Systems Analysis in A nidulans Figure 2. Venn diagram of genes with significant differential regulation in comparison of growth of A. nidulans A4 and phosphoketolase (PHK) over-expressing A. nidulans AR1phkGP74 on four different carbon sources. The over-expressed phosphoketolase, and argB, the transformation marker, are considered to be artefacts and were removed from the central group. Black numbers are the number of differentially regulated genes within each subgroup. Red numbers (below if present) are the number of putative transcription factors identified in that group. doi:10.1371/journal.pone.0003847.g002 glycerol and ethanol. For this reason, one might expect to see a common response between those three sets of experiments. However, these three comparisons only share significant regula- tion of two genes, both of them being putative dehydrogenases. No common response coordinating the increase in biomass on these carbon sources can therefore be elucidated based on transcriptome data. When examining significantly changed expression level of putative transcription factors (Figure 2), it is apparent that these are mostly found in the genes unique to the four carbon sources. This supports that the PHK-response is to a very high degree carbon-source specific. An examination of the expression indices of phk in the wild type strain on the four carbon sources showed that it is, indeed active, and mostly so on glucose and glycerol. Among the genes showing significant differential regulation only on glucose, 24 were transcription factors (22 of them putative) and nine genes were putatively involved in cell growth or cell cycle regulation. The two characterized transcription factors are BrlA, a transcriptional regulator involved in sporulation, inducing the cessation of vegetative growth [14], and CreA, a global carbon repressor [15]. This result suggests that the increased biomass- production on xylose, glycerol and ethanol is in fact not induced by regulation on these media, but rather that phk over-expression directly increases the specific growth rate and Ysx, and this effect is repressed on glucose by a complex array of regulatory factors. To further investigate the effect of over-expressing phk on metabolism, we examined differentially expressed metabolic genes (Supplementary Figure S1, S2, S3, S4). A pathway analysis of all significantly expressed genes (p,0.05) showed the metabolic response to be highly carbon-source dependent. On glycerol and ethanol, no differential regulation is found in the central metabolism (with the exception of one step in the GABA shunt on glycerol). Over-expression of phk on xylose shows regulation around acetaldehyde (connecting pyruvate and ethanol). On glucose, scattered response was found throughout metabolism, including glycolysis, TCA and C5 metabolism, but an especially strong response was found in all steps of the C-2 metabolism pathways connecting acetyl-CoA, acetate, acetaldehyde and ethanol, suggesting strong regulation of the levels of these metabolites (Figure 3). To validate the results statistically, and eliminate the possibility of simply seeing an effect of the higher number of regulated genes in the glucose comparison, we employed the statistical reporter metabolite algorithm [16,17] (Supplementary Table S4, S5, S6, S7). The algorithm identified acetaldehyde and ethanol to be highly significant nodes (among the ten most regulated metabolites, p,0.01) when grown on glucose (acetyl-CoA was not found as a significant reporter metabolite even though all reactions connecting it to the TCA were induced on glucose, since it is involved in a large number of fatty acid reactions with no significant regulation). Regulation around acetaldehyde and ethanol was only observed to a small extent in the gene expression data from growth on xylose medium (among the 60 most regulated metabolites, p,0.05), and not at all on glycerol (p.0.70) and ethanol medium (p.0.97). Since we do not observe increased fluxes around acetyl CoA in the labelling study (Figure 1B), we hypothesise the glucose repression response as having the purpose of homeostasis of acetyl CoA in spite of phk- over-expression. Discussion To the best of our knowledge, we provide the first evidence that the phosphoketolase pathway plays a significant role in the central carbon metabolism of filamentous fungi. The transcriptome analysis showed that phk was expressed in the wild type cultivations on all carbon sources and mostly so on glucose and glycerol. To determine the role of the phosphoketolase in A. nidulans cells, we integrated the gene into the genome under a strong constitutive promoter. Interestingly, this mutant grew with a higher specific PLoS ONE | www.plosone.org 4 December 2008 | Volume 3 | Issue 12 | e3847