Transport and metabolism of 2-deoxy-D-glucose by Rhodotorula glutinis

2-Deoxy-D-glucose transport by Rhodotorula glutinis is an active process. The intracellular concentration of free deoxyglucose after 15 min incubation of Rhodotorula cells with this sugar was 230 times the extracellular concentration. Although cell extracts at this time contained more 2-deoxy-D-glucose 6-phosphate than deoxyglucose, pulse-labelling experiments demonstrated that deoxyglucose is transported as the free sugar and subsequently phosphorylated. After transport, Rhodotorula cells metabolize deoxyglucose. The major metabolites during 30-90 min incubations were determined to be 2-deoxy-D-glucose 6-phosphate, 2-deoxy-D-glucitol, 2-deoxy-D-gluconate and 2,2'-dideoxy-alpha, alpha'-trehalose. Rhodotorula glutinis also degrades deoxyglucose to CO2. The concentrations of intermediates in this pathway were too low to detect and resolve in extracts of control cells. In 2,4-dinitrophenol-poisoned cells, however, it appears that deoxyglucose degradation is restricted largely to loss of C-1 as CO2 and it was possible to identify 1-deoxy-D-ribulose 5-phosphate as an intermediate presumably arising from metabolism of deoxyglucose by the oxidative portion of the hexose monophosphate pathway.     

Transport of gluconate in Rhodotorula glutinis. Inactivation by glucose of the uptake system.

Transport of gluconate has been studied in a wild-type strain of Rhodotorula glutinis and in a mutant derived from it which has acquired the ability to grow on gluconate as the only carbon and energy source. The transport is energy dependent. It shows the same K_m for gluconate (0.1 mm) between pH 4.7 and 7, which suggests that the negatively charged gluconate is the true substrate for the transport system. The rate of gluconate uptake is much lower in the wild type than in the mutant. The mutant grown on gluconate transports gluconate much faster than if grown on other carbon sources. Glucose rapidly and irreversibly inactivates the transport system. This inactivation can also be effected by δ-gluconolactone and to a lesser extent by acetate; it is not prevented by gluconate and occurs also in the presence of cycloheximide.     

Carotenoid Biosynthesis in Rhodotorula glutinis

It was determined that lycopene could be cyclized directly by Rhodotorula glutinis. It was also shown the the temperature effect (i.e., increased beta-carotene synthesis in response to lower incubation temperatures) in R. glutinis was controlled by changes in enzyme concentration.     

Carbohydrate assimilation by clinical and environmental Rhodotorula glutinis strains

This study was carried to determine the carbohydrate assimilation patterns of Rhodotorula strains isolated from clinical and environmental specimens. We have tested the commercial system ID 32C (bioMerieux, France) on 80 different strains of Rhodotorula glutinis: 47 strains from clinical samples and 33 strains from environmental samples. The assimilation percentages obtained in our study for galactose, cellobiose, gluconate and sorbose were lower than those showed in the identification table of the method. However, the assimilation percentages for mannitol and esculin were higher. According to our results, we conclude that the numerical profiles and the identification software of the commercial system present limitations for the characterization of some R. glutinis strains.     

Synthesis of intracellular and extracellular metabolites by Rhodotorula glutinis

A close relationship has been established between the production of extracellular and intracellular lipids and polysaccharides by Rhodotorula glutinis. Their synthesis depended on the pH of the medium. The composition and dynamics of polyol content in the cells, and the presence of acids typical of exolipids in the composition of intracellular lipids under certain conditions, suggest that the components of extracellular lipids are synthesized within the yeast cell.     

Transport und Umsatz von Polyalkoholen bei der Hefe Rhodotorula gracilis (glutinis)

Zusammenfassung Die Polyalkohole Sorbitol, d-Mannitol, Ribitol, Xylitol, d-Arabitol, l-Arabitol und Erythritol werden von der obligat aeroben Hefe Rhodotorula gracilis über einen beweglichen Träger in der Zellmembran aufgenommen. Der Transportmechanismus ist aktiv, das erreichte Akkumulationsverhältnis ist jedoch bei allen Polyalkoholen erheblich geringer als bei Monosacchariden. Es nimmt, wie auch bei Monosacchariden, mit steigender Außenkonzentration ab, sogar auf Werte kleiner als 1.Kinetische Daten weisen darauf hin, daß das Trägersystem für Polyalkohole identisch ist mit dem für Monosaccharide, jedoch für Polyalkohole eine wesentlich geringere Affinität und maximale Geschwindigkeit aufweist. Aufgrund des hohen Affinitätsunterschiedes wird die Polyalkoholaufnahme in der Anwesenheit von Monosacchariden unterbunden.Die aufgenommenen Polyalkohole werden im Zellinneren nicht umgesetzt; eine Ausnahme stellen Ribitol und l-Arabitol dar, in deren Anwesenheit ein Abbausystem für Pentitole induziert wird.

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Transport and utilization of alditols in the yeast Rhodotorula gracilis (glutinis)I. Constitutive transport of alditols

The obligate aerobic yeast Rhodotorula gracilis was found to take up the alditols d-glucitol, d-mannitol, ribitol, xylitol, d-arabinitol, l-arabinitol and erythritol by means of a constitutive mobile membrane carrier. This uptake involved active transport, that is, it was dependent on the supply of metabolic energy, leading to the accumulation of alditols inside the cells. The accumulation ratio (intracellular concentration to extracellular concentration, S_i/S_o) was much lower for alditols than for monosaccharides. As for monosaccharides, this ratio decreased with increasing extracellular concentration, even to values below 1.The kinetic data showed that the carrier system for alditols was identical to that for monosaccharides, though it had a much lower affinity and maximum velocity for alditols. Hence the uptake of alditols was blocked in the presence of monosaccharides.Only ribitol and l-arabinitol were catabolized following enzyme induction. The other alditols were not broken down.

     

Effect of glucose on pyruvate utilization by Rhodotorula glutinis

Summary The utilization of glucose and pyruvate by the yeast Rhodotorula glutinis in a medium containing both carbon sources has been studied. Glucose is readily consumed whereas the uptake of pyruvate is completely blocked by the presence of the sugar.The content of pyruvate kinase and phosphoenolpyruvate carboxykinase in R. glutinis cells growing on glucose plus pyruvate are drastically affected with time by the disappearance of the sugar from the culture medium. After complete exhaustion of glucose, the level of pyruvate kinase drops sharply down to a minimum whereas that of phosphoenolpyruvate carboxykinase rises abruptly up to a maximum.Feeding experiments with labelled compounds show that glucose affects the utilization of the amino acids alanine and aspartate, and conversely that the amino acids influence the utilization of the sugar. Glucose breakdown and its incorporation into polysaccharides is controlled by the amino acids and gluconeogenesis from the amino acids is controlled by the sugar.     

Uptake of disaccharides by the aerobic yeast Rhodotorula glutinis

Sucrose (and raffinose), trehalose, maltose, cellobiose, and lactose were examined for their transport into Rhodotorula glutinis. Melibiose and lactose were found not to be transported at all. Sucrose, raffinose and trehalose are split by periplasmic hydrolases prior to the penetration of their monosaccharide components into cells, the hydrolysis being the rate-limiting factor for the uptake process. Maltose and cellobiose appear to use specific uptake systems. Experiments with protoplasts of Rhodotorula glutinis support the conclusions that sucrose and trehalose are not consumed in the absence of exoenzymes.     

Regulation of ornithine transcarbamylase in Rhodotorula glutinis

The regulation of ornithine transcarbamylase (OTC) of Rhodotorula glutinis has been studied, by growing the yeasts in different carbon and nitrogen sources and estimating the enzyme level in crude yeasts extracts.The results show a nutritional repression of OTC by arginine, when added to the culture media as carbon, nitrogen or carbon and nitrogen sources. On the other hand ornithine does not exert any effect in the same experimental conditions.     

Invertase from a strain of Rhodotorula glutinis

An invertase (beta-D-fructofuranoside fructohydrolase, EC 3.2.1.26) from Rhodotorula glutinis was purified by ammonium sulfate fractionation, gel filtration and anion exchange chromatography. Invertase molecular weight was estimated to be 100 kDa by analytical gel filtration and 47 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Molecular mass determinations indicated that the native enzyme exists as a homodimer. It is a glycoprotein that contains 19% carbohydrate. The enzyme attacks beta-D-fructofuranoside (raffinose, stachyose and sucrose) from the fructose end. It has a K(m) of 0.227 M and a V(max) of 0.096 micromol/min with sucrose as a substrate. Invertase activity is stable between pH 2.6 and 5.5 for 30 min, maximum activity being observed at pH 4.5. The activation energy was 6520 cal/mol. The enzyme is stable between 20 and 60 degrees C. Mg(2+) and Ca(2+) ions stimulated invertase activity 3-fold, while Fe(2+), K(+), Co(2+), Na(+) and Cu(2+) increased activity about 2-fold. The transfructosylation reaction could not be observed. This enzyme is of particular interest since it appears to have a high hydrolytic activity in 1 M sucrose solution. This fact would make the enzymatic hydrolysis process economically efficient for syrup production using by-products with high salt and sugar contents such as sugar cane molasses.     

Kinetics of D-glucose and 2-deoxy-D-glucose transport by Rhodotorula glutinis

The yeast Rhodotorula glutinis (Rhodosporidium toruloides) is capable of accumulative transport of a wide variety of monosaccharides. Initial velocity studies of the uptake of 2-deoxy-D-glucose were consistent with the presence of at least two carriers for this sugar in the Rhodotorula plasma membrane. Non-linear regression analysis of the data returned maximum velocities of 0.8 +/- 0.2 and 2.0 +/- 0.2 nmol/min per mg (wet weight) and Km values of 18 +/- 4 and 120 +/- 20 microM, respectively, for the two carriers. Kinetic studies of D-glucose transport also revealed two carriers with maximum velocities of 1.1 +/- 0.4 and 2.4 +/- 0.4 nmol/min per mg (wet weight) and Km values of 12 +/- 3 and 55 +/- 12 microM. As expected, 2-deoxy-D-glucose was a competitive inhibitor of D-glucose transport. Ki values for the inhibition were 16 +/- 8 and 110 +/- 40 microM. These Ki values were in good agreement with the Km values for 2-deoxy-D-glucose transport. D-Xylose, the 5-deoxymethyl analog of D-glucose, appears to utilize the D-glucose/2-deoxy-D-glucose carriers. This pentose was observed to be a competitive inhibitor of D-glucose (Ki values = 0.14 +/- 0.06 and 5.6 +/- 1.6 mM) and 2-deoxy-D-glucose (Ki values = 0.15 +/- 0.07 and 4.6 +/- 1.2 mM) transport.     

beta-Carotene production in sugarcane molasses by a Rhodotorula glutinis mutant

Several wild strains and mutants of Rhodotorula spp. were screened for growth, carotenoid production and the proportion of -carotene produced in sugarcane molasses. A better producer, Rhodotorula glutinis mutant 32, was optimized for carotenoid production with respect to total reducing sugar (TRS) concentration and pH. In shake flasks, when molasses was used as the sole nutrient medium with 40 g l⁻¹ TRS, at pH 6, the carotenoid yield was 14 mg l⁻¹ and -carotene accounted for 70% of the total carotenoids. In a 14-l stirred tank fermenter, a 20% increase in torulene content was observed in plain molasses medium. However, by addition of yeast extract, this effect was reversed and a 31% increase in -carotene content was observed. Dissolved oxygen (DO) stat fed-batch cultivation of mutant 32 in plain molasses medium yielded 71 and 185 mg l⁻¹ total carotenoids in double- and triple-strength medium, respectively. When supplemented with yeast extract, the yields were 97 and 183 mg l⁻¹ total carotenoid with a 30% increase in -carotene and a simultaneous 40% decrease in torulene proportion. Higher cell mass was also achieved by double- and triple-strength fed-batch fermentation. Journal of Industrial Microbiology & Biotechnology (2001) 26, 327–332. Received 18 September 2000/ Accepted in revised form 02 March 2001     

粘红酵母和酿酒酵母联合处理味精废水

为了解决味精废水中高NH4+浓度抑制油脂微生物的生长和油脂积累问题,采用粘红酵母和酿酒酵母联合处理味精废水的方法:首先利用酿酒酵母降解味精废水(MSG)中NH4+,然后将处理后的废水进一步发酵培养合成油脂。研究结果表明:用经酿酒酵母预处理过的味精废水作为粘红酵母的培养基发酵时,粘红酵母的生物量为33.3 g/L,油脂产率为18.16%,COD降解率为50.6%,NH4+的降解率为93.9%。比粘红酵母单独处理味精废水,NH4+的降解率提高了6.14倍,生物量、油脂产率和COD降解率分别提高了8.1%、30.06%和9.58%。     

Purification of an epoxide hydrolase from Rhodotorula glutinis

The epoxide hydrolase from Rhodotorula glutinis was isolated and initially characterized. The enzyme was membrane associated and could be solubilized by Triton X-100. Purification yielded an enzyme with sp. act. of 66 mol 1,2-epoxyhexane hydrolyzed min^–1 mg^–1 protein. The enzyme was not completely purified to homogeneity but, nevertheless, a major protein was isolated by SDS-PAGE for subsequential amino acid determination of peptide fragments. From sequence alignments to related enzymes, a high homology towards the active site sequences of other microsomal epoxide hydrolases was found. Molecular mass determinations indicated that the native enzyme exists as a homodimer, with a subunit molecular mass of about 45 kDa. Based upon these, this epoxide hydrolase is structurally related to other microsomal epoxide hydrolases.