Introduction

Pyrroloquinoline quinone (PQQ), a redox cofactor, has been reported to occur in dehydrogenases, oxidases, oxygenases, hydratases, and decarboxylases [1]. As PQQ has an orthoquinone structure that is directly responsible for oxidoreduction, the role of these quinoproteins is to catalyze the primary oxidation step of non-phosphorylated substrates, such as alcohols, aldehydes, or aldoses. Consequently, PQQ is considered to the third type of coenzyme, after pyridine nucleotides and flavins, in biological oxidoreduction [2].

PQQ has been found in both prokaryotic and eukaryotic organisms, such as Klebsiella pneumoniae, Methylobacterium extorquens, Pseudomonas aeruginosa, Polyporus versicolor, and Rhus vernicifera [3]. Interestingly, the enteric bacterium, Escherichia coli, is unable to produce PQQ [4]. The pqq genes involved in PQQ synthesis have been identified in several bacterial species. In K. pneumoniae, the PQQ biosynthetic genes are clustered in the pqqABCDEF operon [5]. M. extorquens AM1 contains a pqqABC/DE operon in which the pqqC and pqqD genes are fused, while the pqqFG genes form an operon with three other genes [6–8]. In P. aeruginosa, the pqqABCDE operon is separated from the pqqF operon [9]. The corresponding pqq genes are highly conserved among the various species, with the exception of K. pneumoniae pqqF.

The pqq cluster of Gluconobacter oxydans ATCC 9937 was cloned and sequenced in 2000 [10]. It has five genes, namely, pqqABCDE. In 2005, the sequencing of the whole genome of G. oxydans 621H revealed that it has a pqqABCDE operon that shares high sequence similarity with the pqqABCDE operon from G. oxydans ATCC 9937 [11]. Holscher more recently reported that, in addition to the pqqABCDE cluster, a gene showing high homology to the E. coli tldD gene is also essential for PQQ biosynthesis in G. oxydans 621H. This gene, tldD, from G. oxydans 621H, may have the same function as the pqqF genes found in other PQQ-synthesizing bacteria [12].

PQQ biosynthesis in E. coli has been successfully achieved through expression of the pqq gene clusters of Acinetobacter calcoaceticus and K. pneumoniae [13, 14]. In the former case, PQQ production was distinctly low, but in the latter case, 280 nM PQQ was accumulated in the medium. The most likely explanation is that PQQ biosynthesis takes place in the cytoplasm, and the PQQ is then released into the medium.

We report here the cloning of the pqqABCDE gene cluster from G. oxydans M5. E. coli strains harboring various plasmids containing the pqqABCDE gene cluster were found to accumulate PQQ in large amounts in the medium. We also found that the growth rate of the recombinants was significantly improved in minimal glucose medium.

Materials and methods

Materials

Phenazine methosulfate (PMS), PQQ, and 2,6-dichlorophenolindophenol (DCIP) were purchased from Sigma–Aldrich (St. Louis, MO). LA Taq polymerase and restriction and modification enzymes were purchased from TaKaRa (Dalian, China). d-sorbitol, d-mannitol, d-arabitol, and other sugar alcohols were obtained from Amresco (Shanghai Genebase Co., Shanghai, China). Other chemicals were obtained commercially and were of reagent grade.

Bacterial strains, plasmids and cultivation

G. oxydans M5 was selected in our laboratory to produce l-sorbose from d-sorbitol [15]. The pMD19-T kit was purchased from TaKaRa. G. oxydans M5 was grown in d-sorbitol medium consisting of 20 g d-sorbitol, 3 g yeast extract, 10 g polypeptone, 1 g KH2PO4, and 0.2 g MgSO4–7H2O in 1 l deionized water. E. coli (JM109, BL21) and recombinant strains were grown in Luria–Bertani (LB) medium at 37°C. For the analysis of d-glucose dehydrogenase (GDH) activity, the strains were pre-cultured in LB medium. The pre-culture cells were washed twice with 50 mM phosphate buffer (pH 7.0) and resuspended in the same buffer at the same concentration as in the original pre-culture. The suspension (inoculum size 5%, v/v) was then transferred to 100 ml glucose minimal medium (5 g d-glucose, 2 g citrate, 10 g potassium phosphate dibasic, and 3.5 g sodium ammonium phosphate in 1 l tap water). The pH was adjusted to 7.0. The cultures were grown at 37°C under vigorous agitation, and cells were harvested at the late-exponential phase after about 25 h of incubation. The antibiotics (Sigma–Aldrich) were used in the medium at the following concentrations: kanamycin, 25 μg/ml; ampicillin, 100 μg/ml.

Preparation of membrane fractions

Cells were collected by centrifugation, washed with 50 mM potassium phosphate buffer (pH 7), and resuspended in the same buffer at a concentration of 0.2 g/ml wet cells. Following the addition of a few grains of DNase powder, the suspension was passed twice through a high-pressure laboratory homogenizer (ATS, Italy) and centrifuged at 12,000 g for 20 min to remove intact cells and cell debris. The membranes were precipitated by centrifugation at 120,000 g for 60 min and homogenized with 50 mM potassium phosphate buffer (pH 7).

d-glucose dehydrogenase assay

GDH activities were determined photometrically at 600 nm in a dye-linked system containing DCIP and PMS at 30°C before or after holo-enzyme formation. The reaction mixture contained enzyme solution, phosphate buffer pH 6.4, 200 mM substrate, 0.67 mM PMS, 0.1 mM DCIP, and 4 mM sodium azide. One unit of dehydrogenase activity was defined as the reduction of 1 μmol/min DCIP, corresponding to the oxidation of 1 μmol/min substrate. The millimolar extinction coefficient of DCIP is 12.6 mM−1 cm−1 at pH 6.4.

Protein concentrations were determined as described by Bradford [16], using bovine serum albumin as the standard.

PQQ determination

The presence of PQQ in culture supernatants was determined using crude membranes from E. coli containing apo-glucose dehydrogenase [17]. Holo-enzyme was prepared by incubating 250 μl of membrane fractions (approximately 0.4 mg protein) at 30°C for 30 min in 50 mM potassium phosphate buffer (pH 6.4) containing 250 μl of sample or a specific amount of PQQ standard (0–10 ng) and 10 mM MgSO4, resulting in 500 μl of enzyme solution. GDH activity was measured as described above after a 5-min incubation in 3 ml of a reaction mixture containing 100 μl solution of enzyme and 0.1% Triton X-100. The reaction was started by the addition of d-glucose substrate. The concentration of PQQ was determined from a standard curve prepared with 0–10 ng PQQ in the reaction mixture.

Construction of expression vectors of the pqqABCDE gene cluster

DNA manipulations were performed according to standard protocols [18]. Genomic DNA isolated from G. oxydans M5 was used as a template for the PCR analysis. The pqqABCDE gene cluster operon sequence information in the whole genome sequence of G. oxydans 621H (accession number CP000009) was used to design the primers according to the methods of Hölscher [12]. The primer sequences and restriction enzymes used in this study are listed in Table 1. Amplification of these gene fragments was performed by PCR using LA DNA polymerase (TaKaRa) as follows: an initial denaturation at 95°C for 5 min, followed by 30 cycles of 45 s at 95°C, 45 s at 55°C, and 240 s at 72°C. A 3.7-kb DNA fragment of G. oxydans M5 bearing the pqqABCDE operon and parts of the upstream and downstream open reading frames (ORFs) was amplified using the primers of pqqF and pqqR and then cloned with T-A vector of pMD19-T, resulting in plasmid pMD19-PQQ. For expression of the pqqABCDE gene cluster with different promoters, the open reading fragment of the pqqABCDE gene cluster was amplified from pMD19/PQQ using two pairs of primers (pqqPLF and pqqPLR; pqqPT7F and pqqPT7R), respectively, and subcloned into the plasmid pUC18 between the EcoRI–PstI sites downstream of the LacZ promoter, resulting in the plasmid pUC18-PLPQQ, and into pET28a between the NdeI–EcoRI sites downstream of T7 promoter, resulting in pET28a-T7PQQ, respectively. The insert fragments were sequenced (Invitrogen, Shanghai, China).

Table 1 Sequences of primers used in this study
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Results and discussion

Cloning and sequencing of the pqqABCDE gene cluster

The full-length gene cluster of pqqABCDE (3.7 kb) was amplified from the chromosome of G. oxydans M5 by PCR. The amplified fragment was cloned in plasmid pMD19-T, resulting in pMD19-PQQ. Sequencing of the fragment revealed that it contained five ORFs that are 100% identical with the genome sequence of G. oxydans 621H [11]. This 100% sequence identity between G. oxydans M5 and G. oxydans 621H was unexpected as the G. oxydans 621H strain had never been grown in our laboratory and, therefore, could not have resulted from contamination with the DNA from the 621H strain. The five ORFs were named A, B, C, D, E and consisted of 81, 915, 720, 291, and 1,077 bp, encoding 27, 305, 240, 97, and 359 amino acids, respectively.

Overexpression of the gene cluster in E. coli using different vectors

To improve PQQ synthesis in E. coli, we used standard procedures to construct two expression vectors containing the gene cluster under the control of the LacZ and T7 promoters, respectively. Following the transformation of E. coli with the two vectors, colonies having an ampicillin-resistant phenotype were selected.

When the cultures were grown initially at 37°C, recombinant proteins appeared to be mainly present in inclusion bodies. We therefore reduced both the incubation temperature and the isopropyl-beta-thio galactopyranoside (IPTG) concentration. At a culture temperature of 30°C and 0.5 mM IPTG, we detected a large amount of soluble expressed protein. The predicted protein products of the gene cluster, PqqA, PqqB, PqqC, PqqD, and PqqE, have calculated molecular weights of 2.9, 32.9, 25.9, 10.5, 38.77 kDa, respectively. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) analysis of the soluble fraction resulted in two distinct protein bands with molecular masses of about 40 and 32 kDa, which approximate the calculated molecular weights of PqqB and PqqE (Fig. 1). Under these expression conditions, the presence of the predicted smaller molecular weights proteins could not be detected by SDS–PAGE.

Fig. 1
figure 1

Expression of pqqABCDE in Escherichia coli. Lanes: 1 Supernatant of cell-free extracts of BL21, 2 supernatant of cell-free extracts of recombinant BL21 (DE3) containing pET28a, 3 supernatant of cell-free extracts of recombinant pET28a-T7PQQ, M, molecular mass standard

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Restoration of E. coli GDH activity by expression of the pqqABCDE gene cluster

Glucose dehydrogenase (EC 1.1.99.17) is a widespread quinoprotein present in many species, including members of the Enterobacteriaceae, Pseudomonas, and Acinetobacter. However, in E. coli, which is unable to synthesize PQQ, GDH is present as apo-GDH [4]. In this study, the GHD activity of E. coli was restored by expression of the pqqABCDE gene cluster. The recombinant strains JM109/pUC18-PLPQQ, JM109/pMD19-PQQ, and BL21/pET28a-T7PQQ produced small clear zones surrounding colonies grown on glucose-calcium carbonate agar; in comparison, there were no such zones surrounding colonies of the wild-type strain. In the former,, the glucose in the medium was oxidized to yield gluconate, which can produce a clear zone on a glucose-calcium carbonate agar plate. To quantitatively assess these results, we further assayed GDH activity using membrane fractions prepared as described in the Materials and Methods. These strains were cultured in minimal glucose medium in order to avoid interference from pre-formed PQQ. As shown in Table 2, the recombinants showed higher PQQ–GDH activities than their wild-type counterpart, indicating that PQQ was synthesized in E. coli through the expression of the pqqABCDE gene cluster alone. In an earlier study, Hölscher reported that a gene showing high homology to the E. coli tldD gene, in addition to the pqqABCDE cluster, is also involved in PQQ biosynthesis in G. oxydans 621H [12]. Therefore, this result suggests that the E. coli tldD gene carries out the same function as that of the tldD of G. oxydans.

Table 2 Enzyme activities involved in glucose oxidation
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Effect of the pqqABCDE gene cluster expression in E. coli on growth

The pqqABCDE gene cluster expression in E. coli had a significant effect on growth. When E. coli JM109 and recombinant E. coli JM109/pUC18-PLPQQ or JM109/pMD19-PQQ containing the pqqABCDE gene cluster plasmid were incubated on LB agar plates at 37°C for 14 h, the recombinant colonies were larger than those of the wild type (Fig. 2). Furthermore, when these stains were cultured in minimal d-glucose medium, the length of the lag phase was only 5 h for both of the recombinant stains; in contrast, the lag phase was 10 h for the wild type. This shows that the length of the lag phase in the recombinant lines was markedly reduced compared with the wild type. Similar effects were found for BL21 and its derivatives controlled by the T7 promoter.

Fig. 2
figure 2

Colonial morphology of recombinant E. coli strains JM109/pUC18-PLPQQ(white color) and JM109/pUC18 (blue color). The cells were aerobically grown on LB agar plates at 37°C for 14 h

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These results indicate that PQQ production resulting from the pqqABCDE gene cluster can improve the growth rate of recombinants and reduce the length of the lag phase. We therefore suggest that the oxidative metabolism of glucose in E. coli via PQQ–PDH was activated as a result of PQQ synthesis in recombinants and that gluconate, the product of glucose oxidation, is used as a growth substrate. As such, this provides an alternative route for glucose utilization via the Entner–Doudoroff pathway, in addition to the PTS (glucose phosphotransferase system). The results are comparable to those obtained following the addition of PQQ in vitro [19, 20]. The nutritional value of PQQ as a vitamin or growth factor for eukaryotic cells has been also pointed out [21].

PQQ biosynthesis in E. coli using different expression vectors

To further investigate the effects of pqqABCDE gene cluster expression on PQQ synthesis, we monitored PQQ concentration during the growth of the three recombinants using wild-type E. coli crude membrane fractions as a source of apo-GDH. A vigorously growing exponential culture (inoculum size 1%, v/v) was inoculated into fresh minimal glucose medium, samples were taken at intervals, and the samples were analyzed for PQQ. The results are summarized in Fig. 3. These findings parallel the results obtained during the growth analysis: the wild-type E. coli strain was not able to synthesize PQQ. In contrast, the E. coli JM109/pMD19-PQQ recombinant strain was able to produce PQQ up to 1,100 ng/ml. Even larger amounts of PQQ were found in cultures of the E. coli JM109/pUC18-PLPQQ or BL21/pET28a-T7PQQ recombinant strains. Using these two latter strains, nearly 2,000 ng/ml (6 mM) PQQ had accumulated 38 h after the start of induction by IPTG. This amount of synthesized PQQ is 21.5-fold higher than that observed in previous studies (280 nM, i.e., 92.4 ng/ml) [14] and demonstrates that the rather elaborate biosynthesis of PQQ can be enhanced significantly through the overexpression of the pqqABCDE gene cluster.

Fig. 3
figure 3

PQQ synthesis using the pqqABCDE gene cluster overexpression strains. The time-course of pyrroloquinoline quinone accumulation (PQQ) in the culture supernatant when JM109/pMD19-PQQ (filled square), JM109/pUC18-PLPQQ (filled circle), and BL21/pET28a-T7PQQ (filled triangle) were grown in minimal glucose medium was monitored. Average values from three replicates are shown

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