Materials and Methods Strain construction and plasmids Aspergillus nidulans A4 was used as wild type reference strain. Aspergillus nidulans AR1 [pyrG89; argB2; veA1], IBT 27263, was used for the transformation. In order to over-express phk in A. nidulans AR1, the gene was cloned into an integrative vector derived from pBARGPE1 (M.L. Pall, J.P. Brunelli http://www.fgsc.net/fgn/pall. html), obtained from the FGSC, USA. For the construction of the plasmid, standard molecular biotechnology techniques were applied according to Sambrook et al. [19] and the final construct was verified by sequencing. A. nidulans phk (XP_662517) was amplified from genomic DNA with PCR using the Fphk_FW (59-CGAGT- CGGCCGGCCATGCCAGGAGAAGTCATCG-39) and Fphk_RV (59-TTGGCGCGCCCTAGCTCCAGTTTCCATC- TGC-39) primers, which introduced FseI and AscI restriction sites. The resulting strain was named AR1phkGP74 (over-expressing phk). Media Minimal medium (MM) for transformation: 10 g/L glucose; 10 mM NaNO3; 0.52 g/L KCL; 0.52 g/L MgSO4; 1.52 g/L KH2PO4; 4?1024 g/L CuSO4?5H2O; 4?1025 g/L Na2- B2O7?10H2O; 8?1024 g/L FeSO4?7H2O; 8?1024 g/L MnSO4?2H2O; 8?1024 g/L Na2MoO4?2H2O; 8?1024 g/L ZnSO4?7H2O. Arginine (4 mM) was supplemented in the MM used for the selection of transformants. For bioreactor cultivations a previously described defined medium was used [3]. Carbon sources used were glucose, xylose, glycerol and ethanol (20 g l21). Arginine, 0.7 g/L, was added to all bioreactor cultivations by sterile filtration. Transformation of A. nidulans Genetic transformation of A. nidulans protoplasts was performed as previously described [20], except that protoplasting was achieved by using the enzyme, Glucanex, (Novozymes A/S), at a concentration of 40 mg/ml in protoplasting buffer. PvuI was used for linearization of the plasmid as only one restriction site is present in the vector and which is located in the ampicillin resistance gene. Transformants were purified by streaking out spores to obtain single colonies on selective minimal medium and incubated at 37uC for 3–4 days. The resulting recombinants were further purified twice by streaking out spores on fresh plates with selective medium. Cultivation conditions and analysis of substrates and products To determine the physiological characteristics as well as for sampling for gene expression analysis, cultivations were performed in well-controlled 1.5 l bioreactors with a working volume of 1.2 l. The bioreactors were equipped with two disc-turbine impellers rotating at 350 rpm and sparged with air at a constant flow rate of 1.0 vvm (volume of gas per volume of liquid per minute). The pH was kept constant at 5.5 by addition of 2 M NaOH or HCl and the temperature was maintained at 30uC. For selected experi- ments, iodo acetate was added by sterile filtration to the sterilized growth medium giving a final concentration of 1 mM. Cell dry weight was determined using nitrocellulose filters (pore size 0.45 mm, Gelman Sciences). Fermentation samples were immediately filtered and stored at 220uC until analysis. The concentrations of glucose, xylose, glycerol, acetate, succinate, and pyruvate were determined by HPLC as described previously [3]. Systems Analysis in A nidulans Analysis of fractional 13C enrichments and computational methods For the 13C labelling experiments A. nidulans A4 and AR1phkGP74, respectively, were cultivated with 5 g/L of [1-13C] glucose and an aeration rate of 1.0 vvm. In the late exponential phase, biomass samples from the cultivations were harvested, hydrolyzed, derivatized and analyzed by gas GC-MS for determination of labelling patterns of intracellular metabolites. All fragments were derived from either glucose-6-phosphate or proteinogenic amino acids [21]. Calculation of drain fluxes for biomass formation and metabolic model. The biomass composition of A. nidulans was approximated by the biomass composition of A. oryzae (strain A1560) grown in glucose limited, continuous cultivations (specific growth rate of 0.17 h21) with ammonia as nitrogen source [22]. The simulation results were not sensitive to the biomass composition (simulation results not shown). The metabolic model of Pedersen et al [22] was used to calculate the precursor requirement for biomass formation. The 9 precursors were assumed to be a-ketoglutarate, acetyl-CoA, erythrose-4- phosphate, glucose-6-phosphate, glyceraldehyde-3-phosphate, mannose, oxaloacetate, pyruvate and ribose-5-phosphate. More details on the precursor calculations can be found in Supplementary Table S2. A modified version of the metabolic model of David et al [23] was used for simulation of the metabolic fluxes. In all cases, the reaction for phosphoketolase activity was included in the model (Supplementary Table S3). In the special case, where iodo acetate was added to the medium, the reaction representing glyceralde- hyde 3-phosphate dehydrogenase was removed and the reactions in the glyoxylate cycle were included (Supplementary Table S3). Computational method. The fluxes were determined by an in-house software based on the metabolic network analysis framework developed by Wiechert [24] implemented in MatLab 7.0 (Mathwork Inc., Natick, MA). The fluxes were found by least square minimisation with the non-linear Levenberg-Marquardt algorithm, where the discrepancies between measured and calculated fluxes as well as measured and calculated SFLs were minimised. Multiple initial guesses for the Levenberg-Marquardt algorithm were generated with a genetic algorithm in order to verify the existence of a global minimum and to address the significance of the simulated result. Selected metabolic fluxes are visualised in the Figure 1 while the complete list can be found in Supplementary Table S3. DNA microarrays Harvesting of mycelium and RNA extraction. The cultures were harvested in the late exponential phase at a biomass dry-weight concentration of 6–7 g/L with the exception of the cultivation on ethanol, where the biomass concentration was 3–4 g/L. Mycelium was harvested by filtration through sterile Mira-Cloth (Calbiochem). The mycelium was quickly dried by squeezing, and subsequently frozen in liquid nitrogen. Samples were stored at 280uC until they were used for RNA extraction. Total RNA was isolated using the Qiagen RNeasy Mini Kit, according to the protocol for isolation of total RNA from plant and fungi [11]. Microarray hybridization. Fifteen mg of fragmented biotin- labeled cRNA was prepared from 5 mg of total RNA and hybridized to the 3AspergDTU Affymetrix GeneChip [25] according to the Affymetrix GeneChip Expression Analysis Technical Manual [26]. Analysis of the transcriptome data. The scanned probe array images (.DAT files) were converted into CEL data files using PLoS ONE | www.plosone.org 6 December 2008 | Volume 3 | Issue 12 | e3847