A lowered concentration of cAMP receptor protein caused by glucose is an important determinant for catabolite repression in Escherichia coli

A decreased intracellular concentration of cAMP is insufficient to account for catabolite repression in Escherichia coli. We show that glucose lowers the amount of cAMP receptor protein (CRP) in cells. A correlation exists between CRP and β-galactosidase levels in cells growing under various conditions. Exogenous cAMP completely eliminates catabolite repression in CRP-overproducing cells, while it does not fully reverse the effect of glucose on β-galactosidase expression in wild-type cells. When the CRP concentration is reduced by manipulating the crp gene, β-galactosidase expression decreases in proportion to the concentration of CRP. These findings indicate that the lowered concentration of CRP caused by glucose is one of the major factors for catabolite repression. We propose that glucose causes catabolite repression by lowering the intracellular levels of both CRP and cAMP.     

Elimination of carbon catabolite repression in Klebsiella oxytoca for efficient 2,3-butanediol production from glucose-xylose mixtures

Microbial preference for glucose implies incomplete and/or slow utilization of lignocellulose hydrolysates, which is caused by the regulatory mechanism named carbon catabolite repression (CCR). In this study, a 2,3-butanediol (2,3-BD) producing Klebsiella oxytoca strain was engineered to eliminate glucose repression of xylose utilization. The crp(in) gene, encoding the mutant cyclic adenosine monophosphate (cAMP) receptor protein CRP(in), which does not require cAMP for functioning, was characterized and overexpressed in K. oxytoca. The engineered recombinant could utilize a mixture of glucose and xylose simultaneously, without CCR. The profiles of sugar consumption and 2,3-BD production by the engineered recombinant, in glucose and xylose mixtures, were examined and showed that glucose and xylose could be consumed simultaneously to produce 2,3-BD. This study offers a metabolic engineering strategy to achieve highly efficient utilization of sugar mixtures derived from the lignocellulosic biomass for the production of bio-based chemicals using enteric bacteria.     

Catabolite repression of the colonization factor antigen I (CFA/I) operon ofEscherichia coli

Experiments were performed to study whether the synthesis of the fimbrial colonization factor antigen I (CFA/I) of enterotoxigenicEscherichia coli is affected by glucose. The CFA/I-producing strain H-10407 (O78:H11:CFA/I) was grown in CFA medium containing various concentrations of glucose. Addition of 1% glucose into the medium resulted in a pronounced decrease in CFA/I production by H-10407 as assessed by ELISA, hemagglutination, and electron microscopy. The repressive effect of glucose was reversed by the addition of 10 mM cAMP to the medium. Examination of the promoter sequence of thecfaA gene of the CFA/I operon revealed a consensus binding site for the catabolite activator protein-cAMP complex. With a reporter plasmid containing a fusion of thecfaA promoter, a portion of thecfaA gene, and thelacZ gene, it was shown that the activity of this promoter was influenced by glucose. In a wild-typeE. coli strain, addition of 0.5% glucose to the growth medium diminished the promoter activity more than 70%. ThecfaA promoter also exhibited a lower level of activity incya (adenyl cyclase) andcrp (cAMP receptor protein) mutants than in the wild-type strain. The addition of 10 mM cAMP resulted in a marked increase in the expression from thecfaA promoter in thecya but not in thecrp mutant. These results suggest that the suppressive effect of glucose in the CFA/I system is mediated via the mechanism of catabolite repression through thecfaA promoter of the CFA/I operon.     

Catabolite repression in Escherichia coli mutants lacking cyclic AMP.

Summary The regulation of catabolite repression of -galactosidase has been studied in Escherichia coli mutants deleted for the adenyl cyclase gene (cya ), and thus unable to synthesize cyclic AMP. It has been found that, provided a second mutation occurs either in the crp gene coding for the catabolite gene activator protein (CAP) or in the Lactose region, these mutants exhibit catabolite repression. If the catabolite repression seen in the mutant strains corresponds to the mechanism operating in wild-type cells, the results would suggest that the intracellular concentration of cyclic AMP cannot be the unique regulator of catabolite repression.Jacques Monod was still with us when most of the work described in this and the following paper was accomplished. His constant interest, his unfailing advice, his warm support, were invaluable. It will be difficult for us to ever enjoy a successful experiment without regretting that he cannot share this pleasure with us.     

Coutilization of glucose and glycerol enhances the production of aromatic compounds in an Escherichia coli strain lacking the phosphoenolpyruvate: carbohydrate phosphotransferase system

Background  

Escherichia coli strains lacking the phosphoenolpyruvate: carbohydrate phosphotransferase system (PTS) are capable of coutilizing glucose and other carbon sources due to the absence of catabolite repression by glucose. In these strains, the lack of this important regulatory and transport system allows the coexistence of glycolytic and gluconeogenic pathways. Strains lacking PTS have been constructed with the goal of canalizing part of the phosphoenolpyruvate (PEP) not consumed in glucose transport to the aromatic pathway. The deletion of the ptsHIcrr operon inactivates PTS causing poor growth on this sugar; nonetheless, fast growing mutants on glucose have been isolated (PB12 strain). However, there are no reported studies concerning the growth potential of a PTS^- strain in mixtures of different carbon sources to enhance the production of aromatics compounds.     

The Escherichia coli rhamnose promoter rhaP BAD is in Pseudomonas putida KT2440 independent of Crp–cAMP activation

We developed an expression vector system based on the broad host range plasmid pBBR1MCS2 with the Escherichia coli rhamnose-inducible expression system for applications in Pseudomonas. For validation and comparison to E. coli, enhanced green fluorescent protein (eGFP) was used as a reporter. For further characterization, we also constructed plasmids containing different modifications of the rhaP _BAD promoter. Induction experiments after the successful transfer of these plasmids into Pseudomonas putida KT2440 wild-type and different knockout strains revealed significant differences. In Pseudomonas, we observed no catabolite repression of the rhaP _BAD promoter, and in contrast to E. coli, the binding of cyclic adenosine monophosphate (cAMP) receptor protein (Crp)–cAMP to this promoter is not necessary for induction as shown by deletion of the Crp binding site. The crp ⁻ mutant of P. putida KT2440 lacked eGFP expression, but this is likely due to problems in rhamnose uptake, since this defect was complemented by the insertion of the l-rhamnose-specific transporter rhaT into its genome via transposon mutagenesis. Other global regulators like Crc, PtsN, and CyoB had no or minor effects on rhamnose-induced eGFP expression. Therefore, this expression system may also be generally useful for Pseudomonas and other γ-proteobacteria.     

Regulation of Arabinose and Xylose Metabolism in Escherichia coli

Bacteria such as Escherichia coli will often consume one sugar at a time when fed multiple sugars, in a process known as carbon catabolite repression. The classic example involves glucose and lactose, where E. coli will first consume glucose, and only when it has consumed all of the glucose will it begin to consume lactose. In addition to that of lactose, glucose also represses the consumption of many other sugars, including arabinose and xylose. In this work, we characterized a second hierarchy in E. coli, that between arabinose and xylose. We show that, when grown in a mixture of the two pentoses, E. coli will consume arabinose before it consumes xylose. Consistent with a mechanism involving catabolite repression, the expression of the xylose metabolic genes is repressed in the presence of arabinose. We found that this repression is AraC dependent and involves a mechanism where arabinose-bound AraC binds to the xylose promoters and represses gene expression. Collectively, these results demonstrate that sugar utilization in E. coli involves multiple layers of regulation, where cells will consume first glucose, then arabinose, and finally xylose. These results may be pertinent in the metabolic engineering of E. coli strains capable of producing chemical and biofuels from mixtures of hexose and pentose sugars derived from plant biomass.The transporters and enzymes in many sugar metabolic pathways are conditionally expressed in response to their cognate sugar or a downstream pathway intermediate. While the induction of these pathways in response to a single sugar has been studied extensively (28), far less is known about how these pathways are induced in response to multiple sugars. One notable exception is the phenomenon observed when bacteria are grown in the presence of glucose and another sugar (10, 15). In such mixtures, the bacteria will often consume glucose first before consuming the other sugar, a process known as carbon catabolite repression (27). The classic example of carbon catabolite repression is the diauxic shift seen in the growth of Escherichia coli on mixtures of glucose and lactose, where the cells first consume glucose before consuming lactose. When the cells are consuming glucose, the genes in the lactose metabolic pathway are not induced, thus preventing the sugar from being consumed. A number of molecules participate in this regulation, including the cyclic AMP receptor protein (CRP), adenylate cyclase, cyclic AMP (cAMP), and EIIA from the phosphoenolpyruvate:glucose phosphotransferase system (PTS) (33). In addition to lactose, the metabolic genes for many other sugars are subject to catabolite repression by glucose in E. coli (27). While the preferential utilization of glucose is well known, it is an open question whether additional hierarchies exist among other sugars.Recently, substantial effort has been directed toward developing microorganisms capable of producing chemicals and biofuels from plant biomass (1, 34, 42). After glucose, l-arabinose and d-xylose are the next most abundant sugars found in plant biomass. Therefore, a key step in producing various chemicals and fuels from plant biomass will be the engineering of strains capable of efficiently fermenting these three sugars. However, one challenge concerns catabolite repression, which prevents microorganisms from fermenting these three sugars simultaneously and, as a consequence, may decrease the efficiency of the fermentation process. E. coli cells will first consume glucose before consuming either arabinose or xylose. As in the case of lactose, the genes in the arabinose and xylose metabolic pathways are not expressed when glucose is being consumed. In addition to glucose catabolite repression, a second hierarchy, between arabinose and xylose, appears to exist. Kang and coworkers have observed that the genes in the xylose metabolic pathway were repressed when cells were grown in a mixture of arabinose and xylose (21). Hernandez-Montalvo and coworkers also observed that E. coli utilizes arabinose before xylose (19). While a number of strategies exist for breaking the glucose-mediated repression of arabinose and xylose metabolism (8, 16, 19, 31), none exist for breaking the arabinose-mediated repression of xylose metabolism. Moreover, little is known about this repression beyond the observations made by these researchers.In this work, we investigate how the arabinose and xylose metabolic pathways are jointly regulated. We demonstrate that E. coli will consume arabinose before consuming xylose when it is grown in a mixture of the two sugars. Consistent with a mechanism involving catabolite repression, the genes in the xylose metabolic pathway are repressed in the presence of arabinose. We found that this repression is AraC dependent and is most likely due to binding by arabinose-bound AraC to the xylose promoters, with consequent inhibition of gene expression.     

Glucose lowers CRP* levels resulting in repression of the lac operon in cells lacking cAMP

CRP—cAMP-dependent operons of Escherichia coli can be expressed in cells lacking functional adenylate cyclase when they carry a second-site mutation in the crp gene ( crp* ). It is known that the expression of these operons is repressed by glucose, but the molecular mechanism underlying this cAMP-independent catabolite repression has been a long-standing mystery. Here we address the question of how glucose inhibits the expression of β-galactosidase in the absence of cAMP. We have isolated several mutations in the crp gene that confer a CRP* phenotype. The expression of β-galactosidase is reduced by glucose in cells carrying these mutations. Using Western blotting and/or SDS—PAGE analysis, we demonstrate that glucose lowers the cellular concentration of CRP* through a reduction in crp * mRNA levels. The level of CRP* protein correlates with β-galactosidase activity. When the crp promoter is replaced with the bla promoter, the inhibitory effect of glucose on crp * expression is virtually abolished. These data strongly suggest that the lowered level of CRP* caused by glucose mediates catabolite repression in cya⁻ crp * cells and that the autoregulatory circuit of the crp gene is involved in the down-regulation of CRP* expression by glucose.     

Effect of iclR and arcA deletions on physiology and metabolic fluxes in Escherichia coli BL21 (DE3)

Deletion of both iclR and arcA in E. coli profoundly alters the central metabolic fluxes and decreases acetate excretion by 70%. In this study we investigate the metabolic consequences of both deletions in E. coli BL21 (DE3). No significant differences in biomass yields, acetate yields, CO₂ yields and metabolic fluxes could be observed between the wild type strain E. coli BL21 (DE3) and the double-knockout strain E. coli BL21 (DE3) ΔarcAΔiclR. This proves that arcA and iclR are poorly active in the BL21 wild type strain. Noteworthy, both strains co-assimilate glucose and acetate at high glucose concentrations (10–15 g l⁻¹), while this was never observed in K12 strains. This implies that catabolite repression is less intense in BL21 strains compared to in E. coli K12.     

Catabolite-Like Repression of Extracellular Enzyme Production in Vibrio parahaemolyticus

Production of extracellular amylase and protease in Vibrio parahaemolyticus was repressed by various carbohydrates present in the medium. In addition, the protease production was repressed very strongly by peptones or casamino acids. Cyclic adenosine 3′, 5′-monophosphate (cyclic AMP) added exogenously could reverse the repression of amylase production, but not that of protease production irrespective of the “repressors” used. Mutants of V. parahaemolyticus, which resembled the reported cya (adenylate cyclase) and crp (cyclic AMP receptor protein) mutants of Escherichia coli and related organisms, were examined for the exoenzyme production. Amylase production in the mutants was defective, while their protease production was not defective, but rather accentuated as compared with that in the parental strain. These findings strongly suggest that amylase production is subject to catabolite repression mediated by cyclic AMP, whereas protease production is controlled by a repression mechanism which mimics in part, but may be distinct from catabolite repression.     

Conversion of glucose‐xylose mixtures to pyruvate using a consortium of metabolically engineered Escherichia coli

Two strains of Escherichia coli were engineered to accumulate pyruvic acid from two sugars found in lignocellulosic hydrolysates by knockouts in the aceE, ppsA, poxB, and ldhA genes. Additionally, since glucose and xylose are typically consumed sequentially due to carbon catabolite repression in E. coli, one strain (MEC590) was engineered to grow only on glucose while a second strain (MEC589) grew only on xylose. On a single substrate, each strain generated pyruvate at a yield of about 0.60 g/g in both continuous culture and batch culture. In a glucose‐xylose mixture under continuous culture, a consortium of both strains maintained a pyruvate yield greater than 0.60 g/g when three different concentrations of glucose and xylose were sequentially fed into the system. In a fed‐batch process, both sugars in a glucose‐xylose mixture were consumed simultaneously to accumulate 39 g/L pyruvate in less than 24 h at a yield of 0.59 g/g.     

Interaction of the CRP-cAMP complex with the cea regulatory region

Summary Analysis of the induction of expression of cea-lacZ fusions in cya and crp mutants showed that catabolite repression affects the kinetics of induction and the rate of induced synthesis. In a cya mutant, addition of cAMP reduced the induction lag and increased the amount of -galactosidase produced. The CRP-cAMP complex was found to bind to two sites 5 to the cea promoter, but deletion analysis showed that only one of these was involved in the control of cea. Deletion of this site resulted in a loss of the stimulatory effects of cAMP in a cya mutant.     

Involvement of Hexokinase Hxk1 in Glucose Catabolite Repression of LIP2 Encoding Extracellular Lipase in the Yeast Yarrowia lipolytica

The yeast Yarrowia lipolytica produces an extracellular lipase encoded by the LIP2 gene. However, very little is known about the mechanisms controlling its expression, especially on glucose media. In this work, the involvement of hexokinase Hxk1 in the glucose catabolite repression of LIP2 was investigated in a lipase overproducing mutant less sensitive to glucose repression. This mutant has a reduced capacity to phosphorylate hexose compared with the wild-type strain, but no differences could be observed between the HXK1 sequences in the two isolates. This suggested that the reduced phosphorylating activity of the mutant strain probably resulted from a modification in the level of HXK1 expression. However, overexpression of the HXK1 gene in this mutant led to a decrease of both LIP2 induction and extracellular lipase activity, suggesting that the hexokinase is involved in the glucose catabolite repression of LIP2 in Y lipolytica.