Large‐scale 13C‐flux analysis reveals distinct transcriptional control of respiratory and fermentative metabolism in Escherichia coli

Despite our increasing topological knowledge on regulation networks in model bacteria, it is largely unknown which of the many co‐occurring regulatory events actually control metabolic function and the distribution of intracellular fluxes. Here, we unravel condition‐dependent transcriptional control of Escherichia coli metabolism by large‐scale 13 C‐flux analysis in 91 transcriptional regulator mutants on glucose and galactose. In contrast to the canonical respiro‐fermentative glucose metabolism, fully respiratory galactose metabolism depends exclusively on the phosphoenol‐pyruvate (PEP)‐glyoxylate cycle. While 2/3 of the regulators directly or indirectly affected absolute flux rates, the partitioning between different pathways remained largely stable with transcriptional control focusing primarily on the acetyl‐CoA branch point. Flux distribution control was achieved by nine transcription factors on glucose, including ArcA, Fur, PdhR, IHF A and IHF B, but was exclusively mediated by the cAMP‐dependent Crp regulation of the PEP‐glyoxylate cycle flux on galactose. Five further transcription factors affected this flux only indirectly through cAMP and Crp by increasing the galactose uptake rate. Thus, E. coli actively limits its galactose catabolism at the expense of otherwise possible faster growth. The authors analyze the role transcription plays in regulating bacterial metabolic flux. Of 91 transcriptional regulators studied, 2/3 affect absolute fluxes, but only a small number of regulators control the partitioning of flux between different metabolic pathways. Synopsis The authors analyze the role transcription plays in regulating bacterial metabolic flux. Of 91 transcriptional regulators studied, 2/3 affect absolute fluxes, but only a small number of regulators control the partitioning of flux between different metabolic pathways. Focusing on central carbon metabolism of Escherichia coli , we aim here to systematically identify transcriptional regulators that control the distribution of metabolic fluxes during aerobic growth on hexoses. To assess the condition dependence of transcriptional control of flux, we selected glucose and galactose as two substrates that are highly similar, yet lead to distinct growth rates (Soupene et al , 2003 ), overall metabolic rates (De Anda et al , 2006 ; Samir El et al , 2009 ) and levels of catabolite repression (Hogema et al , 1998 ; Bettenbrock et al , 2007 ). Experimentally determined fluxes (Fischer and Sauer, 2003a )