13C-MFA Reveals Metabolic Flux Redistribution

To investigate if the carbon flux was really redistributed to the newly constructed EP-bifido pathway, 13C-MFA was performed using 100% 1-¹³C₁ glucose as the feeding substrate was added to a concentration of 10 g/L. Cells at the exponential growth phase were harvested by centrifugation at 7000 g for 5 min at 4 °C. The cell pellet was then washed twice with chemical defined medium and hydrolyzed in 6 M HCl for 24 h at 120 °C (Schwender et al. 2006). The resulting proteinogenic acids were derivatized with N-(tert-butyldimethylsilyl)-N-methyl-trifluoroacetamide containing tert-butyldimethylchlorosilane in acetonitrile at 105 °C for 1 h, and then analyzed by a GC–MS [Agilent 7890 A GC and 5975 C Mass Selective Detector (Agilent Technologies, Santa Clara, USA)] equipped with a DB-1column (Agilent Technologies). The data obtained from GC–MS were corrected by reduction of the natural abundance ratio of C, H, O, N, and Si isotopes [30 (link)]. Metabolic fluxes were estimated by minimizing the residual sum of squares between experimentally measured and model predicted ¹³C-enrichment using ¹³C-Flux software obtained from Dr. Wiechert [33 (link)].