The diethyl sulfate (DES) mutagenesis was chosen for the mutagenic treatment to Phellinus igniarius, and the relationship of mutagenesis time and death rate was investigated with 0.5% DES. The differences of mycelial growth speed, liquid fermentation mycelia biomass, morphology and pigment classes of secondary metabolites production speed and antioxidant activities of metabolite products were discussed. The study displayed that DES mutagenesis could change mycelial morphology without obvious effect on mycelium growth, and the DES mutagenesis improved antioxidant activities of the active ingredients of P. igniarius and had more antioxidant activity of hypoxia/sugar PC12 nerve cells than that of P. igniarius.
Basidiomycota ; drug effects ; genetics ; growth & development ; metabolism ; Mutagenesis ; Mutagens ; pharmacology ; Mycelium ; drug effects ; genetics ; growth & development ; metabolism ; Pigments, Biological ; analysis ; metabolism ; Secondary Metabolism ; drug effects ; Sulfuric Acid Esters ; pharmacology

Fatty acid synthase (FAS) catalyses the reaction between acetyl-CoA and malonyl-CoA to produce fatty acids. It is one of the most important enzyme in lipid biosynthesis. FAS of the oleaginous yeast Rhodosporidium toruloides has two acyl carrier protein (ACP) domains and a distinct subunit composition compared with FASs of other species. As ACP is a protein cofactor crucial for fatty acid chain elongation, more ACPs in the FAS may facilitate the reaction. To study the biochemical and structural properties of this novel FAS from R. toruloides, plasmids were constructed and transformed into Escherichia coli BL21 (DE3). The strain ZWE06 harboring plasmids pET22b-FAS1 and pET24b-FAS2 could co-overexpress the two subunits. The recombinant FAS was purified by sequentially using ammonium sulphate precipitation, sucrose density gradient centrifugation and anion exchange chromatography. The specific activity of the recombinant FAS was 548 mU/mg. The purified complex would be used to study enzyme kinetics and protein structure of FAS, and heterogeneous expression and purification will facilitate revealing the mechanism of this novel FAS with double ACPs.
Acyl Carrier Protein ; Basidiomycota ; enzymology ; Chromatography ; Escherichia coli ; metabolism ; Fatty Acid Synthases ; biosynthesis ; genetics ; Fatty Acids ; biosynthesis ; Plasmids ; Recombinant Proteins ; biosynthesis ; genetics

β-carotene has a wide range of application in food, pharmaceutical and cosmetic industries. For microbial production of β-carotene in Saccharomyces cerevisiae, the supply of geranylgeranyl diphosphate (GGPP) was firstly increased in S. cerevisiae BY4742 to obtain strain BY4742-T2 through over-expressing truncated 3-hydroxy-3-methylglutaryl-CoA reductase (tHMGR), which is the major rate-limiting enzyme in the mevalonate (MVA) pathway, and GGPP synthase (GGPS), which is a key enzyme in the diterpenoid synthetic pathway. The β-carotene synthetic genes of Pantoea agglomerans and Xanthophyllomyces dendrorhous were further integrated into strain BY4742-T2 for comparing β-carotene production. Over-expression of tHMGR and GGPS genes led to 26.0-fold increase of β-carotene production. In addition, genes from X. dendrorhous was more efficient than those from P. agglomerans for β-carotene production in S. cerevisiae. Strain BW02 was obtained which produced 1.56 mg/g (dry cell weight) β-carotene, which could be used further for constructing cell factories for β-carotene production.
Basidiomycota ; enzymology ; Farnesyltranstransferase ; genetics ; metabolism ; Hydroxymethylglutaryl CoA Reductases ; genetics ; metabolism ; Metabolic Engineering ; Polyisoprenyl Phosphates ; Saccharomyces cerevisiae ; metabolism ; beta Carotene ; biosynthesis

To evaluate the effectiveness of enzymatic assisted extraction (EAE) of lipid from the oleaginous yeast Rhodosporidium toruloides in the presence of beta-1,3-glucomannanase at a larger scale, we investigated the effects of enzymatic treatment and extraction conditions on lipid extraction yields at 10-L scale by using the broth of R. toruloides Y4 as the feed and ethyl acetate as the solvent. When it was treated for 0.5 h, the lipid extraction yield reached 71.1%, indicating that the enzymatic treatment process reached similar efficiency to that obtained at 10-mL scale. The inhibitory effect of emulsification was greatly reduced by repeated extraction. After extracted for three times, yields of lipid extraction, solvent recovery and total material recovery reached 92.9%, 87.0% and 94.2% respectively. As it can use the lipid production slurry with good extraction efficiency, EAE technology is promising for industrial production of microbial lipids.
Basidiomycota ; metabolism ; Biofuels ; Bioreactors ; Fermentation ; Industrial Microbiology ; Lipids ; biosynthesis ; isolation & purification ; beta-Mannosidase ; metabolism

OBJECTIVETo study the mechanism of plant growing promoted by Mycorrhizal fungus through the difference of proteomes.

METHODThe differential proteomes between uninoculated and inoculated endophytic fungi, Epulorhiza sp. on Anoectochilus roxburghii were analyzed by two-dimensional gel electrophoresis and MALDI-TOF/TOF mass spectrum.

RESULT AND CONCLUSIONTwenty-seven protein spots were analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Twenty-two candidate proteins were identified by database comparisons. The function of these proteins mostly involved in signal transduction, metabolic regulation, as well as photosynthesis and substance metabolism. The results indicate that the regulator control system of plant is influenced by fungi action, and the positive regulation improves substance metabolism and photosynthesis, which results in strong plant and higher resistance. It is also deduced that silent genes may exist in endosymbiosis plants.

Basidiomycota ; physiology ; Electrophoresis, Gel, Two-Dimensional ; Host-Pathogen Interactions ; Mycorrhizae ; physiology ; Orchidaceae ; growth & development ; metabolism ; microbiology ; Plant Proteins ; analysis ; Proteome ; analysis ; Proteomics ; methods ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

The objective of this work is to investigate how dilution rate and carbon-to-nitrogen (C/N) ratio affects lipid accumulation by Rhodosporidium toruloides AS 2.138 9 in continuous culture. Under steady-state conditions, the increase in dilution rate led to the decrease in lipid content and lipid yield. The highest lipid yield and lipid content at D = 0.02 h(-1) were 0.18 g lipid/g sugar and 57.1%, respectively, while the highest lipid productivity and biomass productivity were obtained at D = 0.14 h(-1). The increase in C/N ratio led to the increase in lipid content. The highest lipid content of 38% was obtained at C/N = 237. The highest lipid yield of 0.12 g lipid/g sugar was obtained at C/N = 92. However, the highest lipid productivity of 0.12 g/(L x h) was obtained at C/N = 32. No significant changes were observed in terms of fatty acid composition of the lipid produced under different C/N ratios, and these three fatty acids, palmitic acid, stearic acid and oleic acid, took over 85% in all samples.
Basidiomycota ; growth & development ; metabolism ; Batch Cell Culture Techniques ; Carbon ; metabolism ; Culture Media ; Fatty Acids ; metabolism ; Glucose ; metabolism ; Lipids ; biosynthesis ; Nitrogen ; metabolism ; Oleic Acid ; biosynthesis ; Palmitic Acid ; metabolism

OBJECTIVETo investigate the cytotoxic activity of endophytic fungi isolated from mangrove fungi.

METHODSIn the present study the DNA was isolated and the ITS region of 5.8s rRNA was amplified using specific primers ITS 1 and ITS4 and sequence was determined using automated sequencers. Blast search sequence similarity was found against the existing non redundant nucleotide sequence database thus, identified as Aspergilus flavus, Hyporcaea lixii, Aspergillus niger, Eutorium amstelodami, Irpex hydnoides and Neurospora crassa. Among the seven isolates, one fungi Irpex hydnoides was selected for further studies. The fungi were grown in sabouraud broth for five days and filtrate were separated and subjected to ethyl acetate for further studies.

RESULTSNearly half (49.25%) of the extracts showed activity (IC50 of 125µg/mL). These values were within the cutoff point of the National Cancer Institute criteria for cytotoxicity (IC50<20 µg/mL) in the screening of crude plant extracts. The GC MS analysis revealed that the active principals might be Tetradecane (6.26%) with the RT 8.606.

CONCLUSIONSIt is clear from the present study that mangrove fungi with bioactive metabolites can be expected to provide high quality biological material for high throughout biochemical, anti cancer screening programmes. The results help us conclude that the potential of using metabolic engineering and post genomic approaches to isolate more novel bioactive compounds and to make their possible commercial application is not far off.

Basidiomycota ; chemistry ; classification ; genetics ; metabolism ; Biological Products ; chemistry ; toxicity ; Cell Survival ; drug effects ; DNA, Ribosomal Spacer ; Gas Chromatography-Mass Spectrometry ; Hep G2 Cells ; Humans ; Verbenaceae ; microbiology

We evaluated an astaxanthin overproducing Phaffia rhodozyma JMU-MVP14, and developed astaxanthin high-yielding fermentation process. We analyzed several fermentation parameters, i.e., biomass, astaxanthin and total carotenoids content to compare the characteristics of P rhodozyma JMU-MVP14 and the original strain through flask fermentation experiments. We conducted batch and fed-batch fermentation experiments in 7 L fermentor to investigate the effects of pH controlling models and feeding medium compositions on the production of astaxanthin. We further evaluated the capability and practical value of P rhodozyma JMU-MVP14 by fed-batch cultivation in the 1 m3 fermentor. Flask fermentation experiments revealed that P. rhodozyma JMU-MVP14 produced high yield of astaxanthin and carotenoids with specific productivity of astaxanthin and specific productivity of total carotenoids of 6.01 mg/g and 10.38 mg/g. Results of batch culture experiments in the 7 L fermentor showed that controlling the pH by ammonia auto-feeding was better than discontinuously adjusting pH value at 6.0 with regard to the high productivities of biomasses and astaxanthin. This P. rhodozyma strain synthesized astaxanthin partially linked to the growth with the Ks and pmax of 0.20 h ' and 21.73 g/L, respectively. Results of batch-fed fermentations in 7 L fermentor indicated that the complex feeding medium consisted of 50% glucose, 0.5% yeast extract and 0.3% corn steep syrup had lower astaxanthin productivity than the simple feeding medium containing only 50% glucose, which produced biomass, volumetric productivity of astaxanthin, volumetric productivity of total carotenoids, specific productivity of astaxanthin and total carotenoids at 32.81 g/L, 155.99 mg/L, 4.94 mg/g, 399.99 mg/L and 12.19 mg/g, respectively. As fed-batch cultured in 1 m3 fermentor, P rhodozyma JMU-MVP14 yielded 85.11 g/L of biomass, 279.96 mg/L of volumetric productivity of astaxanthin, 618.01 mg/L of volumetric productivity of total carotenoids, 3.29 mg/g of specific productivity of astaxanthin and 7.26 mg/g of specific productivity of total carotenoids. Additionally, P rhodozyma JMU-MVP14 cell contained 21.54% of protein, 41.34% of carbohydrate and 34.31% of lipid. These comprehensive results suggest that P. rhodozyma JMU-MVPl14 has great practical prosperity related to its strong ability to produce astaxanthin and good value byproducts.
Basidiomycota ; genetics ; growth & development ; metabolism ; Batch Cell Culture Techniques ; Carotenoids ; biosynthesis ; Culture Media ; Fermentation ; Hydrogen-Ion Concentration ; Industrial Microbiology ; Kinetics ; Xanthophylls ; biosynthesis

A comparative study of batch and repeated batch process was carried out for astaxanthin fermentation of Phaffia rhodozyma to develop a more economical method for astaxanthin industrial production. In shaking flask fermentation, the change of biomass and astaxanthin production was studied to compare the five-day cycle with four-day cycle of repeated batch culture of P. rhodozyma. Astaxanthin production increased at first and then decreased subsequently in seven cycles, yet the yield of astaxanthin in the next six cycles remains higher than that of the first cycle. Comparing the average production of astaxanthin in the seven cycles, four-day cycle performed even better than five-day cycle. Subsequently, a repeated fed-batch process was used in a 5-1 bioreactor. The experimental data showed that biomass and astaxanthin production of the second batch could reach the level of the first batch, no matter that the carbon source was glucose or hydrolysis sugar of starch. This result showed that this strain had good stability, and thus repeated batch and fed-batch process could be applied in astaxanthin fermentation for economical purpose.
Basidiomycota ; genetics ; metabolism ; Batch Cell Culture Techniques ; methods ; Bioreactors ; microbiology ; Fermentation ; Industrial Microbiology ; methods ; Xanthophylls ; biosynthesis

OBJECTIVETo observe the proliferation inhibition, apoptosis, and cell proliferation cycle of human lung carcinoma cell line A549 treated with Inotodiol extracts from Inonotus obliquus and explore the possibility of Inotodiol extracts from Inonotus obliquus as a new tumor chemopreventive drug.

METHODSHuman lung cancer cell line A549 was treated with different concentrations of Inotodiol, the effects of Inotodiol on cell apoptosis, the expression of Ki-67, Bcl-2, Bax, and p53 and cell cycle were detected by TUNEL assay, immunohistochemistry, and flow cytometry assay respectively.

RESULTSInotodiol extracts had antiproliferation effect on human lung carcinoma cell line A549. The expression of Ki-67 decreased with the increase of Inotodiol concentration and exposure time (P<0.05), in a dose-dependent and time-dependent manner. The typical characteristics of the apoptosis of A549 cells treated with Inotodiol were observed, and the apoptotic rate of A549 cell at 48 h was the highest by TUNEL assay. Inotodiol arrested A549 cells in the S phase, and apoptotic peak was observed by flow cytometry. Immunocytochemistry indicated that the expression of Bcl-2 protein decreased, while the expression of p53 and Bax proteins increased in A549 cells treated with Inotodiol, compared with the control cells (P<0.05).

CONCLUSIONInotodiol can inhibit proliferation and induce the apoptosis of A549 cells, and its molecular mechanism may be associated with the up-regulating expression of p53 and bax proteins and down-regulating expression of Bcl-2 protein, which arrested A549 cells in S phase.

Adenocarcinoma ; drug therapy ; genetics ; metabolism ; pathology ; Apoptosis ; drug effects ; genetics ; Basidiomycota ; chemistry ; Cell Cycle ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Drug Evaluation, Preclinical ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Gene Expression Regulation, Neoplastic ; drug effects ; Genes, bcl-2 ; drug effects ; Genes, p53 ; drug effects ; Humans ; Ki-67 Antigen ; metabolism ; Lanosterol ; analogs & derivatives ; pharmacology ; therapeutic use ; Lung Neoplasms ; drug therapy ; genetics ; metabolism ; pathology ; Phytotherapy ; bcl-2-Associated X Protein ; genetics