High-level expression of Rhodotorula gracilis d-amino acid oxidase in Pichia pastoris

By combining gene design and heterologous over-expression of Rhodotorula gracilis D-amino acid oxidase (RgDAO) in Pichia pastoris, enzyme production was enhanced by one order of magnitude compared to literature benchmarks, giving 350 kUnits/l of fed-batch bioreactor culture with a productivity of 3.1 kUnits/l h. P. pastoris cells permeabilized by freeze-drying and incubation in 2-propanol (10% v/v) produce a highly active (1.6 kUnits/g dry matter) and stable oxidase preparation. Critical bottlenecks in the development of an RgDAO catalyst for industrial applications have been eliminated.

CTAB, Triton X-100 and freezing-thawing treatments of Candida guilliermondii: Effects on permeability and accessibility of the glucose-6-phosphate dehydrogenase, xylose reductase and xylitol dehydrogenase enzymes

Cells of Candida guilliermondii (ATCC 201935) were permeabilised with surfactant treatment (CTAB or Triton X-100) or a freezing-thawing procedure. Treatments were monitored by in situ activities of the key enzymes involved in xylose metabolism, that is, glucose-6-phosphate dehydrogenase (G6PD), xylose reductase (XR) and xylitol dehydrogenase (XD). The permeabilising ability of the surfactants was dependent on its concentration and incubation time. The optimum operation conditions for the permeabilisation of C. guilliermondii with surfactants were 0.41 mM (CTAB) or 2.78 mM (Triton X-100), 30 degrees C, and pH 7 at 200 rpm for 50 min. The maximum permeabilisation measured in terms of the in situ G6PD activity observed was, in order, as follows: CTAB (122.4 +/- 15.7 U/g(cells)) > freezing-thawing, , (54.3 +/- 1.9 U/g(cells)) > Triton X-100 (23.5 +/- 0.0 U/g(cells)). These results suggest that CTAB surfactant is more effective in the permeabilisation of C. guilliermondii cells in comparison to the freezing-thawing and Triton X-100 treatments. Nevertheless, freezing-thawing was the only treatment that allowed measurable in situ XR activity. Therefore, freezing-thawing permeabilised yeast cells could be used as a source of xylose reductase for analytical purposes or for use in biotransformation process such as xylitol preparation from xylose. The level of in situ xylose reductase was found to be 13.2 +/- 0.1 U/g(cells).

A soluble diacylglycerol acyltransferase is involved in triacylglycerol biosynthesis in the oleaginous yeast Rhodotorula glutinis

The biosynthesis of triacylglycerol (TAG) occurs in the microsomal membranes of eukaryotes. Here, we report the identification and functional characterization of diacylglycerol acyltransferase (DGAT), a member of the 10 S cytosolic TAG biosynthetic complex (TBC) in Rhodotorula glutinis. Both a full-length and an N-terminally truncated cDNA clone of a single gene were isolated from R. glutinis. The DGAT activity of the protein encoded by RgDGAT was confirmed in vivo by the heterologous expression of cDNA in a Saccharomyces cerevisiae quadruple mutant (H1246) that is defective in TAG synthesis. RgDGAT overexpression in yeast was found to be capable of acylating diacylglycerol (DAG) in an acyl-CoA-dependent manner. Quadruple mutant yeast cells exhibit growth defects in the presence of oleic acid, but wild-type yeast cells do not. In an in vivo fatty acid supplementation experiment, RgDGAT expression rescued quadruple mutant growth in an oleate-containing medium. We describe a soluble acyl-CoA-dependent DAG acyltransferase from R. glutinis that belongs to the DGAT3 class of enzymes. The study highlights the importance of an alternative TAG biosynthetic pathway in oleaginous yeasts.

On the population dynamics of Eudiaptomus gracilis Sars and Heterocope borealis Fischer in the Bodensee-Obersee. Part B. Eudiaptomus gracilis Sars. [Translation from: Trudy Instituta Biologii Vnutrennykh Vodnany 12(15) 170-174, 1966.]

Experimental research was conducted to study the development of eggs of Eudiaptomus gracilis Sars. The egg production was studied as well as the population dynamics. Factors like losses in the lake and through the effluent Rhine at Konstanz were considered.

One new flavanoid and Anti-HIV active constituents from boussingaultia gracilis miers var. pseudobaselloides bailey

Boussingaultia gracilis Miers var. pseudobaselloides Bailey (Basellaccae) is a folk medicine used as an analgesic and supplements, only a few research was reported on the chemical constituents of this plant. This paper presented its chemical and anti-HIV

纤细眼虫( Euglena gracilis)核纤层蛋白基因的初步研究

　目的:探讨在纤细眼虫( Euglena gracilis) 中是否存在核纤层蛋白(lamin) 基因,为核纤层 蛋白基因的起源与进化研究提供线索. 方法:以较为低等生物的核纤层蛋白基因cDNA 序列为参 考,设计引物P0010 和P0012 ,以纤细眼虫总DNA 为模板进行PCR ,PCR 产物回收、测序. 由于不能 获取完整序列,所以据测序结果又设计了P0013 和P0014 两条中间引物,组成P0010/ P0013 和 P0014/ P0012 引物对进行PCR ,回收产物测序. 结果:P0010/ P0012、P0010/ P0013 和P0014/ P0012 引物 对进行PCR 分别获得775、400 和395 bp 的特异性PCR 产物,对这3 种产物分别测序,最终获得了 P0010 和P0012 之间的全序列,共775 bp. 结论:在纤细眼虫中存在着核纤层蛋白基因.

The nuclear matrix of Euglena gracilis (Euglenophyta): A stage of nuclear matrix evolution?

Euglena gracilis cell was extracted sequentially with CSK-Triton buffer, RSB-Magik solution and DNase-As solution. DGD embedment-free electron microscopy showed that in the extracted nucleus there was a residual non-chromatin fibrous network. That it could not be removed by hot trichloroacetic acid further supported the idea that it was a non-histone, non-chromatin fibrous protein network, and should be the internal network of the nuclear matrix. After the sequential extraction, the nuclear membrane was removed, leaving behind a layer of lamina; the chromatin was digested and eluted from the dense chromosomes and residual chromosomal structures that should be chromosomal scaffold were revealed. Western blot analysis with antiserum against rat lamins showed that nuclear lamina of the cell possessed two positive polypeptides, a major one and a minor one, which had molecular masses similar to lamin B and lamin A, respectively. Comparing these data with those of the most primitive eukaryote Archezoa and of higher eukaryotes, it was suggested that the lower unicellular eukaryote E. gracillis already had the nuclear matrix structure, and its nuclear matrix (especially the lamina) might represent a stage of evolutionary history of the nuclear matrix. (C) 2000 Editions scientifiques et medicales Elsevier SAS.

Identification, cloning, and characterization of microsatellite DNA in Euglena gracilis

Microsatellite DNA has been developed into one of the most popular genetic markers. We have identified and cloned microsatellite loci in the genome of a free-living protozoan Euglena gracilis FACHB-848, using the random amplified microsatellites method (RAMS). The digoxigenin-labelled oligonucleotides(CT)(10) and (GT)(10) served as probes to detect complementary sequences in the randomly amplified polymorphic DNA (RAPD) fingerprints produced by means of Southern blotting. Subsequently, positive RAPD fragments were cloned. From a total of 31 RAPD primer profiles, eight microsatellite loci of E. gracilis were detected and characterized. Further, six sites (i.e. EGMS1, EGMS3, EGMS4, EGMS5, EGMS6, and EGMS7) showed polymorphisms. We found a GT or CT microsatellite every 10.5 kb in the genome of E. gracilis, and similar to animal genomes, the (GT)(n) motif was much more abundant than the (CT)(n) motif. These polymorphic microsatellite DNA will serve as advantageous molecular markers for studying the genetic diversity and molecular ecology of Euglena.

Residual plastids of bleached mutants of Euglena gracilis and their effects on the expression of nucleus-encoded genes

Bleached mutants of Euglena gracilis were obtained by treatment with ofloxacin (Ofl) and streptomycin (Sm) respectively. As shown by electron microscopy, the residual plastids contain prothylakoids in an Ofl mutant, and the highly developed and tightly stacked membranous structure found in cells of two Sm, mutants. Nine genes of the plastid genome were examined with PCR, showing that ribosomal protein genes and most other plastid genes were lost in all but one Sm mutant. Using differential display and RT-PCR, it was shown that chloroplast degeneration could cause changes in transcription of certain nucleus-encoded genes during heterotrophic growth in darkness.

Biosorption of uranium by chemically modified Rhodotorula glutinis

The present paper reports the biosorption of uranium onto chemically modified yeast cells, Rhodotorula glutinis, in order to study the role played by various functional groups in the cell wall. Esterification of the carboxyl groups and methylation of the amino groups present in the cells were carried out by methanol and formaldehyde treatment, respectively. The uranium sorption capacity increased 31% for the methanol-treated biomass and 11% for the formaldehyde-treated biomass at an initial uranium concentration of 140 mg/L The enhancement of uranium sorption capacity was investigated by Fourier transform infrared (FTIR) spectroscopy analysis, with amino and carboxyl groups were determined to be the important functional groups involved in uranium binding. The biosorption isotherms of uranium onto the raw and chemically modified biomass were also investigated with varying uranium concentrations. Langmuir and Freundlich models were well able to explain the sorption equilibrium data with satisfactory correlation coefficients higher than 0.9. (C) 2010 Elsevier Ltd. All rights reserved.

Metabolic studies on the nitrophile yeast Rhodotorula glutinis DSBCA06

Nitrogen is the most abundant element in atmosphere and fundamental component of proteins, nucleic acids and other essential molecules. In the past century the industrial use of nitrogen compounds has grown exponentially causing widespread pollution. Nitrogen pollution has wide-ranging impacts including contributions to global warming, acid rains and eutrophication. Reduction of nitrogen use in industry and agriculture coupled whit remediation treatments could represent a solution. To this purpose we isolated from environmental samples a nitrophile strain capable of removing nitrogen compounds efficiently from the medium. Through the molecular characterization, we identified the strain as a Rhodotorula glutinis that we called DSBCA06. We examined the main metabolic features of the strain, also to determine the best growing conditions. At the same time, the ability of the strain to grow in presence of high nitrite concentrations was assayed, being a relevant feature poorly studied earlierfor other environmental yeasts. The ability of the strain to grow in presence of heavy metal cations was also tested, showing a noticeable tolerance. The cost of bioremediation treatments is often a problem. One of the way to obviate this is to produce valuable secondary metabolites, capable of positively impact the cost of the processes. In this context the ability of the strain to produce carotenoids, natural molecules with antioxidant properties used for food production, cosmetic and pharmaceutical industry, has been evaluated. The strain Rhodotorula glutinis DSBCA06 showed interesting features suggesting its possible use in bioremediation or industrials process for production of secondary metabolites such as lipids and carotenoids.