Systems Analysis in A nidulans Figure 3. Response of central metabolism to phosphoketolase over-expression during growth on a glucose medium. A green box means that one or more isoenzymes of this reaction is down-regulated in the phosphoketolase over-expressing strain relative to the wild type. A red box indicates up-regulation. A blue box shows that some genes with this function are up-regulated while others are down-regulated. A grey box indicates that a gene could not be assigned to this function. The map was adapted from a map of A. niger metabolism published by Andersen et al [31]. doi:10.1371/journal.pone.0003847.g003 growth rate on xylose, glycerol and ethanol compared to the wild type, while over-expression of phk had no effect on the growth rate on glucose. Faster growth was also followed by a significant higher biomass yield in the mutant compared to the wild type strain. In the PP/glycolytic pathway, 5 mol of ATP are produced in the conversion of 3 moles of xylulose-5-P to 5 moles of pyruvate. Therefore the ATP yield per 1 mol of xylulose-5-P is 1.67 moles. On the other hand, in the cleavage of 1 mol of xylulose-5-P to pyruvate and acetyl-P by the phosphoketolase pathway, 2 mol of ATP are produced and 1 mol is further produced by the conversion of acetyl-P to acetate. Looking more into the total stoichiometry from xylulose-5-P to acetyl-CoA it is obvious that the PHK pathway is an energetically efficient route leading to improved specific growth rates on xylose, glycerol and ethanol. 3 X5PR5 PYR+5 NADH+5 ATPR5 Ac-CoA+5 CO2+5 NADH+5 ATP [PPP+Glycolysis] 3 X5PR3 PYR+3 Ac-CoA+3 NADH+6 ATPR6 Ac- CoA+3 CO2+3 NADH+6 ATP [Phosphoketolase] More interesting is that the less favorable the carbon source is for growth of A. nidulans cells (lower biomass yields), the more profound is the effect of over-expressing phk on the specific growth rate (Table 1). Consequently, this strategy is important for processes where a less energetic carbon source than glucose is needed. However, the question still remains, why over-expression of phk had no effect on the specific growth rate on glucose. While during growth on ethanol the TCA cycle is very active making the extra acetyl-CoA an important source of energy (1 mol of acetyl- CoA yields 4 mol of NADH), the same effect is not expected when cells are growing on glucose. Furthermore, Panagiotou et al [18] have demonstrated that a balance between ATP and the ratio of the redox cofactors NADH/NADPH is crucial for improving the specific growth rate on glucose. In that study, over-expression of a NADH kinase led to a 1.2-fold improvement of the specific growth rate; this large increase was rather surprising since the NADH kinase is catalyzing an ATP dependent reaction, meaning that over-expression of this enzyme is associated with cost of energy. Bridging the two studies, it seems that the specific growth rate of A. nidulans on glucose is not limited by ATP and during growth on glucose there is already an efficient supply of all necessary precursors for biomass production and therefore inducing the phosphoketolase pathway does therefore not have a positive effect. The above hypothesis was confirmed by the transcriptome analysis, where the response to phk over-expression on the glucose medium seems targeted to neutralize the effect by regulating the acetate metabolism and initiating a growth dampening response. On the other three substrates, we observed that the induction of the PHK pathway increases growth without initiating inhibiting responses, thus supporting that it is a pathway that adds flexibility to the central metabolism as observed in the flux study. It also suggests that A. nidulans is not optimized for growth on glycerol or ethanol as sole carbon sources. As these carbon sources, and especially glycerol and ethanol, are seldom found alone in the dead biomass that the saphrophytic A. nidulans grows on in nature, it is not surprising that the cells have not been evolved to grow fast on these carbon sources. It also suggests that production strains that grow faster on these substrates can be evolved or constructed by phk over-expression. The major difference between growth in nature and growth in aerobic, stirred bioreactors is probably that oxygen can be taken up efficiently in the bioreactor, and consequently respiration is expected to be much more efficient in our experiments compared to nature. If this is the case then less carbon is needed for ATP generation and instead carbon can be used for synthesis of biomass. When A. nidulans is growing on xylose, a pentose sugar, then PHK offers a direct way of generating cytosolic acetyl-CoA. When ATP generation is efficient, then PHK is an efficient way of generating acetyl-CoA since the route via pyruvate decarboxylase results in loss of carbon and a corresponding lower biomass yield. There is no requirement for an efficient PHK when growing on glucose. PLoS ONE | www.plosone.org 5 December 2008 | Volume 3 | Issue 12 | e3847