Changes in Size of Intracellular Pools of Coenzyme A and Its Thioesters inEscherichia coliK-12 Cells to Various Carbon Sources and Stresses

Abstract

Intracellular pools of three CoA molecular species of coenzyme A, CoASH, acetyl-CoA, and malonyl-CoA, in Escherichia coli K-12 cells were studied by acyl-CoA cycling method in replacement culture. The sizes and compositions of CoA pools starved for a carbon source changed within minutes after the addition of one of various carbon sources. A large acetyl-CoA pool formed after the addition of D-glucose, D-fructose, D-mannose, glycerol, or sorbitol, but there was little change when L-glucose, sucrose, maltose, succinate, or acetate was added. The beta-anomer of D-glucose was assimilated 10 times faster than the alpha-anomer. Intracellular CoA pools also changed with stress: in the pH, incubation temperature, or with osmotic stress. The sizes and compositions of CoA pools were not affected by pH changing between 4 and 8, but the breakdown of acetyl-CoA and CoASH was greater at pH 9 than at pH 4 to 8. Production of acetyl-CoA was greatest at 40 degrees C, and at 50 degrees C, an acetyl-CoA pool did not form at all and the size of the CoASH pool declined. When the organism was stressed by the addition of NaCl at concentrations of more than 0.6 M, little acetyl-CoA was produced. The total CoA pool (the sum of the concentrations of CoASH, acetyl-CoA, and malonyl-CoA) remained within the limits of 0.83-1.40 nmol/mg of dry cell weight (0.30-0.52 mM). Whenever acetyl-CoA increased, CoASH decreased. Therefore, the acetyl-CoA/CoASH ratio is an important index of facultative anaerobes that reflects the state of carbon and energy metabolism in vivo.

Inhibition of Acetyl Phosphate-dependent Transcription by an Acetylatable Lysine on RNA Polymerase

“…(i) The intracellular pools of both AcP and AcCoA are dynamic. They fluctuate throughout growth and upon exposure to different carbon sources (16,30). Furthermore, both central metabolites can be used as donors for post-translational modification of proteins: AcP by donating its phosphoryl group for protein phosphorylation, and AcCoA by donating its acetyl group for protein acetylation (12,32).…”

“…(ii) The limited supply of CoA would be overwhelmed by exposure to large amounts of glucose (e.g. 4.0%), resulting in a large AcCoA-to-CoA ratio (16,30). (iii) Disruption of the Pta-AckA pathway also would be expected to put pressure on the limited CoA pool.…”

“…For several reasons, we considered the possibility that AcCoA could function as this inhibitor, perhaps by acting as an acetyl-donor. First, the concentration of AcCoA has been shown to rapidly increase when E. coli cells are exposed to glucose (30) and growth in the presence of 0.4% glucose causes a general increase in protein acetylation (15). Second, pta mutants excrete large amounts of pyruvate and lactate in an AcCoA-sensitive manner (31), suggesting that disruption of the Pta-AckA pathway causes a metabolic imbalance that could affect the AcCoA pool.…”

Prokaryotic type III pantothenate kinase enhances coenzyme A biosynthesis in <i>Escherichia coli</i>

“…1), and subject to feedback inhibition by CoA and acyl-CoAs (Vallari et al, 1987). As a result, intracellular CoA content is strictly regulated (Chohnan et al, 1997(Chohnan et al, , 1998. On the other hand, Vadali and coworkers (2004a) successfully increased the intracellular CoA pool by expressing E. coli pantothenate kinase from a high-copy number plasmid.…”

Cofactor Engineering Enhances the Physiological Function of an Industrial Strain

“…Consistent with this result, an increase in the CoA/acetyl-CoA ratio was observed. Furthermore, it was found that the specific activity of CoA synthase decreased with a shift from glucose to acetate [65] . This result demonstrated that CoA synthase is not sensitive to acetyl-CoA.…”