Systems Analysis Unfolds the Relationship between the Phosphoketolase Pathway and Growth in Aspergillus nidulans Gianni Panagiotou1., Mikael R. Andersen1., Thomas Grotkjær2., Torsten B. Regueira1, Gerald Hofmann1, Jens Nielsen1¤, Lisbeth Olsson1¤* 1 Center for Microbial Biotechnology, Department of Systems Biology, Technical University of Denmark, Lyngby, Denmark, 2 Fluxome Sciences A/S, Lyngby, Denmark Abstract Background: Aspergillus nidulans is an important model organism for studies on fundamental eukaryotic cell biology and on industrial processes due to its close relation to A. niger and A. oryzae. Here we identified the gene coding for a novel metabolic pathway in A. nidulans, namely the phosphoketolase pathway, and investigated the role of an increased phosphoketolase activity. Methodology/Principal Findings: Over-expression of the phosphoketolase gene (phk) improved the specific growth rate on xylose, glycerol and ethanol. Transcriptome analysis showed that a total of 1,222 genes were significantly affected by over- expression of the phk, while more than half of the affected genes were carbon source specific. During growth on glucose medium, the transcriptome analysis showed that the response to phk over-expression is targeted to neutralize the effect of the over-expression by regulating the acetate metabolism and initiate a growth dampening response. Conclusions/Significance: Metabolic flux analysis using 13C-labelled glucose, showed that over-expression of phospho- ketolase added flexibility to the central metabolism. Our findings further suggests that A. nidulans is not optimized for growth on xylose, glycerol or ethanol as the sole carbon sources. Citation: Panagiotou G, Andersen MR, Grotkjær T, Regueira TB, Hofmann G, et al. (2008) Systems Analysis Unfolds the Relationship between the Phosphoketolase Pathway and Growth in Aspergillus nidulans. PLoS ONE 3(12): e3847. doi:10.1371/journal.pone.0003847 Editor: Julian Rutherford, Newcastle University, United Kingdom Received September 2, 2008; Accepted November 3, 2008; Published December 4, 2008 Copyright: 2008 Panagiotou et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This study was partly financed by the Villum Kann Rasmussen and Lundbeck foundations (GP) and the Danish Research Agency for Technology and Production (MRA). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: lo@bio.dtu.dk ¤ Current address: Department of Chemical and Biological Engineering, Chalmers University of Technology, Gothenburg, Sweden . These authors contributed equally to this work. Introduction Filamentous fungi are prominent users of the sugar xylose, but currently little work has been devoted to study the metabolism of this sugar. This is in sharp contrast to the large and continued commercial interest for xylose utilization in bioethanol production and as cheap raw material in industrial fermentations. It is known that xylose enters the pentose phosphate (PP) pathway, but it is not clear how it interacts with the remaining part of the metabolism. Phosphoketolases (EC 4.1.2.9, EC 4.1.2.22) are a class of key enzymes of the phosphoketolase pathway of heterofer- mentative and facultatively homofermentative lactic acid bacteria, as well as the D-fructose 6-phosphate shunt of bifidobacteria, and xylose fermenting yeast [1,2]. The first type of phosphoketolase (PHK, EC 4.1.2.9) catalyses an irreversible thiamine diphosphate dependent phosphorolytic reaction: in the presence of inorganic phosphate, D-xylulose-5-phosphate is cleaved into acetyl phosphate and glyceraldehyde-3-phosphate. The study of this PHK has been hampered by the fact that the substrate D-xylulose-5-phosphate initially was expensive, and since the beginning of 2001 it has been withdrawn from the commercial market. Recently, our group [3,4] provided evidence of the PHK pathway existence in Aspergillus nidulans, which could be induced under certain cultivation conditions. Induction of PHK in Aspergilli increases the carbon flux towards the precursors acetyl-CoA or acetate, and consequently there is a potential to improve the yields and productivities of a long series of industrially important secondary metabolites derived from acetyl-CoA. For a comprehensive evaluation of genes that are transcriptionally modulated during growth in the presence of an active PHK pathway, we constructed a strain that constitutively expressed the gene putatively encoding PHK (phk, AN4913.3) and performed a large-scale analysis of gene expression in A. nidulans by using DNA microarrays. Despite the wide use of DNA microarrays, only a few studies have been reported on transcriptome profiling in Aspergilli, e.g. A. oryzae [5], A. flavus [6–8], A. nidulans [9–11]. In order to accurately determine the effects of over-expressing phk on transcription, we grew the cells on a broad range of carbon sources (glucose, xylose, glycerol and ethanol) in well controlled bioreactors. To further understand the effects on glucose, we performed labelling experiments with [1-13C] glucose to quantify the metabolic fluxes in the central carbon metabolism. Quantifi- PLoS ONE | www.plosone.org 1 December 2008 | Volume 3 | Issue 12 | e3847