Characterization and Engineering of a Novel Pyrroloquinoline Quinone Dependent Glucose Dehydrogenase from <Emphasis Type="Italic">Sorangium cellulosum</Emphasis> So ce56

A novel pyrroloquinoline quinone dependent glucose dehydrogenase like enzyme (PQQ GDH) was isolated from Sorangium cellulosum So ce56. The putative coding region was cloned, over expressed in E. coli and the resulting enzyme was characterized. The recombinant protein has a relative molecular mass of 63 kDa and shows 43% homology to PQQ GDH-B from Acinetobacter calcoaceticus. In the presence of PQQ and CaCl₂ the enzyme has dehydrogenase activity with the substrate glucose as well as with other mono- and disaccharides. The thermal stability and its pH activity profile mark the enzyme as a potential glucose biosensor enzyme. In order to decrease the activity on maltose, which is unwanted for a potential application in biosensors, the protein was rationally modified at three specified positions. The best variant showed a 59% reduction in activity on maltose compared to the wild type enzyme. The catalytic efficiency (k _cat/K _M) was reduced fivefold but the specific activity still amounted to 63% of the wild type activity.     

Properties of a chimeric glucose dehydrogenase improved by site directed mutagenesis

Glucose dehydrogenase, a membrane bound enzyme oxidizing glucose to gluconic acid in the periplasmic space of Gram-negative bacteria plays a key role in mineral phosphate solubilization and is also an industrially important enzyme, being used as a glucose biosensor. A chimeric glucose dehydrogenase (ES chimera) encoding the N-terminal transmembrane domain from Escherichia coli and the C-terminal periplasmic domain from Serratia marcescens was constructed and the expression was studied on MacConkey glucose medium. The phosphate solubilizing ability of the chimeric GDH was also evaluated, substantiating the role of GDH in mineral phosphate solubilization (MPS). Four mutants of ES chimeric GDH were generated by site directed mutagenesis and the enzyme properties studied. Though the substrate affinity was unaltered for E742K and Y771M, the affinity of H775A and EYH/KMA to glucose and galactose decreased marginally and the affinity to maltose increased. Though Y771M showed a decreased GDH activity there was an increase in the heat tolerance. All the mutants showed an increase in the EDTA tolerance. The triple mutant EYH/KMA showed improved heat and EDTA tolerance and also an increase in affinity to maltose over the ES chimeric GDH.     

Transconjugation studies in <Emphasis Type="Italic">Azospirillum</Emphasis> sp. negative to mineral phosphate solubilization

It has been previously shown that certain gram-negative bacteria do not have the ability to solubilize insoluble phosphates due to the lack of pyrroloquinoline-quinone synthesis genes (pqq). PQQ is required as cofactor for the assembly of the glucose dehydrogenase (GDH) holoenzyme, which acts in the oxidation of glucose to gluconic acid. In this context the transconjugation and expression of pqq genes in Azospirillum sp. was studied using the construct pMCG 898. pMCG 898 containing pqq gene/s was mobilized into an Azospirillum strain negative to mineral phosphate solubilization by biparental mating. The presence of the construct was also confirmed by minipreps of the transconjugants. The transconjugants were able to solubilize dicalcium phosphate while the wild type was not able to do so. The nitrogen-fixing ability of the transconjugants was also examined and they retained the ability to fix nitrogen. Further detailed studies are required to confirm the utility of such strains in releasing inorganic P from fixed phosphates in soil.     

Variation in the Nature of Organic Acid Secretion and Mineral Phosphate Solubilization by Citrobacter sp. DHRSS in the Presence of Different Sugars

A novel phosphate solubilizing bacterium (PSB) was isolated from the rhizosphere of sugarcane and is capable of utilizing sucrose and rock phosphate as the sole carbon and phosphate source, respectively. This PSB exhibited mineral phosphate solubilizing (MPS) phenotype on sugars such as sucrose and fructose, which are not substrates for enzyme glucose dehydrogenase (GDH), along with GDH substrates, viz., glucose, xylose, and maltose, as carbon sources. PCR amplification of the rRNA gene and sequence analysis identified this bacterium as Citrobacter sp. DHRSS. On sucrose and fructose Citrobacter sp. DHRSS liberated 170 and 100 μM free phosphate from rock phosphate and secreted 49 mM (2.94 g/L) and 35 mM (2.1 g/L) acetic acid, respectively. Growth of Citrobacter sp. DHRSS on sucrose is mediated by an intracellular inducible neutral invertase. Interestingly, in the presence of GDH substrates like glucose and maltose, Citrobacter sp. DHRSS produced approximately 20 mM (4.36 g/L) gluconic acid and phosphate released was 520 and 570 μM, respectively. Citrobacter sp. DHRSS GDH activity was found when grown on GDH and non-GDH substrates, indicating that it is constitutive and could act on a wide range of aldose sugars. This study demonstrates the role of different organic acids in mineral phosphate solubilization by rhizobacteria depending on the nature of the available carbon source.     

The Klebsiella aerogenes glutamate dehydrogenase (gdhA) gene: cloning, high-level expression and hybrid enzyme formation in Escherichia coli

Summary The NADP-dependent glutamate dehydrogenase gene of Klebsiella aerogenes was cloned in E. coli in the expression plasmid pRK9. The cloned gene shows a high level of expression in E. coli in the hybrid plasmid pKG3 and such expression is independent of the vector promoter, as shown by experiments in which the promoter was deleted. Active hybrid GDH hexamers were shown in cell-free extracts of an E. coli strain carrying cloned gdhA genes of both E. coli and K. aerogenes. The nucleotide sequence of the N-terminal coding region of the K. aerogenes gdhA gene was determined and found to be strongly homologous with that of E. coli.Abbreviations GDH glutamate dehydrogenase - PMS phenazine methosulphate - MTT 3-(4,5-Dimethylthiazolyl-2)-2,5-diphenyltetrazolium-bromide - PMSF phenylmethylsulphonylfluoride - SSC standard saline citrate - DTT dithiothreitol - bp base pairs - kbp kilo base pairs - dNTP deoxynucleoside triphosphate     

High-level expression of recombinant glucose dehydrogenase and its application in NADPH regeneration

Two glucose dehydrogenase (E.C. 1.1.1.47) genes, gdh223 and gdh151, were cloned from Bacillus megaterium AS1.223 and AS1.151, and were inserted into pQE30 to construct the expression vectors, pQE30-gdh223 and pQE30-gdh151, respectively. The transformant Escherichia coli M15 with pQE30-gdh223 gave a much higher glucose dehydrogenase activity than that with the plasmid pQE30-gdh151. Thus it was used to optimize the expression of glucose dehydrogenase. An proximately tenfold increase in GDH activity was achieved by the optimization of culture and induction conditions, and the highest productivity of glucose dehydrogenase (58.7 U/ml) was attained. The recombinant glucose dehydrogenase produced by E. coli M15 (pQE30-gdh223) was then used to regenerate NADPH. NADPH was efficiently regenerated in vivo and in vitro when 0.1 M glucose was supplemented concomitantly in the reaction system. Finally, this coenzyme-regenerating system was coupled with a NADPH-dependent bioreduction for efficient synthesis of ethyl (R)-4-chloro-3-hydroxybutanoate from ethyl 4-chloro-3-oxobutanoate.     

Engineering of recombinant E. coli cells co‐expressing P450pyrTM monooxygenase and glucose dehydrogenase for highly regio‐ and stereoselective hydroxylation of alicycles with cofactor recycling

E. coli (P450pyrTM‐GDH) with dual plasmids, pETDuet containing P450pyr triple mutant I83H/M305Q/A77S (P450pyrTM) and ferredoxin reductase (FdR) genes and pRSFDuet containing glucose dehydrogenase (GDH) and ferredoxin (Fdx) genes, was engineered to show a high activity (12.7 U g^?1 cdw) for the biohydroxylation of N‐benzylpyrrolidine 1 and a GDH activity of 106 U g^?1 protein. The E. coli cells were used as efficient biocatalysts for highly regio‐ and stereoselective hydroxylation of alicyclic substrates at non‐activated carbon atom with enhanced productivity via intracellular recycling of NAD(P)H. Hydroxylation of N‐benzylpyrrolidine 1 with resting cells in the presence of glucose showed excellent regio‐ and stereoselectivity, giving (S)‐N‐benzyl‐3‐hydroxypyrrolidine 2 in 98% ee as the sole product in 9.8 mM. The productivity is much higher than that of the same biohydroxylation using E. coli (P450pyrTM)b without expressing GDH. E. coli (P450pyrTM‐GDH) was found to be highly regio‐ and stereoselective for the hydroxylation of N‐benzylpyrrolidin‐2‐one 3 , improving the regioselectivity from 90% of the wild‐type P450pyr to 100% and giving (S)‐N‐benzyl‐4‐hydroxylpyrrolidin‐2‐one 4 in 99% ee as the sole product. A high activity of 15.5 U g^?1 cdw was achieved and (S)‐ 4 was obtained in 19.4 mM. E. coli (P450pyrTM‐GDH) was also found to be highly regio‐ and stereoselective for the hydroxylation of N‐benzylpiperidin‐2‐one 5 , increasing the ee of the product (S)‐N‐benzyl‐4‐hydroxy‐piperidin‐2‐one 6 to 94% from 33% of the wild‐type P450pyr. A high activity of 15.8 U g^?1 cdw was obtained and (S)‐ 6 was produced in 3.3 mM as the sole product. E. coli (P450pyrTM‐GDH) represents the most productive system known thus far for P450‐catalyzed hydroxylations with cofactor recycling, and the hydroxylations with E. coli (P450pyrTM‐GDH) provide with simple and useful syntheses of (S)‐ 2 , (S)‐ 4 , and (S)‐ 6 that are valuable pharmaceutical intermediates and difficult to prepare. Biotechnol. Bioeng. 2013; 110: 363–373. © 2012 Wiley Periodicals, Inc.     

Co-expression of P450 BM3 and glucose dehydrogenase by recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis> and its application in an NADPH-dependent indigo production system

P450 BM3 mutant can catalyze indole to indoxyl, and indoxyl can dimerize to form indigo. But the reaction catalyzed by P450 BM3 requires NADPH, as coenzyme regeneration is very important in this system. As we know, when glucose dehydrogenase oxidizes glucose to glucolactone, NADH or NADPH can be formed, which can contribute to NADPH regeneration in the reaction catalyzed by P450 BM3. In this paper, a recombinant Escherichia coli BL21 (pET28a (+)-P450 BM3-gdh0310) was constructed to co-express both P450 BM3 gene and glucose dehydrogenase (GDH) gene. To improve the expression level of P450 BM3 and GDH in E. coli and to avoid the complex and low-efficiency refolding operation in the purification procedure, the expression conditions were optimized. Under the optimized conditions, the maximum P450 BM3 and GDH activities amounted to 8173.13 and 0.045 U/mg protein, respectively. Then bioconversion of indole to indigo was carried out by adding indole and glucose to the culture after improved expression level was obtained under optimized conditions, and 2.9 mM (760.6 mg/L) indigo was formed with an initial indole concentration of 5 mM.     

Expression of two kinds of recombinant glutamate dehydrogenase from Aeropyrum pernix with different N-terminal sequence length in Escherichia coli

Two recombinant Aeropyrum pernix glutamate dehydrogenase (GDH) enzymes with different length N-termini were cloned and expressed in Escherichia coli: sGDH begins with the amino acid sequence of the extracted native enzyme (M-Q-P-T-D-P-L-E-E), whereas lGDH begins with the sequence of the predicted ORF (M-E-V-L-A-L-Q-P-T-D) and is longer than sGDH by five amino acids (M-E-V-L-A). Purified recombinant lGDH was more stable than sGDH, indicating that the N-terminal extension, containing mostly hydrophobic residues, affected the overall stability of recombinant lGDH. This stabilising effect of extending the N-terminal sequence on an oligomeric enzyme such as GDH is novel; factors affecting stabilisation have previously only been discussed in the context of the contribution of internal amino acids. Electronic Publication     

Synthesis of optically pure <Emphasis Type="Italic">S</Emphasis>-sulfoxide by <Emphasis Type="Italic">Escherichia coli</Emphasis> transformant cells coexpressing the P450 monooxygenase and glucose dehydrogenase genes

A cytochrome P450 monooxygenase (P450SMO) from Rhodococcus sp. can catalyze asymmetric oxygenation of sulfides to S-sulfoxides. However, P450SMO-catalyzed biotransformations require a constant supply of NAD(P)H, the expense of which constitutes a great hindrance for this enzyme application. In this study, we investigated the asymmetric oxygenation of sulfide to S-sulfoxide using E. coli cells, which co-express both the P450SMO gene from Rhodococcus sp. and the glucose dehydrogenase (GDH) gene from Bacillus subtilis, as a catalyst. The results showed that the catalytic performance of co-expression systems was markedly improved compared to the system lacking GDH. When using recombinant E. coli BL21 (pET28a-P450-GDH) whole cell as a biocatalyst, NADPH was efficiently regenerated when glucose was supplemented in the reaction system. A total conversion of 100% was achieved within 12 h with 2 mM p-chlorothioanisole substrate, affording 317.3 mg/L S-sulfoxide obtained. When the initial sulfide concentration was increased to 5 mM, the substrate conversion was also increased nearly fivefold: S-sulfoxide amounted to 2.5 mM (396.6 mg/L) and the ee value of sulfoxide product exceeded 98%. In this system, the effects of glucose concentration and substrate concentration were further investigated for efficient biotransformation. This system is highly advantageous for the synthesis of optically pure S-sulfoxide.     

Characterisation of a secondary alcohol dehydrogenase from Xanthomonas campestris DSM 3586

The chromosomal locus NP_636946 of Xanthomonas campestris DSM 3586 (ATCC 33913) which was earlier presumed to encode a quinoprotein glucose dehydrogenase has been cloned, expressed in Escherichia coli and the recombinant enzyme has been characterised. It was found to have no glucose dehydrogenase activity but to be active on many different polyols and diols, aliphatic alcohols, certain aldonic acids and amino-sugars. The product of d-gluconic acid oxidation was 5-keto-d-gluconic acid. The enzyme differs from polyol/gluconate dehydrogenases found in Gluconobacter by its single-chain architecture, different substrate specificity and much higher (20- to 30-fold) expression level in E.coli.     

Influence of Calcium Ion on Peeling of Satsuma Mandarin Segments

5-((R)-1-Hydroxyethyl)-furo[2,3-c]pyridine ((R)-FPH) is a useful chiral building block in the synthesis of pharmaceuticals. An NADH-dependent alcohol dehydrogenase (AFPDH) isolated from Candida maris catalyzed the reduction of 5-acetylfuro[2,3-c]pyridine (AFP) to (R)-FPH with 100% enantiomeric excess. The gene encoding AFPDH was cloned and sequenced. The AFPDH gene comprises 762 bp and encodes a polypeptide of 27,230 Da. The deduced amino acid sequence showed a high degree of similarity to those of other members of the short-chain alcohol dehydrogenase superfamily. The AFPDH gene was overexpressed in Escherichia coli under the control of the lac promoter. One L of the cultured broth of an E. coli transformant coexpressing AFPDH and the glucose dehydrogenase (GDH) gene reduced 250 g of AFP to (R)-FPH in an organic solvent two-phase system. Under coupling with NADH regeneration using 2-propanol, 1 L of the cultured broth of an E. coli transformant expressing the AFPDH gene reduced 150 g of AFP to (R)-FPH. The optical purity of the (R)-FPH formed was 100% enantiomeric excess under both reaction conditions.     

Stereoselective synthesis of (<Emphasis Type="Italic">R</Emphasis>)-3-quinuclidinol through asymmetric reduction of 3-quinuclidinone with 3-quinuclidinone reductase of <Emphasis Type="Italic">Rhodotorula rubra</Emphasis>

A novel nicotinamide adenine dinucleotide phosphate-dependent carbonyl reductase, 3-quinuclidinone reductase, was isolated from Rhodotorula rubra JCM3782. The enzyme catalyzes the asymmetric reduction of 3-quinuclidinone to (R)-3-quinuclidinol. The gene encoding the enzyme was also cloned and sequenced. A 819-bp nucleotide fragment was confirmed to be the gene encoding the 3-quinuclidinone reductase by agreement of the internal amino acid sequences of the purified enzyme. The gene encodes a total of 272 amino acid residues, and the deduced amino acid sequence shows similarity to those of several short-chain dehydrogenase/reductase family proteins. An expression vector, pWKLQ, which contains the full length 3-quinuclidinone reductase gene was constructed. Using Escherichia coli cells coexpressing the 3-quinuclidinone reductase and glucose dehydrogenase (cofactor regeneration enzyme) genes, 618 mM 3-quinuclidinone was almost stiochiometrically converted to (R)-3-quinuclidinol with an >99.9% enantiomeric excess within 21 h of reaction.     

Molecular and biochemical characterisation of a Teladorsagia circumcincta glutamate dehydrogenase

A full length cDNA encoding glutamate dehydrogenase was cloned from Teladorsagia circumcincta (TcGDH). The TcGDH cDNA (1614 bp) encoded a 538 amino acid protein. The predicted amino acid sequence showed 96% and 93% similarity with Haemonchus contortus and Caenorhabditis elegans GDH, respectively. A soluble N-terminal 6xHis-tagged GDH protein was expressed in the recombinant Escherichia coli strain BL21 (DE3) pGroESL, purified and characterised. The recombinant TcGDH had similar kinetic properties to those of the enzyme in homogenates of T. circumcincta, including greater activity in the aminating than deaminating reaction. Addition of 1 mM ADP and ATP increased activity about 3-fold in the deaminating reaction, but had no effect in the reverse direction. TcGDH was a dual co-factor enzyme that operated both with NAD⁺ and NADP⁺, GDH activity was greater in the deaminating reaction with NADP⁺ as co-factor and more with NADH in the aminating reaction.     

Gamma-radiation induced modifications in substrate specificity of glucose dehydrogenase and carbon source utilization pattern of phosphate-solubilizing <Emphasis Type="Italic">Pantoea</Emphasis> strains

Glucose, maltose, and mannose as sole carbon sources, induced synthesis of glucose dehydrogenase (GDH) in three strains of Pantoea with specific activities from 0.14 to 0.6 U/mg proteins. Utilization of lactose indicated that the enzyme belongs to GDH type B isozyme. Of mutant clones, developed through radiation mutagenesis, P2-M2 utilized ribose with GDH specific activity of 0.57 U/mg protein, P4-M3 grown on glucose gave 1.5 U/mg protein and P4-M5 had high activities, when grown on galactose, maltose, and lactose. Clones P3-M2 and P2-M5 had versatile utilization of sugars and released higher amounts of P from tri-calcium phosphate and can be efficiently used for biofertilization.     

Glutamate dehydrogenase from the hyperthermophilic bacterium Thermotoga maritima: molecular characterization and phylogenetic implications

The hyperthermophilic bacterium Thermotoga maritima, which grows at up to 90°C, contains an L-glutamate dehydrogenase (GDH). Activity of this enzyme could be detected in T. maritima crude extracts, and appeared to be associated with a 47-kDa protein which cross-reacted with antibodies against purified GDH from the hyperthermophilic archaeon Pyrococcus woesei. The single-copy T. maritima gdh gene was cloned by complementation in a glutamate auxotrophic Escherichia coli strain. The nucleotide sequence of the gdh gene predicts a 416-residue protein with a calculated molecular weight of 45852. The gdh gene was inserted in an expression vector and expressed in E. coli as an active enzyme. The T. maritima GDH was purified to homogeneity. The NH₂-terminal sequence of the purified enzyme was PEKSLYEMAVEQ, which is identical to positions 2–13 of the peptide sequence derived from the gdh gene. The purified native enzyme has a size of 265 kDa and a subunit size of 47 kDa, indicating that GDH is a homohexamer. Maximum activity of the enzyme was measured at 75°C and the pH optima are 8.3 and 8.8 for the anabolic and catabolic reaction, respectively. The enzyme was found to be very stable at 80°C, but appeared to lose activity quickly at higher temperatures. The T. maritima GDH shows the highest rate of activity with NADH (V _max of 172U/mg protein), but also utilizes NADPH (V _max of 12U/mg protein). Sequence comparisons showed that the T. maritima GDH is a member of the family II of hexameric GDHs which includes all the GDHs isolated so far from hyperthermophiles. Remarkably, phylogenetic analysis positions all these hyperthermophilic GDHs in the middle of the GDH family II tree, with the bacterial T. maritima GDH located between that of halophilic and thermophilic euryarchaeota. Received: 15 July 1996 / Accepted: 12 October 1996     

Cloning and Expression of Pyrroloquinoline Quinone (PQQ) Genes from a Phosphate-Solubilizing Bacterium <Emphasis Type="Italic">Enterobacter intermedium</Emphasis>

A grass rhizosphere bacterium, Enterobacter intermedium (60-2G), has a strong ability to solubilize insoluble phosphate. Certain phosphate-solubilizing bacteria secrete gluconic acid for this process. The gluconic acid is produced by direct extracellular oxidation of glucose by a glucose dehydrogenase equipped with pyrroloquinoline quinone (PQQ) as a cofactor. A pqq gene cluster producing PQQ was detected in E. intermedium and this sequence conferred phosphate-solubilizing activity to Escherichia coli DH5. The 6,783-bp pqq sequence had six open reading frames (pqqA, B, C, D, E, and F) and showed 50–95% homology to pqq genes of other bacteria. E. coli DH5 expressing the E. intermedium pqq genes solubilized phosphate from hydroxyapatite after a pH drop to pH 4.0, which paralleled in time the secretion of gluconic acid. We speculate that production of PQQ in E. coli DH5 expressing the pqq cluster activates an endogenous glucose dehydrogenase to permit gluconic acid secretion that solubilizes the insoluble phosphate.     

Effect of anaerobic promoters on the microaerobic production of polyhydroxybutyrate (PHB) in recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis>

Nine anaerobic promoters were cloned and constructed upstream of PHB synthesis genes phbCAB from Ralstonia eutropha for the micro- or anaerobic PHB production in recombinant Escherichia coli. Among the promoters, the one for alcohol dehydrogenase (P _adhE ) was found most effective. Recombinant E. coli JM 109 (pWCY09) harboring P _adhE and phbCAB achieved a 48% PHB accumulation in the cell dry weight after 48 h of static culture compared with only 30% PHB production under its native promoter. Sixty-seven percent PHB was produced in the dry weight (CDW) of an acetate pathway deleted (Δpta deletion) E. coli JW2294 harboring the vector pWCY09. In a batch process conducted in a 5.5-l NBS fermentor containing 3 l glucose LB medium, E. coli JW2294 (pWCY09) grew to 7.8 g/l CDW containing 64% PHB after 24 h of microaerobic incubation. In addition, molecular weight of PHB was observed to be much higher under microaerobic culture conditions. The high activity of P _adhE appeared to be the reason for improved micro- or anaerobic cell growth and PHB production while high molecular weight contributed to the static culture condition.     

Efficient synthesis of (R)-3-hydroxypentanenitrile in high enantiomeric excess by enzymatic reduction of 3-oxopentanenitrile

(R)-3-Hydroxypentanenitrile (HPN) is an important intermediate in the synthesis of an immunosuppressive inosine 5′-monophosphate dehydrogenase inhibitor. An efficient enzymatic procedure for the synthesis of (R)-HPN with over 99 % enantiomeric excess using a novel acetoacetyl-CoA reductase (AdKR) from Achromobacter denitrificans was successfully established. Many microorganisms are known to reduce 3-oxopentannitrile (KPN) to (R)-HPN. An enzyme from A. denitrificans partially purified using ion exchange chromatography reduced KPN to (R)-HPN with high enantioselectivity. The AdKR gene was cloned and sequenced and found to comprise 738 bp and encode a polypeptide of 26,399 Da. The deduced amino acid sequence showed a high degree of similarity to those of other putative acetoacetyl-CoA reductases and putative 3-ketoacyl-ACP reductases. The AdKR gene was singly expressed and coexpressed together with a glucose dehydrogenase (GDH) as a coenzyme regenerator in Escherichia coli under the control of the lac promoter. (R)-HPN was synthesized with over 99 % e.e. using a cell-free extract of recombinant E. coli cells coexpressing AdKR and GDH.     

Quantitative aspects of glucose metabolism by Escherichia coli B/r,grown in the presence of pyrroloquinoline quinone

Escherichia coli B/r was grown in chemostat cultures under various limitations with glucose as carbon source. Since E. coli only synthesized the glucose dehydrogenase (GDH) apo-enzyme and not the appropriate cofactor, pyrroloquinoline quinone (PQQ), no gluconate production could be observed. However, when cell-saturating amounts of PQQ (nmol to mol range) were pulsed into steady state glucose-excess cultures of E. coli, the organisms responded with an instantaneous formation of gluconate and an increased oxygen consumption rate. This showed that reconstitution of GDH in situ was possible.Hence, in order to examine the influence on glucose metabolism of an active GDH, E. coli was grown aerobically in chemostat cultures under various limitations in the presence of PQQ. It was found that the presence of PQQ indeed had a sizable effect: at pH 5.5 under phosphate- or sulphate- limited conditions more than 60% of the glucose consumed was converted to gluconate, which resulted in steady state gluconate concentrations up to 80 mmol/l. The specific rate of gluconate production (0.3–7.6 mmol·h^-1·(g dry wt cells)^-1) was dependent on the growth rate and the nature of the limitation. The production rate of other overflow metabolites such as acetate, pyruvate, and 2-oxoglutarate, was only slightly altered in the presence of PQQ. The fact that the cells were now able to use an active GDH apparently did not affect apo-enzyme synthesis.Abbreviations HEPES N-2-hydroxy-ethylpiperazine-N-2-ethane sulphonic acid - MES 2-morpholinoethane sulphonic acid - PQQ pyrroloquinoline quinone (systematic name: 2,7,9-tricarboxy-1H-pyrrolo-(2,3-f)-quinoline-4,5-dione) - WB Wurster's Blue (systematic name: 1,4-bis-(dimethylamino)-benzene perchlorate