Cloning, Characterization, and Functional Expression ofacs, the Gene Which Encodes Acetyl Coenzyme A Synthetase inEscherichia coli (original) (raw)
Related papers
Regulation of Acetyl Coenzyme A Synthetase in Escherichia coli
Journal of Bacteriology, 2000
Updated information and services can be found at: These include: REFERENCES http://jb.asm.org/content/182/15/4173#ref-list-1 at: This article cites 45 articles, 26 of which can be accessed free CONTENT ALERTS more» articles cite this article), Receive: RSS Feeds, eTOCs, free email alerts (when new 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:
Frontiers in Microbiology, 2020
Acetate is a characteristic by-product of Escherichia coli K-12 growing in batch cultures with glucose, both under aerobic as well as anaerobic conditions. While the reason underlying aerobic acetate production is still under discussion, during anaerobic growth acetate production is important for ATP generation by substrate level phosphorylation. Under both conditions, acetate is produced by a pathway consisting of the enzyme phosphate acetyltransferase (Pta) producing acetyl-phosphate from acetyl-coenzyme A, and of the enzyme acetate kinase (AckA) producing acetate from acetyl-phosphate, a reaction that is coupled to the production of ATP. Mutants in the AckA-Pta pathway differ from each other in the potential to produce and accumulate acetyl-phosphate. In the publication at hand, we investigated different mutants in the acetate pathway, both under aerobic as well as anaerobic conditions. While under aerobic conditions only small changes in growth rate were observed, all acetate mutants showed severe reduction in growth rate and changes in the by-product pattern during anaerobic growth. The AckA − mutant showed the most severe growth defect. The glucose uptake rate and the ATP concentration were strongly reduced in this strain. This mutant exhibited also changes in gene expression. In this strain, the atoDAEB operon was significantly upregulated under anaerobic conditions hinting to the production of acetoacetate. During anaerobic growth, protein acetylation increased significantly in the ackA mutant. Acetylation of several enzymes of glycolysis and central metabolism, of aspartate carbamoyl transferase, methionine synthase, catalase and of proteins involved in translation was increased. Supplementation of methionine and uracil eliminated the additional growth defect of the ackA mutant. The data show that anaerobic, fermentative growth of mutants in the AckA-Pta pathway is reduced but still possible. Growth reduction can be explained by the lack of an important ATP generating pathway of mixed acid fermentation. An ackA deletion mutant is more severely impaired than pta or ackA-pta deletion mutants. This is most probably due to the production of acetyl-P in the ackA mutant, leading to increased protein acetylation.
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.
Biochimie, 1989
n Growth of Escherichia coil on acetate as the sole source of carbon and energy requires operation of the glyoxylate bypass in connection with the expression of the polycistronic ace operon. The structural organization of this operon is presented, including the 3 structural genes coding respectively for malate synthase (aceB), isocitrate lyase (aceA) and isocitrate dehydrogenase kinase/phosphatase (aceK), and the surrounding genes iclR and metA. In addition, the differential expression of genes aceB, aceA, and aceK has been tested both in vivo in a minicell system and in vitro in a plasmiddirected transcription-translation coupled system. Moreover, the codon usage and adaptation to transfer RNA frequencies during translation of the corresponding messenger RNAs have been measured.
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.
Expression of Two Escherichia coli Acetyl-CoA Carboxylase Subunits Is Autoregulated
Journal of Biological Chemistry, 2003
Acetyl-CoA carboxylase (ACC) catalyzes the first step of fatty acid biosynthesis, the synthesis of malonyl-CoA from acetyl-CoA using ATP and bicarbonate. In Escherichia coli and most other bacteria, ACC is composed of four subunits encoded by accA, accB, accC, and accD. Prior work from this laboratory showed that the in vivo expression of the accBC operon had a strikingly nonlinear response to gene copy number (Li, S.-J, and Cronan, J. E., Jr. (1993) J. Bacteriol. 175, 332-340) in that the presence of 50 or more copies of the accBC operon resulted in only a 2-3-fold increase in AccB and AccC. We now report that AccB functions to negatively regulate transcription of the accBC operon. Expression of a chimeric protein consisting of the N terminus of E. coli AccB and the C-terminal bioinylation domain of Bacillus subtilis AccB down-regulated transcription of the E. coli accBC operon. A truncated form of AccB consisting of the N-terminal 68 amino acids of E. coli AccB was sufficient to negatively regulate the accBC operon. In vivo bypass of acetyl-CoA carboxylase activity by expression of a malonyl-CoA synthase from Rhizobium trifolii allowed construction of strain deleted for the accA and accB genes. Unexpectedly, the ⌬accB mutation could not be resolved from the ⌬accA mutation. Transcription of the accBC operon in the ⌬accB ⌬accA strain continued well into stationary phase under growth conditions that normally result in greatly decreased transcription. These data support a model in which AccB acts as an autoregulator of accBC operon transcription.
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
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...