Medium contained per litre: 15 g glucose, 20 mL salt solution (50× with 180 g/L NaNO3, 10 g/L KCI, 30 g/L KH2PO4, 10 g/L MgSO4.7H2O) and 1 mL trace element solution (1000× with 10 g/L EDTA, 4.4 g/L ZnSO4·7H2O, 1.5 g/L MnCl2.4H2O, 0.32 g/L CuSO4.5H2O, 7.5 g/L FeSO4·7H2O, 0.32 g/L CoCl2·6H2O, 1.47 g/L CaCl2·2H2O and 0.22 g/L (NH4)6Mo7O24.4H2O). In 13C-labeling experiments, glucose was replaced by an equimolar amount of 13C-labeled glucose. To resolve the metabolic fluxes of interest two parallel set-ups were chosen for the labeling studies (Wittmann and Heinzle 2002). This included one set-up with [1-13C] glucose (99%, Cambridge Isotope Laboratories, Andover, USA) and one set-up with a 1:1 mixture of [13C6] glucose (99%, Cambridge Isotope Laboratories, Andover, USA) and naturally labeled glucose. See manuscript for more details.

Labeling analysis of proteinogenic amino acids by GC–MS - cells of A. niger were harvested during mid-exponential growth by filtration of 5mL culture broth through a cellulose acetatefilter (pore size 20 µm, Sartorius, Goettingen, Germany). After removal of excess medium by ... two washing steps with sterile 0.9% NaCI solution, the cells were frozen in liquid nitrogen and then lyophilized at -60 ºC (Alpha 1-4 LD, Christ GmbH, Osterode, Germany). For protein hydrolysis 10mg of lyophilized biomass was incubated in 400 mL of 6M HCl at 100 ºC for 24h. After pH adjustment to 7.0 by 6M NaOH, the hydrolysate was clarified (0.2 µm, Ultrafree MC, Millipore, Bedford, MA, USA) and again lyophilized. The contained proteinogenic amino acids were then converted into t-butyl-di-methyl-silyl derivates [1]. Their labeling pattern was quantified by GC–MS (HP6890, M5973, Agilent Technologies, Waldbronn, Germany) as described previously [2]. All samples were measured first in scan mode, there with excluding isobaric overlay [3]. The relative fractions of the mass isotopomers of interest were then determined in duplicate in selective ion monitoring (SIM) mode.
Metabolic reaction network - a compartmented metabolic network model of A. niger was constructed on basis of validated models recently described [4-7].
Metabolic flux calculation - the estimation of metabolic fluxes was performed with the MATLAB-based program FiatFLUX 1.67, which was kindly provided by the authors [8].

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