cation of metabolic fluxes is one of the most direct measures on operation of metabolic networks, and it is especially valuable in connection with studies of metabolite production where the aim is to direct carbon from the substrate into the metabolic product, e.g. secondary metabolites [12]. Results Construction of the recombinant strain over-expressing phk An A. nidulans mutant, which constitutively over-expresses phk (AR1phkGP74), was constructed. The vector that contained the corresponding gene harboured the strong constitutive glycolytic promoter from glyceraldehyde-3-phosphate dehydrogenase (gpdA) of A. nidulans. Northern blot analysis clearly indicated a strong over-expression of phk in the transformant compared to the reference strain (data not shown). To further verify that the over- expressed gene actually is coding for PHK, the wild type (A4) and the mutant (AR1phkGP74) strains were cultivated on glucose in the presence of 1 mM of iodo acetate, a specific and strong inhibitor of glyceraldehyde-3-phosphate dehydrogenase [13]. While iodo acetate prohibited growth of the A4 cells (specific growth rate of approximately 0.02 h21) it had only a minor effect on the specific growth rate of the AR1phkGP74 mutant (specific growth rate of 0.19 h21). However, growth of the AR1phkGP74 mutant ceased after consumption of half the carbon source, probably due to accumulation of glyceraldehyde-3 phosphate or drain of intracel- lular free phosphate. Functional significance of phosphoketolase in A. nidulans A set of experiments was carried out to investigate the metabolism of the A. nidulans wild type (A4) and the A. nidulans AR1phkGP74 strains on various carbon sources (glucose, xylose, glycerol and ethanol). Substrate and product concentrations in the medium were determined for all cultivations as well as the maximum specific growth rate and the biomass yield on carbon source during the fully aerobic growth phase (Table 1). When glucose was used as carbon source, the A4 strain and the AR1phkGP74 strain had similar maximum specific growth rates and biomass yields on glucose (Ysx). In contrast, over-expression of phk significantly increased the specific growth rate and Ysx to a lesser extent when the cells were cultivated on xylose and glycerol. In the cultivations on ethanol, over- expression of phk surprisingly led to a significant increase in both the specific growth rate and Ysx. These results show that over- expression of phk significantly improves growth performance on all substrates except from glucose. Growth performance was not expected to be improved on the C-2 compound ethanol since acetyl- Table 1. Specific growth rates and biomass yields of A. nidulans A4 (wild type) and AR1phkGP74 (mutant) cultivated in four different carbon sources. Aspergillus nidulans A4/AR1phkGP74 Aspergillus nidulans A4/AR1phkGP74 Ysx (g g21) 0.47/0.48 0.46/0.47 0.42/0.44 0.23/0.35 Systems Analysis in A nidulans CoA is formed as an intermediate during assimilation without the use of phosphoketolase. In vivo characterization using metabolic network analysis The impact of phosphoketolase in glucose cultivations was further studied with 13C labelling experiments in three conditions: strains A4, AR1phkGP74, and AR1phkGP74 in the presence of the glyceraldehyde-3-phosphate dehydrogenase inhibitor iodo acetate (1 mM). The mass spectra obtained from the GC-MS analysis of the hydrolysed biomass were converted into summed fractional labellings (SFL) (Supplementary Table S1). The SFL of a molecule or a fragment hereof reflects the enrichment of 13C, i.e. the enrichment of 13C in each carbon atom in a given fragment. For instance, if a fragment contains three carbon atoms which each contain 20% 13C and 80% 12C then the corresponding SFL is 60% (Figure 1A). It can be observed that addition of iodo acetate results in a significantly lower enrichment of 13C in the fragments. This observation can be explained by a higher flux through the PP pathway, which results in a direct and significant loss of 13C labelled carbon dioxide and formation of 2 moles NADPH. Consequently, the remaining fragments will not be enriched by 13C to the same degree. When iodo acetate is added and blocks the lower part of the Embden-Meyerhoff-Parnas (EMP) pathway, an efficient PHK and glyoxylate cycle are required for growth. All metabolites in the lower part of the EMP pathway and the TCA cycle must be formed from acetyl-CoA, and consequently the glyoxylate cycle is needed to form C-4 compounds from acetyl-CoA. Using the SFL data, we estimated the fluxes in the central carbon metabolism by considering a simple non-compartmental- ised metabolic model of A. nidulans with the EMP pathway, the PP pathway and the TCA cycle. We also estimated the fluxes with a more detailed metabolic model which included compartmental- isation of acetyl-CoA, pyruvate and oxaloacetate. However, the SFL data were only slightly better fitted to the larger model. In order to avoid over-fitting of the limited number of data points, we chose the simpler but more robust version of the two models (Supplementary Table S2, S3). The measured and simulated SFLs are listed in the Suppl. Table. S1, while major fluxes are depicted in Figure 1B. In general, a good fit between measured and calculated SFL, were found though the fragments Asp188 and Thr175 had a higher deviation (,15%) between measured and simulated fragments in all experiments. This discrepancy may be explained by the simplicity of the model since oxaloacetate, the precursor of Asp188 and Thr175, is not compartmentalised. However, the value of these fragments only had minor impact on the calculated flux distribution (simulation results not shown). The simulation results showed that the wild type strain (A4) had a relative flux into the oxidative branch of the PP pathway of 56.9 compared to 44.4 when phk was over-expressed (Figure 1). Despite a reduction of the flux into the oxidative branch of the PP pathway, the flux through PHK remained largely constant (29.2 vs. 32.8 when phk was over-expressed). Since the specific growth rate, as well as the biomass yield on glucose, was similar for the two strains, there must be an alternative source of NADPH production in the strain with over-expressed phk (AR1phkGP74). This may be achieved through a switch in isoenzyme preference, e.g. the use of NADP-dependent isocitrate dehydrogenase rather than the corresponding NAD-dependent enzyme or a higher flux through malic enzyme. In either case, the results demonstrate that this A. nidulans strain has a high degree of metabolic flexibility with respect to fluxes in the central carbon metabolism. It is likely that over-expression of phk enhances this metabolic flexibility as it opens for an alternative route from sugars to C-2 and C-3 compounds. The metabolic network analysis demonstrates that Carbon source m (h21) glucose xylose glycerol ethanol 0.23/0.23 0.16/0.19 0.11/0.14 0.12/0.17 The standard deviation was below 3% in all cultivations (triplicates). doi:10.1371/journal.pone.0003847.t001 PLoS ONE | www.plosone.org 2 December 2008 | Volume 3 | Issue 12 | e3847