Monitoring novel metabolic pathways using metabolomics and machine learning: induction of the phosphoketolase pathway in Aspergillus nidulans cultivations G. Panagiotou, a I. Kouskoumvekaki, b S.O. Jo´nsdo´ttir, b and L. Olsson a, * a Center for Microbial Biotechnology, Biocentrum-DTU, Technical University of Denmark, Building 223, DK-2800 Kgs. Lyngby, Denmark b Center for Biological Sequence Analysis, BioCentrum-DTU, Technical University of Denmark, Building 208, DK-2800 Kgs. Lyngby, Denmark Received 31 January 2007; Accepted 23 April 2007 The physiological phenotype of Aspergillus nidulans was determined under different environmental conditions through the quantification of the intracellular and extracellular metabolite pools and clear evidence of the presence of a novel fungal metabolic pathway, the phosphoketolase pathway, was obtained. Induction of the phosphoketolase pathway resulted after blocking the EMP pathway through the deactivation of glyceraldehyde-3-P dehydrogenase (G3PD). Deactivation of G3PD in cultivations of A. nidulans on glucose and xylose led to a 10-fold decrease in the specific growth rate; however, growth could still be sustained solely through the phosphoketolase pathway. Metabolomics and machine learning tools were successfully used to monitor the alteration caused by the inhibition of G3PD in the metabolism of A. nidulans grown on glucose, xylose, acetate as well as mixtures of glucose or xylose with acetate. This is the first study that demonstrates in vivo that the fungal central carbon metabolism includes an active phosphoketolase pathway. KEY WORDS: Aspergillus nidulans; phosphoketolase; metabolite profile; Self Organizing Maps (SOM). 1. Introduction Although there is currently a great deal of interest for the utilization of xylose as a fermentation substrate, there is no clear evidence for that xylose metabolism in fungi relies on the pentose phosphate pathway alone. Phosphoketolases (EC 4.1.2.9, EC 4.1.2.22) are key enzymes of the phosphoketolase pathway of heterofer- mentative and facultatively homofermentative lactic acid bacteria and of the D-fructose 6-phosphate shunt of bifidobacteria (Rohr et al., 2002). Phosphoketolase EC 4.1.2.9 catalyses an irreversible thiamine diphosphate (ThDP) dependent phosphorolytic reaction splitting D- xylulose 5-phosphate in the presence of inorganic phosphate to yield acetyl phosphate and glycerol 3- phosphate (figure 1). The exact interaction mechanism of the ThDP moiety with its binding partners has not been investigated, but it has been assumed to be similar to that of transketolases (Schellenberger, 1998). The study of its presence, specificity and role in other microbes has been hampered by the fact that the sub- strate D-xylulose 5-phosphate, was quite expensive when it was commercially available, and since the beginning of 2001 is no longer in the market. Evans and Ratledge have examined a number of yeasts for the presence of phosphoketolase and they found that the enzyme was present in 19 out of 25 tested species (Evans et al., 1984). However, the in vitro activity of phosphoketolase in filamentous fungi has only recently been reported in anaerobic batch cultivations of F. oxysporum by using a coupled assay, but metabolic network analysis suggested that the flux through phosphoketolase was below the detection limit (Panagiotou et al., 2006). One of the most interesting features of filamentous fungi is their capacity for producing a great variety of secondary metabolites. Several of these compounds are currently used for industrial purposes, such as various antibiotics and vitamins. Biosynthesis of these com- pounds in fungal cells occurs after sugar depletion and in many cases requires the induction of key enzymes of the central carbon metabolism in order to increase the pool of the necessary precursor molecules (Amor et al., 2000). Aspergilli are a group of filamentous fungi that play an important role as cell factories for the produc- tion of organic acids (Kubicek and Rohr, 1986), enzymes (Carlsen and Nielsen, 2001) and polyketides, for example statins by Aspergillus terreus (Manzoni and Rollini, 2002). Polyketides are one of the most impor- tant groups of natural products, due to their huge structural variety and versatile biological activities. The polyketide synthases (PKS) are the responsible enzymes for producing an array of natural products with differ- ent biological activities. Despite the structural diversity of polyketides, the PKS share a common chemical strategy for the assembly of polyketide scaffolds, by * To whom correspondence should be addressed. E-mail: lo@biocentrum.dtu.dk. Metabolomics, Vol. 3, No. 4, December 2007 (Ó 2007) DOI: 10.1007/s11306-007-0061-7 503 1573-3882/07/1200-0503/0 Ó 2007 Springer ScienceþBusiness Media, LLC