Systems Analysis in A nidulans Figure 1. A) Principles of metabolic network analysis with [1-13C] glucose. The 13C labelling pattern of pyruvate is dependent on the active metabolic pathways. For instance, pyruvate formed in the EMP pathway contains 13C carbon in position 3, while activity of the PP pathway results in loss of labelled carbon. Pyruvate is the precursor of valine, which is incorporated into biomass. Based on the labelling pattern of valine and other proteinogenic amino acids the fluxes in the central carbon metabolism can be resolved. B) Major metabolic fluxes of the A. nidulans strains investigated. The two strains, A4 (WT) and AR1phkGP74 (PHK). IA indicates the presence of iodo acetate in the cultivation medium. All fluxes are relative to a glucose uptake rate of 100 moles (arbitrary number). doi:10.1371/journal.pone.0003847.g001 NADPH mainly is provided through the PP pathway, but there are alternative sources. Addition of iodo acetate to the medium had a dramatic impact on the fluxes for the strain over-expressing phk, and consequently a large deviation from the typical flux distribution was anticipated. Initial simulations demonstrated that the flux through glyceralde- hyde-3-phosphate dehydrogenase was negligible and could be omitted (results not shown). When glycolysis is blocked, acetyl- CoA must be formed exclusively through PHK, and consequently is a six-fold increase in the flux expected. The flux through the TCA cycle increased from 82 to 99 and the glyoxylate cycle was very active (Figure 1). As mentioned in previous section, growth ceased after consumption of half the carbon source, and consequently the specific growth rate and the biomass yield could not be determined accurately. However, a lower biomass yield was expected since inhibition of glyceraldehyde 3-phosphate dehydro- genase blocks the lower part of glycolysis and results in low ATP production and accumulation of glyceraldehyde-3-phosphate. In conclusion, with over-expression of phk it is possible to obtain quite high conversion yields of sugars to secondary metabolites originating from acetyl-CoA at the expense of a lower flux through glycolysis (either with chemical or genetic inhibition). Transcriptome analysis The strains A. nidulans A4 and AR1phkGP74 were grown on the four carbon (glucose, xylose, glycerol or ethanol) sources in triplicates. From each of the 24 cultivations samples were taken for transcriptome analysis. Statistical analysis of the data was done in order to identify genes that exhibited different expression between the two strains in the four conditions. Figure 2 reveals that surprisingly few genes shared regulation between the sources of carbon when phk was over-expressed. A total of 1,222 unique genes were affected by the up-regulation of phk. Only four genes were significant on all four carbon-sources (AN0158.3, AN2165.3, AN2555.3 and AN6798.3), and only 29 genes appeared to be significant between three carbon sources. Indeed, for all carbon sources more than 50% of the regulated genes are specific to that carbon source. On the glucose medium, this percentage is as high as 86%. It is thus apparent that the response to the over-expression of phk, is highly dependent on the carbon source used for growth with glucose having the highest number of responding genes. Analysis of the 1,222 regulated genes revealed several interesting facts on the response to the phk-over-expression. The phk-over-expressing strain had an increased specific growth rate and Ysx compared to the wild type during growth on xylose, PLoS ONE | www.plosone.org 3 December 2008 | Volume 3 | Issue 12 | e3847