Glucose stock solutions were prepared at 20 wt% in distilled water. The optimized minimum medium contained (per liter of medium): 0.50 g K2HPO4, 0.30 g KH2PO4, 0.50 g NH4Cl, 0.50 g NaCl, 0.20 g MgCl2.6H2O, 0.04 g CaSO4.2H2O, 40 mL of 1 M Tris, 5 mL of Wolfe׳s minerals, and 5 mL of Wolfe׳s vitamins.

13C-Metabolic flux analysis - 13C –MFA was performed using the Metran software [1,2], which is based on the elementary metabolite units (EMU) framework [3,4]. Fluxes were estimated by minimizing the variance-weighted sum of squared residuals (SSR) between the experimentally ... measured and model predicted mass isotopomer distributions of amino acids using non-linear least-squares regression [5]. Flux estimation was repeated 10 times starting with random initial values for all fluxes to find a global solution. At convergence, accurate 95% confidence intervals were computed for all estimated fluxes by evaluating the sensitivity of the minimized SSR to flux variations [5]. Metabolic network model - A metabolic network model of T. thermophilus central carbon metabolism was constructed for 13C–MFA based on KEGG and PathwayTools metabolic pathway databases [6,7,8]. The model includes all major metabolic pathways of central carbon metabolism. A lumped biomass reaction was used to describe the drain of precursor metabolites and co-factors required for cell growth. The complete model is provided in the original paper.

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