Comment[ArrayExpressAccession] E-GEOD-48864 MAGE-TAB Version 1.1 Public Release Date 2013-07-15 Investigation Title Expression analysis of Aspergillus oryzae grown on different carbon sources Comment[Submitted Name] Expression analysis of Aspergillus oryzae grown on different carbon sources Experiment Description In this study, we focused on chemically defined inducers or substrates to drive expression of cellulases, hemicellulases and accessory enzymes in the model filamentous fungus Aspergillus oryzae. Cellohexaose (O-CHE), mannohexaose (O-MHE), xylopentaose (O-XPE), arabinoheptaose (O-AHP), 1,3:1,4-M-NM-2-glucohexaose (O-BGHEXA), 63-M-NM-1-D-glucosyl-maltotriosyl-maltotriose (O-GMH), 61-M-NM-1-D-galactosyl-mannotriose (O-GM3), xyloglucan (X3Glc4-borohydride reduced; O-X3G4R), turanose (TYR) and sophorose (SOP) were used to induce the plant polysaccharide degradation machinery of A. oryzae. The strain used in this study was the A. oryzae sequenced strain RIB40, obtained from IBT culture collection at Technical University of Denmark. To obtain a global view of the A. oryzae transcriptome activated for plant biomass conversion, mRNA from growth after 2 h on 10 different carbohydrate active enzyme inducers (di- and M-bM-^@M-^Soligo saccharides) was subjected to custom-designed Agilent microarray analysis. Term Source Name ArrayExpress EFO Term Source File http://www.ebi.ac.uk/arrayexpress/ http://www.ebi.ac.uk/efo/efo.owl Person Last Name Udatha Topakas Udatha Khodaie Salazar Olsson Andersen Smith Person First Name D.B.R.K. Gupta Evangelos D Melanie Margarita Lisbeth Mikael John Person Mid Initials G Z R A Person Email gupta.udatha@outlook.com Person Affiliation University of Gothenburg Person Address Department of Chemistry and Molecular Biology, University of Gothenburg, Medicinaregatan 9C, Gothenburg, Sweden, Sweden Person Roles submitter Protocol Name P-GSE48864-1 P-GSE48864-4 P-GSE48864-5 P-GSE48864-2 P-GSE48864-3 P-GSE48864-6 Protocol Description The scanned probe array images were converted into data files using the Agilent software. ID_REF = VALUE = normalized signal Standard Agilent protocol cRNA was quantified in a spectrophotometer. cRNA quality was assessed using a BioAnalyzer. A GeneChip Fluidics Station FS-400 (fluidics protocol FS450_001) and a GeneChip Scanner 3000 were used for hybridization and scanning. The composition of the batch cultivation medium was the following (in g L-1): 20 g glucose monohydrate, 2.0 g MgSO4.7H2O, 2 g K2SO4, 2.0 g KH2PO4, 5.0 g (NH4)2SO4, 0.5 ml L-1 pluronic acid (PE-6100) (BASF SE, Ludwigshafen, Germany) and 0.6 ml L-1 of trace elements solution. Trace elements solution composition (in g L-1): 14.3 g ZnSO4.7H2O, 8.5 g MnSO4.H2O, 13.8 g FeSO4.7H2O, 2.5 g CuSO4.5H2O, 3 g citric acid monohydrate (as a chelating agent) and 0.5 g NiCl2.6H2O. A. oryzae spore propagation medium (Cove-N-Gly) (in g L-1): 218 g sorbitol, 10 g glycerol 99.5%, 2.02 g KNO3, 25 g agar and 50 ml L-1 of salt solution. Cove-N-Gly salt solution (in g L-1): 26 g KCl, 26 g MgSO4.7H2O, 76 g KH2PO4 and 50 ml L-1 of trace elements solution. Cove-N-Gly trace elements solution (in mg L-1): 40 mg Na2B4O7.10H2O, 400 mg CuSO4.5H2O, 1,200 mg FeSO4.7H2O, 700 mg MnSO4.H2O, 800 mg Na2MoO4.2H2O and 10 g ZnSO4.7H2O. A. oryzae RIB40 batch fermentations were inoculated with spores propagated on Cove-N-Gly spore propagation medium plates incubated for 5 days at 30 C. To obtain enough mycelia with well dispersed filamentous growth grown under the same conditions to start enzyme induction with ten different protein inducing oligosaccharides in 250 mL shake flask cultivations, two consecutive batch cultivations were carried out. A. oryzae batch fermentations were performed in 3.6 L Infors bioreactors with a working volume of 2.8 L. Reactors were equipped with two Rushton four-blade disc turbines, no baffles, pH and temperature control. The temperature was maintained at 30 C and the pH was controlled by automatic addition of by addition of 10% H3PO4 or 10% NH3 solution. The pH was set to 3.5 to prevent spore aggregation and kept constant throughout the cultivation. The stirring speed was initially set at 200 rpm and the aeration rate to 0.1 vvm (volume of gas per volume of liquid per minute). After germination, these parameters were increased to 1000 rpm and 1 vvm and kept steady throughout all the rest of the fermentation. The batch cultivation was used to harvest biomass for consecutive induction by any of the different oligosaccharides at the final concentration listed in brackets: 1,3:1,4-M-NM-2-glucohexaose (66.67 mg L-1), Glc-Maltotriosyl-maltotriose (333.33 mg L-1), 1,4-M-NM-2-D-xylopentaose (66.67 mg L-1), 1,5-alpha-L-Arabinoheptaose (100 mg L-1), Galactosyl-mannotriose (133.33 mg L-1), sophorose (66.67 mg L-1), D-turanose (333.33 mg L-1), D-melibiose (333.33 mg L-1), 1,4-M-NM-2-D-mannohexaose (133.33 mg L-1), cellohexaose (200 mg L-1), xyloglucan reduced (X3GLC4) (333.33 mg L-1) and glucose (as control, 20 g L-1). Unless otherwise stated, the oligosaccharides used were obtained from Megazyme (Megazyme, International Ireland Ltd, Wicklow, Ireland); solely sophorose, D-melibiose and D-turanose were obtained from Sigma (Sigma-Aldrich Inc., St. Louis, MO, United States). Oligosaccharides were very well dissolved in water, filtered sterilized and added to each of the three shake flask replicates. Mycelium was harvested after ~32 h cultivation in mid-exponential phase. Mycelium was washed with sterile mineral media without carbon source, squeezed and weighed under sterile conditions. 800 mg of wet weighed mycelia were used for inoculation of each shake flask cultivation. For gene expression analysis, mycelium was harvested after 2 h cultivation with the respective inducer from each of the three biological replicates. The cultures were filtered through sterile Miracloth (Calbiochem, San Diego, CA, USA) and washed with a suitable amount of 0.9 % NaCl solution. The mycelium was quickly dried by squeezing and subsequently frozen in liquid nitrogen. Samples were stored at -80M-0C until RNA extraction. A. oryzae total RNA was extracted using the Qiagen RNeasy Mini Kit (QIAGEN Nordic, Ballerup, Denmark), according to the protocol for isolation of total RNA from plant and fungi. For the purpose, approximately 500 mg of frozen mycelium was ground to powder using a ceramic mortar and pestle. Mycelium was kept in liquid nitrogen throughout the grinding processing. All samples were inspected for good quality of total RNA extracted with a BioAnalyzer (2100 BioAnalyzer, Agilent Technologies Inc., Santa Clara, CA, USA). RNA quantification was performed in a spectrophotometer (Biophotometer 6131, Eppendorf AG, Hamburg, Germany) and total RNA was stored at -80 oC until further processing. Arrays were washed and stained using a GeneChip Fluidics Station FS-400, and scanned on an Agilent GeneArray Scanner 3000. Protocol Type normalization data transformation protocol labelling protocol hybridization protocol growth protocol nucleic acid extraction protocol array scanning protocol Experimental Factor Name SUBSTRATE DESCRIPTION GROWN ON SUBSTRATE Experimental Factor Type substrate description grown on substrate Comment[SecondaryAccession] GSE48864 Comment[GEOReleaseDate] 2013-07-15 Comment[ArrayExpressSubmissionDate] 2013-07-15 Comment[GEOLastUpdateDate] 2013-07-15 Comment[AEExperimentType] transcription profiling by array Comment[AdditionalFile:Data1] GSE48864_ctrl_vs_I1.csv Comment[AdditionalFile:Data2] GSE48864_ctrl_vs_I10.csv Comment[AdditionalFile:Data3] GSE48864_ctrl_vs_I11.csv Comment[AdditionalFile:Data4] GSE48864_ctrl_vs_I2.csv Comment[AdditionalFile:Data5] GSE48864_ctrl_vs_I3.csv Comment[AdditionalFile:Data6] GSE48864_ctrl_vs_I4.csv Comment[AdditionalFile:Data7] GSE48864_ctrl_vs_I5.csv Comment[AdditionalFile:Data8] GSE48864_ctrl_vs_I6.csv Comment[AdditionalFile:Data9] GSE48864_ctrl_vs_I7.csv Comment[AdditionalFile:Data10] GSE48864_ctrl_vs_I9.csv SDRF File E-GEOD-48864.sdrf.txt