to vegetable oils used in biodiesel industry with C16 and C18 FA (95-99%) as the major components, and contain mainly oleic (C18:1), palmitic (C16:0), and linoleic (C18:2) acids, which are suitable for biodiesel synthesis. Estimated fuel properties for biodiesel produced by R15, R4, and R48 satisfied all the criteria established by ASTM D6751 and EN 14214 with good cetane number, iodine value, and oxidation stability. An improvement in biodiesel quality of R15 was observed with the glucose increase. The best global properties of biodiesel from R4 were obtained with 30 g L-1 of glucose. CONCLUSIONS: Rhodotorula spp. from Antarctica are promising candidates for third-generation biodiesel synthesis.

Lipid Production by Rhodotorula glutinis in Continuous Cultivation with a Gravity Sedimentation System.

Lipid accumulation is generally believed to be a partially growth-coupled biochemical process that results in differences in lipid content between different cells. To separate lipid-rich cells and increase the cellular biomass in bioreactors during the cultivation of the oleaginous yeasts, a gravity sedimentation system (GSS) is coupled to a bioreactor. The dilution rate (D) and the ratio of the outflow rate from the outlet of the GSS to the inflow rate into the bioreactor (B) were evaluated. The biomass in the bioreactor with GSS increased by 16.3% and 30.6% at D values of 0.05 h-1 (B = 0.25) and 0.02 h-1 (B = 0.5), respectively. Interestingly, cells containing 39.3% lipids were obtained from the outlet of the GSS (D = 0.02 h-1, B = 0.5), and the lipid content increased by 7.8% compared to that of the bioreactor. The results indicated that use of a GSS is a very effective method for increasing the cell concentration and separation of lipid-rich cells.

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.

Improved stereoselective bioreduction of t-butyl 6-cyano-(5R)-hydroxy-3-oxohexanoate by Rhodotorula glutinis through heat treatment.

Optically pure t-butyl 6-cyano-(3R, 5R)-dihydroxyhexanoate ((R)-1b) is the key precursor for atorvastatin calcium, the most widely used cholesterol-lowering drug. In this work, a strain ZJB-09224 capable of asymmetrically reducing t-butyl 6-cyano-(5R)-hydroxy-3-oxohexanoate (1a) to corresponding optically pure (R)-1b was successfully isolated from soil sample, identified belonging to Rhodotorula glutinis based on the morphology, physiological tests, and the 18S rDNA sequence analysis. It was found that heat treatment of cell suspension at 45 °C for 25 Min significantly improved R. glutinis ZJB-09224 stereoselectivity. The asymmetric bioreduction of 1a was most efficient at pH 7.5, 35 °C, 50 mM (15.0 g L-1 ) substrate concentration, 40.0 g DCW L-1 cell loading size, 0.54 M (60.0 g L-1 ) sodium lactate acting as co-substrate. Under these optimal conditions, 0.046 M (R)-1b was produced with de (diastereomeric excess) value of 99.2% after 40 H conversion. Moreover, R. glutinis ZJB-09224 has a broad substrate spectrum, making it a potential tool for some valuable chiral alcohol pharmaceutical intermediates synthesis.

Optimization of pineapple pulp residue hydrolysis for lipid production by Rhodotorula glutinis TISTR5159 using as biodiesel feedstock.

The higher lipid productivity of Rhodotorula glutinis TISTR5159 was achieved by optimizing the pineapple pulp hydrolysis for releasing the high sugars content. The sequential simplex method operated by varied; solid-to-liquid ratio, sulfuric acid concentration, temperature, and hydrolysis time were successfully applied and the highest sugar content (83.2 g/L) evaluated at a solid-to-liquid ratio of 1:10.8, 3.2% sulfuric acid, 105 °C for 13.9 min. Moreover, the (NH4)2SO4 supplement enhanced the lipid productivity and gave the maximum yields of biomass and lipid of 15.2 g/L and 9.15 g/L (60.2%), respectively. The C16 and C18 fatty acids were found as main components included oleic acid (55.8%), palmitic acid (16.6%), linoleic acid (11.9%), and stearic acid (7.8%). These results present the possibility to convert the sugars in pineapple pulp hydrolysate to lipids. The fatty acid profile was also similar to vegetable oils. Thus, it could be used as potential feedstock for biodiesel production.