Analysis of metabolic fluxes in batch and continuous cultures of Bacillus subtilis

Abstract

It is well recognized that metabolic fluxes are the key variables that must be determined in order to understand metabolic regulation and patterns. However, owing to difficulties in measuring the flux values, evaluation of metabolic fluxes has not been an integral part of the most metabolic studies. Flux values for metabolites of glycolysis, tricarboxylic acid (TCA) cycle, and hexose monophosphate (HMP) pathway were obtained for batch and glucose-limited continuous cultures of Bacillus subtilis by combining the information from the stoichiometry of key biosynthetic reactions with the experimental data on concentrations of glucose and metabolic by-products, CO(2) evolution, and oxygen uptake rates. The results indicate that (1) the metabolic fluxes and energetic yield as well as the extent of flux mismatch in metabolic activity of glycolysis and the TCA cycle reactions can be accurately quantified; (2) the flux through the TCA cycle in continuous culture is much in excess of cell energetic and biosynthetic demands for precursors; (3) for the range of growth rates examined the TCA cycle flux increases almost in proportion to growth rate and is significantly repressed only at very high growth rates of batch cultures; and (4) for continuous cultures the isocitrate dehydrogenase catalyzed reaction of the TCA cycle is the major source of the reduced form of nicotinamide-adenine dinucleotide phosphate (NADPH) used in biosynthesis.

Proposed mechanism of acetate accumulation in two recombinantEscherichia coli strains during high density fermentation

“…To solve the metabolites' balanced equations, least squares programming techniques were implemented. The total flows (mmol/L) were determined instead of the actual fluxes (mmol/g/h) because they provide a better average picture for batch culture (Goel et al, 1993). Incorporating both pathways for a complete picture of the carbon pathway could not be done because of the singularity arising by incorporating both pathways in the network (Vallino and Stephanopoulos, 1990).…”

“…One way to lower acetate accumulation is to reduce the growth rate and, consequently, the glucose uptake rate by supplying the glucose slowly. Using this strategy, the TCA cycle can handle all the acetyl CoA produced by the glycolytic pathway, thus eliminating acetate formation (Goel et al, 1993;Riesenberg et al, 1991). When K12 derivatives of E. coli were grown under these conditions, less acetate was produced and higher levels of isocitrate lyase (ICL) were detected (Kleman and Strohl, 1994).…”

Metabolic Analysis of Escherichia coli in the Presence and Absence of the Carboxylating Enzymes Phosphoenolpyruvate Carboxylase and Pyruvate Carboxylase

“…Flux analysis of a metabolic system typically involves calculation of the intracellular fluxes based on measured excretion fluxes and the stoichiometry of the reactions involved in the metabolic network. Previous studies have applied this technique to a wide variety of fermentations (1,8,12,22,26,33,34,36).In order to improve our understanding of anaerobic succinate production in E. coli, we analyzed carbon flux distributions in response to genetic perturbations affecting the activities of PPC and PYC. In this study, we investigated the metabolic shifts in E. coli resulting from a null mutation in the ppc gene, overexpression of PPC and overexpression of PYC.…”

“…Flux analysis of a metabolic system typically involves calculation of the intracellular fluxes based on measured excretion fluxes and the stoichiometry of the reactions involved in the metabolic network. Previous studies have applied this technique to a wide variety of fermentations (1,8,12,22,26,33,34,36).…”

Comparison of dynamic responses of cellular metabolites in Escherichia coli to pulse addition of substrates

“…The pH of the culture was maintained at 7.0 by adding 2.0 M NaOH or 2.0 M HCl. An agitation speed of 500 rpm with 1.0 L/min of air flow ensured a DO concentration higher than 30% of air saturation (Goel et al 1993;Sauer et al 1999;Dauner et al 2001;Hua et al 2003;Yang et al 2003;Sarker 2008).…”