The Entner-Doudoroff pathway in Escherichia coli is induced for oxidative glucose metabolism via pyrroloquinoline quinone-dependent glucose dehydrogenase.

The Entner-Doudoroff pathway was shown to be induced for oxidative glucose metabolism when Escherichia coli was provided with the periplasmic glucose dehydrogenase cofactor pyrroloquinoline quinone (PQQ). Induction of the Entner-Doudoroff pathway by glucose plus PQQ was established both genetically and biochemically and was shown to occur in glucose transport mutants, as well as in wild-type E. coli. These data complete the body of evidence that proves the existence of a pathway for oxidative glucose metabolism in E. coli. PQQ-dependent oxidative glucose metabolism provides a metabolic branch point in the periplasm; the choices are either oxidation to gluconate followed by induction of the Entner-Doudoroff pathway or phosphotransferase-mediated transport. The oxidative glucose pathway might be important for survival of enteric bacteria in aerobic, low-phosphate, aquatic environments.     

Nutritional complementation of oxidative glucose metabolism in Escherichia coli via pyrroloquinoline quinone-dependent glucose dehydrogenase and the Entner-Doudoroff pathway.

Two glucose-negative Escherichia coli mutants (ZSC113 and DF214) were unable to grow on glucose as the sole carbon source unless supplemented with pyrroloquinoline quinone (PQQ). PQQ is the cofactor for the periplasmic enzyme glucose dehydrogenase, which converts glucose to gluconate. Aerobically, E. coli ZSC113 grew on glucose plus PQQ with a generation time of 65 min, a generation time about the same as that for wild-type E. coli in a defined glucose-salts medium. Thus, for E. coli ZSC113 the Enter-Doudoroff pathway was fully able to replace the Embden-Meyerhof-Parnas pathway. In the presence of 5% sodium dodecyl sulfate, PQQ no longer acted as a growth factor. Sodium dodecyl sulfate inhibited the formation of gluconate from glucose but not gluconate metabolism. Adaptation to PQQ-dependent growth exhibited long lag periods, except under low-phosphate conditions, in which the PhoE porin would be expressed. We suggest that E. coli has maintained the apoenzyme for glucose dehydrogenase and the Entner-Doudoroff pathway as adaptations to an aerobic, low-phosphate, and low-detergent aquatic environment.     

Nutritional complementation of oxidative glucose metabolism in Escherichia coli via pyrroloquinoline quinone-dependent glucose dehydrogenase and the Entner-Doudoroff pathway.

Two glucose-negative Escherichia coli mutants (ZSC113 and DF214) were unable to grow on glucose as the sole carbon source unless supplemented with pyrroloquinoline quinone (PQQ). PQQ is the cofactor for the periplasmic enzyme glucose dehydrogenase, which converts glucose to gluconate. Aerobically, E. coli ZSC113 grew on glucose plus PQQ with a generation time of 65 min, a generation time about the same as that for wild-type E. coli in a defined glucose-salts medium. Thus, for E. coli ZSC113 the Enter-Doudoroff pathway was fully able to replace the Embden-Meyerhof-Parnas pathway. In the presence of 5% sodium dodecyl sulfate, PQQ no longer acted as a growth factor. Sodium dodecyl sulfate inhibited the formation of gluconate from glucose but not gluconate metabolism. Adaptation to PQQ-dependent growth exhibited long lag periods, except under low-phosphate conditions, in which the PhoE porin would be expressed. We suggest that E. coli has maintained the apoenzyme for glucose dehydrogenase and the Entner-Doudoroff pathway as adaptations to an aerobic, low-phosphate, and low-detergent aquatic environment.     

Glucose and Gluconate Metabolism in a Mutant of Escherichia coli Lacking Gluconate-6-phosphate Dehydrase

A mutant lacking gluconate-6-phosphate dehydrase (the first enzyme of the Entner-Doudoroff pathway) was isolated after ethyl methane sulfonate mutagenesis of Escherichia coli. Other enzymes of gluconate metabolism (gluconokinase, gluconate-6-phosphate dehydrogenase, and 2-keto-3-deoxygluconate-6-phosphate aldolase) were present in the mutant. When the mutant was grown on gluconate-1-(14)C, alanine isolated from protein was unlabeled, showing that the dehydrase was absent in vivo and that the sole pathway of gluconate metabolism in the mutant was the hexose monophosphate shunt. The mutant grew on gluconate with a doubling time of 155 min, compared with the parent strain's 56 min. On glucose and fructose it grew with normal doubling times. Thus, in E. coli, the Entner-Doudoroff pathway is used for gluconate metabolism but not for glucose metabolism.     

Identification of Pantoea ananatis gene encoding membrane pyrroloquinoline quinone (PQQ)-dependent glucose dehydrogenase and pqqABCDEF operon essential for PQQ biosynthesis

Pantoea ananatis accumulates gluconate during aerobic growth in the presence of glucose. Computer analysis of the P. ananatis SC17(0) sequenced genome revealed an ORF encoding a homologue (named gcd) of the mGDH (EC 1.1.99.17) apoenzyme from Escherichia coli and a putative pyrroloquinoline quinone (PQQ) biosynthetic operon homologous to pqqABCDEF from Klebsiella pneumoniae. Construction of Δgcd and Δpqq mutants of P. ananatis confirmed the proposed functions of these genetic elements. The P. ananatis pqqABCDEF was cloned in vivo and integrated into the chromosomes of P. ananatis and E. coli according to the Dual In/Out strategy. Introduction of a second copy of pqqABCDEF to P. ananatis SC17(0) doubled the accumulation of PQQ. Integration of the operon into E. coli MG1655ΔptsGΔmanXY restored the growth of bacteria on glucose. The obtained data show the essential role of pqqABCDEF in PQQ biosynthesis in P. ananatis and E. coli. We propose that the cloned operon could be useful for an efficient phosphoenolpyruvate-independent glucose consumption pathway due to glucose oxidation and construction of E. coli strains with the advantage of phosphoenolpyruvate-derived metabolite production.     

Research on the metabolic engineering of the direct oxidation pathway for extraction of phosphate from ore has generated preliminary evidence for PQQ biosynthesis in Escherichia coli as well as a possible role for the highly conserved region of quinoprotein dehydrogenases

The ability of some bacteria to dissolve poorly soluble calcium phosphates (CaPs) has been termed 'mineral phosphate solubilizing' (MPS). Since most microorganisms and plants must assimilate P via membrane transport, biotransformation of CaP into soluble phosphate is considered an essential component of the global P cycle. In many Gram-negative bacteria, strong organic acids produced in the periplasm via the direct oxidation pathway have been shown to dissolve CaP in the adjacent environment. Therefore, the quinoprotein glucose dehydrogenase (PQQGDH) may function in the ecophysiology of many soil bacteria. There is interest in using MPS bacteria for industrial bioprocessing of rock phosphate ore (a substituted fluroapatite) or even for direct inoculation of soils as a 'biofertilizer' analogous to nitrogen fixation. Our laboratory has spent 20 years studying superior MPS bacteria. Screening genomic libraries in the appropriate E. coli genetic background can 'trap' PQQ or GDH genes from these bacteria via functional complementation. In setting the 'trap' for PQQ genes, we have identified DNA fragments that apparently induce PQQGDH activity in E. coli with no sequence homology to known PQQ genes. These data suggest that E. coli may have an alternative, inducible PQQ biosynthesis pathway. Finally, a novel protein engineering strategy to increase the catalytic rate of PQQGDH has emerged and will be discussed.     

The role of glucose limitation in the regulation of the transport of glucose, gluconate and 2-oxogluconate, and of glucose metabolism in Pseudomonas aeruginosa.

The pathway of glucose metabolism in Pseudomonas aeruginosa was regulated by the availability of glucose and related compounds. On changing from an ammonium limitation to a glucose limitation, the organism responded by adjusting its metabolism substantially from the extracellular direct oxidative pathway to the intracellular phosphorylative route. This change was achieved by repression of the transport systems for gluconate and 2-oxogluconate and of the associated enzymes for 2-oxogluconate metabolism and gluconate kinase, while increasing the levels of glucose transport, hexokinase and glucose 6-phosphate dehydrogenase. The role of gluconate, produced by the action of glucose dehydrogenase, as a major inhibitory factor for glucose transport, and the possible significance of these regulatory mechanisms to the organism in its natural environment, are discussed.     

Functions of amino acid residues in the active site of Escherichia coli pyrroloquinoline quinone-containing quinoprotein glucose dehydrogenase

Several mutants of quinoprotein glucose dehydrogenase (GDH) in Escherichia coli, located around its cofactor pyrroloquinoline quinone (PQQ), were constructed by site-specific mutagenesis and characterized by enzymatic and kinetic analyses. Of these, critical mutants were further characterized after purification or by different amino acid substitutions. H262A mutant showed reduced affinities both for glucose and PQQ without significant effect on glucose oxidase activity, indicating that His-262 occurs very close to PQQ and glucose, but is not the electron acceptor from PQQH(2). W404A and W404F showed pronounced reductions of affinity for PQQ, and the latter rather than the former had equivalent glucose oxidase activity to the wild type, suggesting that Trp-404 may be a support for PQQ and important for the positioning of PQQ. D466N, D466E, and K493A showed very low glucose oxidase activities without influence on the affinity for PQQ. Judging from the enzyme activities of D466E and K493A, as well as their absorption spectra of PQQ during glucose oxidation, we conclude that Asp-466 initiates glucose oxidation reaction by abstraction of a proton from glucose and Lys-493 is involved in electron transfer from PQQH(2).     

Different degradation pathways for glucose and fructose in Rhodopseudomonas capsulata

In Rhodopseudomonas capsulata the enzymes of the Entner-Doudoroff pathway and the Embden-Meyerhof pathway have been examined. Fructose-grown cells contained inducible activities of phosphoenolpyruvate-fructosephospho-transferase and 1-phosphofructokinase and only low levels of fructokinase and 6-phosphofructokinase. Although fructose-grown cells contained, in addition, all the enzymes of the Entner-Doudoroff pathway together with fructose-1,6-diphosphatase and phosphoglucose isomerase, the Entner-Doudoroff pathway was not operative in fructose catabolism and served only the degradation of glucose. The functional separation of glucose and fructose catabolism via the Entner-Doudoroff and a modified Embden-Meyerhof pathway, respectively, was confirmed by different approaches: 1. Radiorespirometric experiments with glucose and fructose labelled in positions 1, 2, 3, 3+4 and 6 have been carried out. The pattern of 14CO2-evolution from position-labelled glucose was characteristic for the Entner-Doudoroff pathway, that from position-labelled fructose for the Embden-Meyerhof pathway. 2. In the presence of arsenite up to 50% of glucose- and fructose-carbon was excreted as pyruvate. Using 1-14C-glucose, 86% of the pyruvate was labelled in the carboxyl group, whereas using 1-14C-fructose only 19% of the pyruvate was labelled in the carboxyl group. 3. A glucose-6-phosphate dehydrogenase-deficient mutant was isolated which lacked a functional Entner-Doudoroff pathway but which was unaltered in its ability to grow on fructose.     

The influence of the culture pH value on the direct glucose oxidative pathway in Klebsiella pneumoniae NCTC 418

Klebsiella pneumoniae NCTC 418 was cultured aerobically in chemostat cultures (D=0.3 h^-1; 35°C) under respectively carbon-, phosphate-, potassium-, sulphate-, and ammonia-limited conditions with glucose as the sole carbon and energy source. The effect of the external pH value on glucose metabolism and on the enzymes of the direct glucose oxidative pathway was examined. The pH value of the medium had a profound influence on both the activity and the synthesis of the glucose dehydrogenase and the gluconate dehydrogenase. At pH values ranging from pH 5.5 to pH 6.0 maximal activity and synthesis of these enzymes resulted in a more than 80% conversion of the glucose consumed into gluconate and 2-ketogluconate under potassium-or phosphate-limited conditions. On the other hand, no gluconate and/or 2-ketogluconate production could be detected when K. pneumoniae was cultured at pH 8.0. Whereas the synthesis of gluconate dehydrogenase seemingly was completely repressed, still some glucose dehydrogenase was present. The lack of glucose dehydrogenase activity at pH 8.0 was shown not to be due to the dissociation of the cofactor PQQ from the enzyme.Abbreviations DCIP dichlorophenol indophenol - PQQ pyrroloquinoline quinone [2,7,9-tricarboxy-1H-pyrrolo (2,3-f) quinoline-4,5-dione] - WB Wurster's Blue [1,4-bis-(dimethylamino)-benzene perchlorate]     

Responses of the central metabolism in Escherichia coli to phosphoglucose isomerase and glucose-6-phosphate dehydrogenase knockouts

The responses of Escherichia coli central carbon metabolism to knockout mutations in phosphoglucose isomerase and glucose-6-phosphate (G6P) dehydrogenase genes were investigated by using glucose- and ammonia-limited chemostats. The metabolic network structures and intracellular carbon fluxes in the wild type and in the knockout mutants were characterized by using the complementary methods of flux ratio analysis and metabolic flux analysis based on [U-(13)C]glucose labeling and two-dimensional nuclear magnetic resonance (NMR) spectroscopy of cellular amino acids, glycerol, and glucose. Disruption of phosphoglucose isomerase resulted in use of the pentose phosphate pathway as the primary route of glucose catabolism, while flux rerouting via the Embden-Meyerhof-Parnas pathway and the nonoxidative branch of the pentose phosphate pathway compensated for the G6P dehydrogenase deficiency. Furthermore, additional, unexpected flux responses to the knockout mutations were observed. Most prominently, the glyoxylate shunt was found to be active in phosphoglucose isomerase-deficient E. coli. The Entner-Doudoroff pathway also contributed to a minor fraction of the glucose catabolism in this mutant strain. Moreover, although knockout of G6P dehydrogenase had no significant influence on the central metabolism under glucose-limited conditions, this mutation resulted in extensive overflow metabolism and extremely low tricarboxylic acid cycle fluxes under ammonia limitation conditions.     

Carbohydrate metabolism in the mosquito pathogen Bacillus sphaericus 2362.

Bacillus sphaericus 2362 is pathogenic for mosquito larvae and is being considered for large-scale production as a larvicide. The inability of the bacteria to metabolize carbohydrates requires that they be grown on proteinaceous media. This bacterium was found to be unable to transport glucose or sucrose into the cell, and it lacked glucokinase and hexokinase activity. In addition, it lacked phosphoglucose isomerase, phosphofructokinase, and glucose 6-phosphate dehydrogenase, which are early enzymes of the Embden-Myerhof-Parnas and hexose monophosphate pathways. The presence of other enzymes in these pathways was indicated by assay, by the metabolism of glycerol to acetate, and by growth on acetate and gluconate as sole carbon sources. Critical enzymes of the Entner-Doudoroff pathway were also shown to be absent.     

Altered glucose transport and shikimate pathway product yields in E. coli

Different glucose transport systems are examined for their impact on phosphoenolpyruvate availability as reflected by the yields of 3-dehydroshikimic acid and byproducts 3-deoxy-d-arabino-heptulosonic acid, 3-dehydroquinic acid, and gallic acid synthesized by Escherichia coli from glucose. 3-Dehydroshikimic acid is an advanced shikimate pathway intermediate in the syntheses of a spectrum of commodity, pseudocommodity, and fine chemicals. All constructs carried plasmid aroF(FBR) and tktA inserts encoding, respectively, a feedback-insensitive isozyme of 3-deoxy-d-arabino-heptulosonic acid 7-phosphate synthase and transketolase. Reliance on the native E. coli phosphoenolpyruvate:carbohydrate phosphotransferase system for glucose transport led in 48 h to the synthesis of 3-dehydroshikimic acid (49 g/L) and shikimate pathway byproducts in a total yield of 33% (mol/mol). Use of heterologously expressed Zymomonas mobilis glf-encoded glucose facilitator and glk-encoded glucokinase resulted in the synthesis in 48 h of 3-dehydroshikimic acid (60 g/L) and shikimate pathway byproducts in a total yield of 41% (mol/mol). Recruitment of native E. coli galP-encoded galactose permease for glucose transport required 60 h to synthesize 3-dehydroshikimic acid (60 g/L) and shikimate pathway byproducts in a total yield of 43% (mol/mol). Direct comparison of the impact of altered glucose transport on the yields of shikimate pathway products synthesized by E. coli has been previously hampered by different experimental designs and culturing conditions. In this study, the same product and byproduct mixture synthesized by E. coli constructs derived from the same progenitor strain is used to compare strategies for increasing phosphoenolpyruvate availability. Constructs are cultured under the same set of fermentor-controlled conditions.     

Carbohydrate metabolism in the mosquito pathogen Bacillus sphaericus 2362

Bacillus sphaericus 2362 is pathogenic for mosquito larvae and is being considered for large-scale production as a larvicide. The inability of the bacteria to metabolize carbohydrates requires that they be grown on proteinaceous media. This bacterium was found to be unable to transport glucose or sucrose into the cell, and it lacked glucokinase and hexokinase activity. In addition, it lacked phosphoglucose isomerase, phosphofructokinase, and glucose 6-phosphate dehydrogenase, which are early enzymes of the Embden-Myerhof-Parnas and hexose monophosphate pathways. The presence of other enzymes in these pathways was indicated by assay, by the metabolism of glycerol to acetate, and by growth on acetate and gluconate as sole carbon sources. Critical enzymes of the Entner-Doudoroff pathway were also shown to be absent.     

Distribution of the phosphoenolpyruvate:glucose phosphotransferase system in fermentative bacteria.

A number of selected fermentative bacteria were surveyed for the presence of the phosphoenolpyruvate:glucose phosphotransferase system, with particular attention to those organisms which ferment glucose by pathways other than the Embden-Meyerhof-Parnas pathway. The phosphoenolpyruvate:glusoe phosphotransferase system was found in all homofermentative lactic acid bacteria tested that ferment glucose via the Embden-Meyerhof-Parnas pathway, but in none of a group of heterofermentative species of Lactobacillus or Leuconostoc, which ferment glucose via the phosphoketolase pathway. A phosphoenolpyruvate:glucose phosphotransferase system was also absent in Zymomonas mobilis, which ferments glucose via an anaerobic Entner-Doudoroff pathway. It thus appears that the phosphotransferase mode of glucose transport is limited to bacteria with the Embden-Meyerhof-Parnas mode of glucose fermentation.     

Promiscuity in the part-phosphorylative Entner-Doudoroff pathway of the archaeon Sulfolobus solfataricus

The hyperthermophilic archaeon Sulfolobus solfataricus metabolises glucose and galactose by a 'promiscuous' non-phosphorylative variant of the Entner-Doudoroff pathway, in which a series of enzymes have sufficient substrate promiscuity to permit the metabolism of both sugars. Recently, it has been proposed that the part-phosphorylative Entner-Doudoroff pathway occurs in parallel in S. solfataricus as an alternative route for glucose metabolism. In this report we demonstrate, by in vitro kinetic studies of D-2-keto-3-deoxygluconate (KDG) kinase and KDG aldolase, that the part-phosphorylative pathway in S. solfataricus is also promiscuous for the metabolism of both glucose and galactose.     

Cloning of Klebsiella pneumoniae pqq genes and PQQ biosynthesis in Escherichia coli

We have cloned genes from Klebsiella pneumoniae which are required for pyrroloquinoline quinone (PQQ) biosynthesis. The cloned 6.7 kb fragment can complement several chromosomal pqq mutants. Escherichia coli strains are unable to synthesize PQQ but E. coli strains containing the cloned 6.7 kb K. pneumoniae fragment can synthesize PQQ in large amounts and E. coli pts mutants can be complemented on minimal glucose medium by this clone.     

Participation of the Entner-Doudoroff pathway in Escherichia coli strains with an inactive phosphotransferase system (PTS- Glc+) in gluconate and glucose batch cultures

The activity of the enzymes of the central metabolic pathways has been the subject of intensive analysis; however, the Entner-Doudoroff (ED) pathway has only recently begun to attract attention. The metabolic response to edd gene knockout in Escherichia coli JM101 and PTS- Glc+ was investigated in gluconate and glucose batch cultures and compared with other pyruvate kinase and PTS mutants previously constructed. Even though the specific growth rates between the strain carrying the edd gene knockout and its parent JM101 and PTS- Glc+ edd and its parent PTS- Glc+ were very similar, reproducible changes in the specific consumption rates and biomass yields were obtained when grown on glucose. These results support the participation of the ED pathway not only on gluconate metabolism but on other metabolic and biochemical processes in E. coli. Despite that gluconate is a non-PTS carbohydrate, the PTS- Glc+ and derived strains showed important reductions in the specific growth and gluconate consumption rates. Moreover, the overall activity of the ED pathway on gluconate resulted in important increments in PTS- Glc+ and PTS- Glc+ pykF mutants. Additional results obtained with the pykA pykF mutant indicate the important contribution of the pyruvate kinase enzymes to pyruvate synthesis and energy production in both carbon sources.