Metabolic Control Analysis of Aspergillus niger L-Arabinose Catabolism Marco J. L. de Groot,* ,†,‡,§ Wai Prathumpai, | Jaap Visser, ‡,⊥ and George J. G. Ruijter ‡,# Fungal Genomics and Section of Molecular Genetics of Industrial Microorganisms, Wageningen University, Dreijenlaan 2, NL-6703HA Wageningen, The Netherlands, and Center for Process Biotechnology, BioCentrum-DTU, Technical University of Denmark, DK-2800 Kgs, Lyngby, Denmark A mathematical model of the L-arabinose/D-xylose catabolic pathway of Aspergillus niger was constructed based on the kinetic properties of the enzymes. For this purpose L-arabinose reductase, L-arabitol dehydrogenase and D-xylose reductase were purified using dye-affinity chromatography, and their kinetic properties were characterized. For the other enzymes of the pathway the kinetic data were available from the literature. The metabolic model was used to analyze flux and metabolite concentration control of the L-arabinose catabolic pathway. The model demonstrated that flux control does not reside at the enzyme following the intermediate with the highest concentra- tion, L-arabitol, but is distributed over the first three steps in the pathway, preceding and following L-arabitol. Flux control appeared to be strongly dependent on the intracellular L-arabinose concentration. At 5 mM intracellular L-arabinose, a level that resulted in realistic intermediate concentrations in the model, flux control coefficients for L-arabinose reductase, L-arabitol dehydrogenase and L-xylulose reductase were 0.68, 0.17 and 0.14, respectively. The analysis can be used as a guide to identify targets for metabolic engineering aiming at either flux or metabolite level optimization of the L-arabinose catabolic pathway of A. niger. Faster L-arabinose utilization may enhance utilization of readily available organic waste containing hemicelluloses to be converted into industrially interesting metabolites or valuable enzymes or proteins. Introduction Arabinan is a polysaccharide found in plant cell wall heteropolysaccharides as a side chain of pectin (1). Aspergillus niger is able to degrade arabinan extra- cellularly to the pentose L-arabinose using its arabi- nanolytic system. To date three arabinanases have been characterized: two R-L-arabinofuranosidases (ABFA and ABFB) and an arabinan 1,5-R-L-arabinanase (ABNA) (2). L-Arabinose can be used by the fungus as a carbon source via the L-arabinose catabolic pathway. This pathway (Figure 1) converts L-arabinose to D-xylulose 5-phosphate, which is further metabolized via the non-oxidative pen- tose phosphate pathway. Of the L-arabinose/D-xylose catabolic pathway the genes encoding D-xylulose kinase (xkiA) and D-xylose reductase (xyrA) have previously been cloned in A. niger (3)(4). Of the L-arabinose specific part of the pathway recently the L-arabitol dehydrogenase (LAD) and L-xylulose reductase (LXR) encoding genes were cloned and characterized in Trichoderma reesii (5, 6). * To whom correspondence should be addressed. Ph: +31 (0)- 30 2515871. FAX: +31 (0)30 2518219. E-mail: mjl_degroot@ kimarco.nl. † Fungal Genomics, Wageningen University. ‡ Section of Molecular Genetics of Industrial Microorganisms, Wageningen University. § Present address: Center for Biomedical Genetics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Sorbonnelaan 16, NL-3584CA Utrecht, The Netherlands. | Technical University of Denmark. ⊥ Present address: Fungal Genetics and Technology Consul- tancy, PO Box 396, NL-6700AJ Wageningen, The Netherlands. # Present address: Metabolic Diseases Laboratory, Leiden University Medical Centre, PO Box 9600, NL-2300RC Leiden, The Netherlands. Figure 1. L-Arabinose/D-xylose catabolic pathway. The arrows represent the directions of the rate equations used in the model. ARD ) L-arabinose reductase, XYR ) D-xylose reductase, LAD ) L-arabitol dehydrogenase, LXR ) L-xylulose reductase, XDH ) xylitol dehydrogenase and XKI ) D-xylulose kinase. 1610 Biotechnol. Prog. 2005, 21, 1610-1616 10.1021/bp050189o CCC: $30.25 © 2005 American Chemical Society and American Institute of Chemical Engineers Published on Web 10/20/2005