Regulation of Acetyl Coenzyme A Synthetase in Escherichia coli (original) (raw)
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Scientific Reports
Escherichia coli excretes acetate upon growth on fermentable sugars, but the regulation of this production remains elusive. Acetate excretion on excess glucose is thought to be an irreversible process. However, dynamic 13 C-metabolic flux analysis revealed a strong bidirectional exchange of acetate between E. coli and its environment. The Pta-AckA pathway was found to be central for both flux directions, while alternative routes (Acs or PoxB) play virtually no role in glucose consumption. Kinetic modelling of the Pta-AckA pathway predicted that its flux is thermodynamically controlled by the extracellular acetate concentration in vivo. Experimental validations confirmed that acetate production can be reduced and even reversed depending solely on its extracellular concentration. Consistently, the Pta-AckA pathway can rapidly switch from acetate production to consumption. Contrary to current knowledge, E. coli is thus able to co-consume glucose and acetate under glucose excess. These metabolic capabilities were confirmed on other glycolytic substrates which support the growth of E. coli in the gut. These findings highlight the dual role of the Pta-AckA pathway in acetate production and consumption during growth on glycolytic substrates, uncover a novel regulatory mechanism that controls its flux in vivo, and significantly expand the metabolic capabilities of E. coli. More than a century ago, Harden reported that the enterobacterium Escherichia coli excretes acetate when growing on excess fermentable sugars 1. This phenomenon has been extensively investigated due to its physiological and applicative importance 2-7. In E. coli, the main, constitutive, pathway of acetate production involves a combination of the phosphate acetyl-transferase (Pta) and acetate kinase (AckA). This way, acetyl-coA is converted into acetyl-phosphate then into acetate which is excreted 7. Another route to form acetate is through oxidative decarboxylation of pyruvate by pyruvate oxidase PoxB 8,9. E. coli is also able to consume acetate as a carbon and energy source to support growth. Acetate can be metabolized by two alternative pathways: the reversible Pta-AckA pathway (a low affinity route with a K M for acetate of 7-10 mM) 2,10 , or the high affinity, irreversible acetyl-coA synthetase, Acs (with a K M for acetate of 200 μ M) 2,11,12. Both pathways lead to the formation of acetyl-CoA (Fig. 1). E. coli cells growing on excess glucose produce acetate but consume it only after the glucose is totally consumed 7. This diauxic behavior is due to the catabolite repression exerted by glucose on acetate utilization. When glucose is in excess, the EIIA component of the phosphoenolpyruvate-carbohydrate phosphotransferase system PTS (the main glucose transport system in E. coli), mostly exists in its unphosphorylated form. This leads to the inhibition of adenylyl cyclase. Therefore cAMP levels are low and the transcriptional activator cAMP receptor protein (CRP), which is needed to transcribe acs, is inactive. The repression of acs expression prevents acetate consumption during the period of growth on glucose. In the absence of glucose, cAMP is produced and binds to CRP, which leads to acs expression and allows cells to consume acetate. Consistent with the control of acs expression, simultaneous consumption of acetate and glucose is observed when catabolite repression is partially impaired 13 or weakened 14-17. In these conditions, acs is expressed and acetyl-CoA synthetase (Acs) is active, enabling acetate consumption to occur. The activity of Acs, in concert with the constitutive activity of Pta and AckA, results in setting up a metabolic cycle (Pta-AckA-Acs cycle) in which the acetate produced from glucose by Pta-AckA can be utilized by Acs 15-17. This cycle leads to the simultaneous production and consumption of acetate. Due to catabolite repression, the simultaneous consumption of glucose and acetate is normally expected not to occur. However, it was recently observed that acetate can be taken up and metabolized during exponential growth of wild-type E. coli K-12 strains on a mixture of glucose and acetate 18. This observation was made in conditions
Journal of Bacteriology, 2007
Although acetyl phosphate clearly signals through two-component response regulators, it remains unclear whether acetyl phosphate acts as a direct phospho donor or functions through an indirect mechanism. We used two-dimensional thin-layer chromatography to measure the relative concentrations of acetyl phosphate, acetyl coenzyme A, ATP, and GTP over the course of the entire growth curve. We estimated that the intracellular concentration of acetyl phosphate in wild-type cells reaches at least 3 mM, a concentration sufficient to activate two-component response regulators via direct phosphoryl transfer.
Enhanced Isoamyl Acetate Production upon Manipulation of the Acetyl-CoA Node in Escherichia coli
Biotechnology Progress, 2004
Coenzyme A (CoA) and its thioester derivative acetyl-Coenzyme A (acetyl-CoA) participate in over 100 different reactions in intermediary metabolism of microorganisms. Earlier results indicated that overexpression of upstream rate-limiting enzyme pantothenate kinase with simultaneous supplementation of precursor pantothenic acid to the culture media increased intracellular CoA levels significantly (∼10-fold). The acetyl-CoA levels also increased (∼5-fold) but not as much as that of CoA, showing that the carbon flux from the pyruvate node is rate-limiting upon an increase in CoA levels. In this study, pyruvate dehydrogenase was overexpressed under elevated CoA levels to increase carbon flux from pyruvate to acetyl-CoA. This coexpression did not increase intracellular acetyl-CoA levels but increased the accumulation of extracellular acetate. The production of isoamyl acetate, an industrially useful compound derived from acetyl-CoA, was used as a model reporter system to signify the beneficial effects of this metabolic engineering strategy. In addition, a strain was created in which the acetate production pathway was inactivated to relieve competition at the acetyl-CoA node and to efficiently channel the enhanced carbon flux to the ester production pathway. The synergistic effect of cofactor CoA manipulation and pyruvate dehydrogenase overexpression in the acetate pathway deletion mutant led to a 5-fold increase in isoamyl acetate production. Under normal growth conditions the acetate pathway deletion mutant strains accumulate intracellular pyruvate, leading to excretion of pyruvate. However, upon enhancing the carbon flux from pyruvate to acetyl-CoA, the excretion of pyruvate was significantly reduced.
FEMS Microbiology Letters, 2019
Acinetobacter bacteria preferentially use gluconeogenic substrates instead of hexoses or pentoses. Accordingly, Acinetobacter schindleri ACE reaches a high growth rate on acetate but is unable to grow on glucose, xylose or arabinose. In this work, we compared the physiology of A. schindleri ACE and Escherichia coli JM101 growing on acetate as the carbon source. In contrast to JM101, ACE grew on acetate threefold faster, had a twofold higher biomass yield, and a 45% higher specific acetate consumption rate. Transcriptional analysis revealed that genes like ackA, pta, aceA, glcB, fumA, tktA and talA were overexpressed while acsA, sfcA, ppc and rpiA were underexpressed in ACE relative to JM101. This transcriptional profile together with carbon flux balance analysis indicated that ACE forms acetyl-CoA preferentially by the AckA-Pta (acetate kinase-phosphotransacetylase) pathway instead of Acs (acetyl-CoA synthetase) and that the glyoxylate shunt and tricarboxylic acid cycle are more act...
Microbial Cell Factories, 2009
Background: Acetate metabolism in Escherichia coli plays an important role in the control of the central metabolism and in bioprocess performance. The main problems related to the use of E. coli as cellular factory are i) the deficient utilization of carbon source due to the excretion of acetate during aerobic growth, ii) the inhibition of cellular growth and protein production by acetate and iii) the need for cofactor recycling (namely redox coenzymes and free CoASH) to sustain balanced growth and cellular homeostasis.
1995
Updated information and services can be found at: These include: CONTENT ALERTS more» cite this article), Receive: RSS Feeds, eTOCs, free email alerts (when new articles http://journals.asm.org/site/misc/reprints.xhtml Information about commercial reprint orders: http://journals.asm.org/site/subscriptions/ To subscribe to to another ASM Journal go to: on October 17, 2013 by guest Acetyl coenzyme A synthetase (Acs) activates acetate to acetyl coenzyme A through an acetyladenylate intermediate; two other enzymes, acetate kinase (Ack) and phosphotransacetylase (Pta), activate acetate through an acetyl phosphate intermediate. We subcloned acs, the Escherichia coli open reading frame purported to encode Acs (F. R. Blattner, V. Burland, G. Plunkett III, H. J. Sofia, and D. L. Daniels, Nucleic Acids Res. 21:5408-5417, 1993)
Acetate metabolism regulation in Escherichia coli: carbon overflow, pathogenicity, and beyond
Applied microbiology and biotechnology, 2016
Acetate is ubiquitously found in natural environments. Its availability in the gut is high as a result of the fermentation of nutrients, and although it is rapidly absorbed by intestinal mucosa, it can also be used as carbon source by some members of gut microbiota. The metabolism of acetate in Escherichia coli has attracted the attention of the scientific community due to its role in central metabolism and its link to multiple physiological features. In this microorganism, acetate is involved directly or indirectly on the regulation of functional processes, such as motility, formation of biofilms, and responses to stress. Furthermore, it is a relevant nutrient in gut, where it serves additional roles, which regulate or, at least, modulate pathophysiological responses of E. coli and other bacteria. Acetate is one of the major by-products of anaerobic (fermenting) metabolism, and it is also produced under fully aerobic conditions. This acetate overflow is recognized as one of the maj...
Journal of Bacteriology, 2008
Although a whole arsenal of mechanisms are potentially involved in metabolic regulation, it is largely uncertain when, under which conditions, and to which extent a particular mechanism actually controls network fluxes and thus cellular physiology. Based on 13 C flux analysis of Escherichia coli mutants, we elucidated the relevance of global transcriptional regulation by ArcA, ArcB, Cra, CreB, CreC, Crp, Cya, Fnr, Hns, Mlc, OmpR, and UspA on aerobic glucose catabolism in glucose-limited chemostat cultures at a growth rate of 0.1 h ؊1 . The by far most relevant control mechanism was cyclic AMP (cAMP)-dependent catabolite repression as the inducer of the phosphoenolpyruvate (PEP)-glyoxylate cycle and thus low tricarboxylic acid cycle fluxes. While all other mutants and the reference E. coli strain exhibited high glyoxylate shunt and PEP carboxykinase fluxes, and thus high PEP-glyoxylate cycle flux, this cycle was essentially abolished in both the Crp and Cya mutants, which lack the cAMP-cAMP receptor protein complex. Most other mutations were phenotypically silent, and only the Cra and Hns mutants exhibited slightly altered flux distributions through PEP carboxykinase and the tricarboxylic acid cycle, respectively. The Cra effect on PEP carboxykinase was probably the consequence of a specific control mechanism, while the Hns effect appears to be unspecific. For central metabolism, the available data thus suggest that a single transcriptional regulation process exerts the dominant control under a given condition and this control is highly specific for a single pathway or cycle within the network.