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Methylobacterium sp. CLZ was isolated from soil contaminated with chemical wastewater. This strain simultaneously synthesizes Pyrroloquinoline quinone (PQQ), Coenzyme Q10 (CoQ10), and carotenoids by utilizing methanol as a carbon source. Comparative genomic analysis was performed for five Methylobacterium strains. As per the outcomes, the Methylobacterium CLZ strain showed the smallest genome size and the lowest number of proteins. Thus, it can serve as an ideal cell model for investigating the biological process of Methylobacterium and constructing genetically engineered Methylobacterium. The Methylobacterium CLZ strain's pqqL gene, which does not occur in other Methylobacterium strains but plays a crucial role in PQQ synthesis. This was a surprising finding for the study of PQQ biosynthesis in Methylobacterium. Methylobacterium sp. NI91 strain was generated by random mutagenesis of CLZ strain, and NI91 strain showed a 72.44% increase in PQQ yield. The mutation in the mxaJ gene involved in the methanol dehydrogenase (MDH) synthesis was identified through comparative genomic analysis of the whole genome of mutant strain NI91 and wild-type strain CLZ. The mxaJ gene was found to be upregulated in the NI91 strain. Thus, the up-regulation of the mxaJ gene could be correlated with the high yield of PQQ, and it could provide valuable clues for strain engineering to improve PQQ production.

Pyrroloquinoline quinone (PQQ) has been recognized as the third class of redox cofactors in addition to the well-known nicotinamides (NAD(P) + ) and flavins (FAD, FMN). It plays important physiological roles in various organisms and has strong antioxidant properties. The biosynthetic pathway of PQQ involves a gene cluster composed of 4-7 genes, named pqqA-G, among which pqqA is a key gene for PQQ synthesis, encoding the precursor peptide PqqA. To produce recombinant PqqA in E. coli, fusion tags were used to increase the stability and solubility of the peptide, as well simplify the scale-up of the fermentation process. In this paper, pqqA from Gluconobacter oxydans 621H was expressed in E. coli BL21 (DE3) as a fusion protein with SUMO and purified using a hexahistidine (His6) tag. The SUMO fusion protein and His6 tag were specifically recognized and cleaved by the SUMO specific ULP protease, and immobilized-metal affinity chromatography was used to obtain high-purity precursor peptide PqqA. Expression and purification of target proteins was confirmed by Tricine-SDS-PAGE. Finally, the synthesis of PQQ in a cell-free enzymatic reaction in vitro was confirmed by LC-MS., (Copyright © 2020 Elsevier Inc. All rights reserved.)

6-(N-hydroxyethyl)-amino-6-deoxy-l-sorbofuranose (6NSL), a key precursor in the synthesis of miglitol, is produced from N-2-hydroxyethyl-glucamine (NHEG) by the regioselective oxidation of Gluconobacter oxydans. The limitation of PQQ biosynthesis became a bottleneck for improvement of PQQ-dependent D-sorbitol dehydrogenase (mSLDH) activity. Five expression plasmids were constructed for the co-expression of the pqqABCDE gene cluster and the tldD gene on the basis of pBBR1-gHp0169-sldAB in G. oxydans to increase the biosynthesis of PQQ. The G. oxydans/pGA004, in which pqqABCDE and tldD were expressed as a cluster under the control of gHp0169 promoter, showed the optimal performance. The intracellular PQQ concentration and specific activity of mSLDH in cells increased by 79.3 % and 53.7 %, respectively, compared to that in G. oxydans/pBBR-sldAB. Then, the repeated batch biotransformation of NHEG to 6NSL by G. oxydans/pGA004 was carried out. Up to 75.0 ± 3.0 g/L of 6NSL production with 94.5 ± 3.6 % of average conversion rate of NHEG to 6NSL was achieved after four cycles of run. These results indicated that G. oxydans/pGA004 with high productivity had great potential for 6NSL production in industrial bioprocess., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Oxygen is a vital parameter for pyrroloquinoline quinone (PQQ) biosynthesis. In this study, the effects of oxygen supply on the biosynthesis of PQQ were first investigated systematically with Hyphomicrobium denitrificans FJNU-6. Following a kinetic analysis of the specific cell growth rate (μ x ) and specific PQQ formation rate (μ p ) in 5 L benchtop fermentation systems at various oxygen supply levels ranging from 0 to 60%, a novel, two-stage oxygen supply strategy was developed for enhancing PQQ production and productivity. Moreover, the transcription of genes involved in methanol oxidation and PQQ biosynthesis was analyzed throughout the process to outline the effect of oxygen supply on cell metabolism. Furthermore, with constant feeding of methanol at 0-1 g/L after the initial methanol was consumed completely, the PQQ concentration and productivity reached 1070 mg/L and 7.64 mg/L/h, respectively, after 140 h in a 5-L fermenter. The two-stage oxygen supply strategy developed in this study provides an effective and economical strategy for the industrial production of PQQ.Key Points• A novel, two-stage oxygen supply strategy was developed for enhancing PQQ production and productivity.•The transcription of genes involved in methanol oxidation and PQQ biosynthesis was regulated by changes in oxygen supply.• This study offers an effective and economical strategy for industrial or large-scale production of PQQ.

Pyrroloquinoline quinone (PQQ), an important redox enzyme cofactor, has many physiological and biochemical functions, and is widely used in food, medicine, health and agriculture industry. In this study, PQQ production by recombinant Gluconobacter oxydans was investigated. First, to reduce the by-product of acetic acid, the recombinant strain G. oxydans T1 was constructed, in which the pyruvate decarboxylase (GOX1081) was knocked out. Then the pqqABCDE gene cluster and tldD gene were fused under the control of endogenous constitutive promoter P0169, to generate the recombinant strain G. oxydans T2. Finally, the medium composition and fermentation conditions were optimized. The biomass of G. oxydans T1 and G. oxydans T2 were increased by 43.02% and 38.76% respectively, and the PQQ production was 4.82 and 20.5 times higher than that of the wild strain, respectively. Furthermore, the carbon sources and culture conditions of G. oxydans T2 were optimized, resulting in a final PQQ yield of (51.32±0.899 7 mg/L), 345.6 times higher than that of the wild strain. In all, the biomass of G. oxydans and the yield of PQQ can be effectively increased by genetic engineering.

Plant-growth promoting rhizobacteria benefit crop health and growth through various mechanisms including phosphate and potassium solubilisation, and antimicrobial activity. Previously, we sequenced the genome of bacterial strain Burkholderia cenocepacia CR318, which was isolated from the roots of the starch corn (Zea mays L.) in London, Ontario, Canada. In this work, the species identity of this isolate is confirmed by recA phylogeny and in silico DNA-DNA hybridization (isDDH), and its plant-growth promoting characteristics are described. B. cenocepacia CR318 exhibited strong activity of inorganic phosphate and potassium solubilization. It significantly promoted the growth of corn plants and roots by solubilizing inorganic tricalcium phosphate under greenhouse conditions. Functional analysis of the complete B. cenocepacia CR318 genome revealed genes associated with phosphate metabolism such as pstSCAB encoding a high affinity inorganic phosphate-specific transporter, and the pqqABCDE gene cluster involved in the biosynthesis of pyrroloquinoline quinone (PQQ), which is a required cofactor for quinoprotein glucose dehydrogenase (Gdh). However, it appears that B. cenocepacia CR318 lacks the quinoprotein Gdh which can produce gluconic acid to solubilize inorganic phosphate. Overall, these findings provide an important step in understanding the molecular mechanisms underlying the plant growth promotion trait of B. cenocepacia CR318., Competing Interests: Declaration of Competing Interest No conflict of interest declared., (Crown Copyright © 2019. Published by Elsevier GmbH. All rights reserved.)

6-(N-hydroxyethyl) amino-6-deoxy-l-sorbofuranose (6NSL) is the direct precursor of miglitol for diabetes therapy. The regio- and stereo-selective dehydrogenation offered by the membrane-bound d-sorbitol dehydrogenase (mSLDH) from Gluconobacter oxydans provides an elegant enzymatic method for 6NSL production. In this study, two subunits sldA and sldB of mSLDH were introduced into G. oxydans ZJB-605, and the specific enzyme activity of mSLDH towards NHEG was enhanced by 2.15-fold. However, the endogenous PQQ level was dramatically reduced in the recombinant strain and became a bottleneck to support the holo-enzyme activity. A combined supplementation of four amino acids (Glu, Ile, Ser, Arg) involved in biosynthesis of PQQ in conventional media effectively increased extracellular accumulation of PQQ by 1.49-fold, which further enhanced mSLDH activity by 1.33-fold. The synergic improvement of mSLDH activity provided in this study supports the superior high dehydrogenate activity towards substrate N-2-hydroxyethyl-glucamine, 184.28 g·L -1 of 6NSL was produced after a repeated bioconversion process catalyzed by the resting cells of G. oxydans/pBB-sldAB, all of which presenting a great potential of their industrial application in 6NSL biosynthesis., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Pyrroloquinoline quinone (PQQ) is a bacterial dehydrogenase coenzyme. PQQ can promote body growth and regulate the function of free radical level of the body. It could be applied in food, medicine and other fields. Due to the extremely high cost of chemical synthesis, the production of PQQ by microbial fermentation attracted more and more attention. At present, the production titer of PQQ by fermentation method is too low to achieve industrial application. Due to the lack of a thorough understanding of the PQQ biosynthesis and its regulation mechanisms, and the lack of necessary genetic engineering modification methods for wild type strains, metabolic engineering of microorganisms to enhance PQQ production still lacks essential requirements. In this study, a PQQ-producing bacterium, Methylobacterium extorquens I-F2, was employed as a model strain. By integration of Atmospheric and room temperature plasma (ARTP) mutagenesis, flow cytometry sorting and high-throughput screening strategies, optimization of sample preparation and flow sorting process, a high-titer PQQ mutant strain was obtained. The titer of PQQ was increased by 98.02% compared with that of M. extorqunens I-F2. The process described here showed that the combination of the flow cytometry with high-throughput screening method can be used to obtain high-titer mutants more simply and rapidly, compared with genetic engineering and traditional screening methods.

PqqE is the first enzyme in the biosynthetic pathway of the redox cofactor pyrroloquinoline quinone (PQQ), catalyzing the formation of a carbon-carbon bond in the precursor peptide PqqA. PqqE is a radical S-adenosyl-l-methionine (SAM) (RS) enzyme, a family of enzymes that use the reductive cleavage of a [4Fe-4S] cluster-bound SAM molecule to generate a 5'-deoxyadenosyl radical. This radical is then used to initiate an array of reactions that otherwise would be unlikely to occur. PqqE is a founding member of a subset family of RS enzymes that, additionally to the SAM [4Fe-4S] cluster, have a SPASM domain containing additional, auxiliary Fe-S clusters. Most radical SAM enzymes are highly sensitive to oxygen, which destroys their Fe-S clusters. This can pose several limitations when working with these enzymes, since most of the work has to be done under anaerobic conditions. Here, we summarize the methods developed in our lab for the expression and purification of PqqE. We also highlight the several methods we have used for the characterization of the enzyme., (© 2018 Elsevier Inc. All rights reserved.)

The effects of pH control strategy and fermentative operation modes on the biosynthesis of pyrroloquinoline quinine (PQQ) were investigated systematically with Methylobacillus sp. CCTCC M2016079 in the present work. Firstly, the shake-flask cultivations and benchtop fermentations at various pH values ranging from 5.3 to 7.8 were studied. Following a kinetic analysis of specific cell growth rate (μ x ) and specific PQQ formation rate (μ p ), the discrepancy in optimal pH values between cell growth and PQQ biosynthesis was observed, which stimulated us to develop a novel two-stage pH control strategy. During this pH-shifted process, the pH in the broth was controlled at 6.8 to promote the cell growth for the first 48 h and then shifted to 5.8 to enhance the PQQ synthesis until the end of fermentation. By applying this pH-shifted control strategy, the maximum PQQ production was improved to 158.61 mg/L in the benchtop fermenter, about 44.9% higher than that under the most suitable constant pH fermentation. Further fed-batch study showed that PQQ production could be improved from 183.38 to 272.21 mg/L by feeding of methanol at the rate of 11.5 mL/h in this two-stage pH process. Meanwhile, the productivity was also increased from 2.02 to 2.84 mg/L/h. In order to support cell growth during the shifted pH stage, the combined feeding of methanol and yeast extract was carried out, which brought about the highest concentration (353.28 mg/L) and productivity (3.27 mg/L/h) of PQQ. This work has revealed the potential of our developed simple and economical strategy for the large-scale production of PQQ.

Biosynthesis of the ribosomally synthesized and post-translationally modified peptide (RiPP), pyrroloquinoline quinone (PQQ), is initiated when the precursor peptide, PqqA, is recognized and bound by the RiPP precursor peptide recognition element (RRE), PqqD, for presentation to the first enzyme in the pathway, PqqE. Unlike other RiPP-producing, postribosomal peptide synthesis (PRPS) pathways in which the RRE is a component domain of the first enzyme, PqqD is predominantly a separate scaffolding protein that forms a ternary complex with the precursor peptide and first tailoring enzyme. As PqqD is a stable, independent RRE, this makes the PQQ pathway an ideal PRPS model system for probing RRE interactions using nuclear magnetic resonance (NMR). Herein, we present both the solution NMR structure of Methylobacterium extorquens PqqD and results of 1 H- 15 N HSQC binding experiments that identify the PqqD residues involved in binding the precursor peptide, PqqA, and the enzyme, PqqE. The reported structural model for an independent RRE, along with the mapped binding surfaces, will inform future efforts both to understand and to manipulate PRPS pathways.

Objective: Antioxidants, chelating agents, and probiotics are used to manage the toxic effects of cadmium (Cd) and mercury (Hg). The aim of this study was to investigate the combined effects of antioxidants, chelating agents, and probiotics against heavy metal toxicity., Method: Genetically modified probiotic Escherichia coli Nissle 1917 (EcN-20) producing a potent water soluble antioxidant pyrroloquinoline quinone (PQQ) was supplemented with oral citric acid and compared with another genetically modified probiotic EcN-21 producing PQQ and citric acid against oxidative stress induced by Cd and Hg. Rats were independently given 100 ppm Cd and 80 ppm Hg in drinking water for 4 wk., Results: EcN-20 was found to be more effective than EcN-2 (EcN strain with genomic integration of vgb and gfp genes) with orally given PQQ against oxidative stress induced by Cd and Hg. EcN-20 supplemented with oral citric acid was more effective against Cd and Hg toxicity compared with EcN-2+citric acid (oral), EcN-2+PQQ (oral), EcN-2+PQQ (oral)+citric acid (oral), EcN-20, and EcN-21. However, protection shown by EcN-21 was similar to EcN-20., Conclusion: The combination therapy involving probiotic EcN-20 producing PQQ with citric acid given orally was found to be a moderately effective strategy against toxicity induced by Cd and Hg, whereas the protective effect of EcN-21 was the same as EcN-20., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Objective: To analyze the effect of high 2-keto-L-gulonic acid (2-KLG) on important dehydrogenase, cofactor and transport proteins involved in 2-KLG synthesis., Methods: First, the growth of Gluconobacter oxydans under high 2-KLG was observed. The real-time PCR was used to detect the expression of key sorbitol dehydrogenase gene sldAB, pyrroloquinoline quinone (PQQ) biosynthesis gene cluster pqqABCDE, and five genes encoding hypothetic PQQ transport proteins., Results: According to results of the growth of G. oxydans under different 2-KLG concentration, 40, 80 and 120 g/L 2-KLG were decided to stimulate strains. Real-time PCR showed that PQQ synthesis genes pqqABCDE were not affected by high 2-KLG, but sorbitol dehydrogenase genes sldAB and part of genes encoding PQQ transport proteins were down-regulated under high 2-KLG stress., Conclusion: The expression of sorbitol dehydrogenase genes was restrained by high 2-KLG, PQQ transport was probably inhibited, but PQQ synthesis was not affected.

As a novel cofactor of oxidoreductase, pyrroloquinoline quinone (PQQ) has a great potential of application in medicine, food industries. In order to improve the efficiency of the PQQ production by Methylobacterium extorquens AM1, the strain was treated by atmospheric and room temperature plasma (ARTP). Positive mutants with changes in PQQ yield were obtained based on a high-throughput screening approach. After ARTP treatment, analysis data show that the positive mutation rate was 31.6%. Furthermore, we obtained an excellent positive mutant M. extorquens AM1 (E-F3) with the yield of 54.0 mg/L PQQ, which was approximately 3 times as much compared with that of the wild-type strain. The robust high-throughput screening method for mutagenesis by ARTP improves PQQ production. In addition, this method also provides a new strategy for further strain improvement.

Methylobacterium extorquens AM1 is an aerobic facultative methylotroph known to secrete pyrroloquinoline quinone (PQQ), a cofactor of a number of bacterial dehydrogenases, into the culture medium. To elucidate the molecular mechanism of PQQ biosynthesis, we are focusing on PqqE which is believed to be the enzyme catalysing the first reaction of the pathway. PqqE belongs to the radical S-adenosyl-l-methionine (SAM) superfamily, in which most, if not all, enzymes are very sensitive to dissolved oxygen and rapidly inactivated under aerobic conditions. We here report that PqqE from M. extorquens AM1 is markedly oxygen-tolerant; it was efficiently expressed in Escherichia coli cells grown aerobically and affinity-purified to near homogeneity. The purified and reconstituted PqqE contained multiple (likely three) iron-sulphur clusters and showed the reductive SAM cleavage activity that was ascribed to the consensus [4Fe-4S](2+) cluster bound at the N-terminus region. Mössbauer spectrometric analyses of the as-purified and reconstituted enzymes revealed the presence of [4Fe-4S](2+) and [2Fe-2S](2+) clusters as the major forms with the former being predominant in the reconstituted enzyme. PqqE from M.extorquens AM1 may serve as a convenient tool for studying the molecular mechanism of PQQ biosynthesis, avoiding the necessity of establishing strictly anaerobic conditions., (© The Authors 2015. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.)

Pyrroloquinoline quinone is the third redox cofactor after nicotinamide and flavin in bacteria, and its biosynthesis pathway comprise five steps initiated from a precursor peptide PqqA coded by pqqA gene. Methylovorus sp. MP688 is equipped with five copies of pqqA genes. Herein, the transcription of pqqA genes under different conditions by real-time quantitative PCR and β-galactosidase reporter genes are reported. Multiple pqqA genes were proved to play significant roles and contribute differently in PQQ synthesis. pqqA1, pqqA2, and pqqA4 were determined to be dominantly transcribed over the others, and correspondingly absence of any of the three genes caused a decrease in PQQ synthesis. Notably, pqqA was up-regulated in low pH and limited oxygen environment, and it is pqqA2 promoter that could be induced when bacteria were transferred from pH 7.0 to pH 5.5. Deletion analysis revealed a region within pqqA2 promoter inhibiting transcription. PQQ concentration was increased by overexpression of pqq genes under control of truncated pqqA2 promoter. The results not only imply there exist negative transcriptional regulators for pqqA2 but also provide us a new approach to achieve higher PQQ production by deleting the target binding sequence., (Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The actinomycetes isolates (128) which were taken from agricultural soil samples and collected near a rock phosphate processing unit were screened for mineral phosphate-solubilizing (MPS) ability. A significant MPS activity was observed for 30 isolates on various phosphate sources when grown in the National Botanical Research Institute's phosphate broth. CTM396 and CTM397 strains which showed the highest MPS abilities were identified by 16S rDNA sequencing as members of the genus Streptomyces. Their MPS activity was proved to be concomitant with a drop in pH due to the secretion of gluconic acid (GA). This was correlated with the simultaneous detection by PCR of genes gdh [encoding the glucose dehydrogenase (GDH) responsible for GA production from glucose] and pqq (involved in biosynthesis of the pyrroloquinoline quinone cofactor of GDH), as well as the highlighting of GHD enzyme activity, for the first time in a Streptomyces sp. strain producing GA. Furthermore, the 0.05% of humic acids proved to have a stimulatory effect on the growth and the ability of CTM396 to solubilize Gafsa rock phosphate. According to this study, it is possible to use humic acids and Gafsa rock phosphate in association with spores of ad hoc Streptomyces strains as natural and efficient amendments to improve plant growth with no need of costly and pollutant transformation of Gafsa rock phosphate., (© FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

In Pseudomonas species, production of secondary metabolites and exoenzymes is regulated by the GacS/GacA two-component regulatory system. In Pseudomonas fluorescens SBW25, mutations in the Gac-system cause major transcriptional changes and abolished production of the lipopeptide viscosin and of an exoprotease. In contrast to many other Pseudomonas species and strains, inactivation of the Gac-system in strain SBW25 significantly enhanced its antimicrobial activities against oomycete, fungal and bacterial pathogens. Here, random plasposon mutagenesis of the gacS mutant led to the identification of seven mutants with reduced or loss of antimicrobial activity. In four mutants, the plasposon insertion was located in genes of the pyrroloquinoline quinone (PQQ) biosynthesis pathway. Genetic complementation, ectopic expression, activity bioassays and Reversed-phase high-performance liquid chromatography (RP-HPLC) analyses revealed that a gacS mutation in SBW25 leads to enhanced expression of pqq genes, resulting in an increase in gluconic and 2-ketogluconic acid production, which in turn acidified the extracellular medium to levels that inhibit growth of other microorganisms. We also showed that PQQ-mediated acidification comes with a growth penalty for the gacS mutant in the stationary phase. In conclusion, PQQ-mediated acidification compensates for the loss of several antimicrobial traits in P. fluorescens SBW25 and may help gac mutants to withstand competitors., (© 2014 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Rahnella aquatilis strain HX2 has the ability to promote maize growth and suppress sunflower crown gall disease caused by Agrobacterium vitis, A. tumefaciens, and A. rhizogenes. Pyrroloquinoline quinone (PQQ), a cofactor of aldose and alcohol dehydrogenases, is required for the synthesis of an antibacterial substance, gluconic acid, by HX2. Mutants of HX2 unable to produce PQQ were obtained by in-frame deletion of either the pqqA or pqqB gene. In this study, we report the independent functions of pqqA and pqqB genes in relation to PQQ synthesis. Interestingly, both the pqqA and pqqB mutants of R. aquatilis eliminated the ability of strain HX2 to produce antibacterial substance, which in turn, reduced the effectiveness of the strain for biological control of sunflower crown gall disease. The mutation also resulted in decreased mineral phosphate solubilization by HX2, which reduced the efficacy of this strain as a biological fertilizer. These functions were restored by complementation with the wild-type pqq gene cluster. Additionally, the phenotypes of HX2 derivatives, including colony morphology, growth dynamic, and pH change of culture medium were impacted to different extents. Our findings suggested that pqqA and pqqB genes individually play important functions in PQQ biosynthesis and are required for antibacterial activity and phosphorous solubilization. These traits are essential for R. aquatilis efficacy as a biological control and plant growth promoting strain. This study enhances our fundamental understanding of the biosynthesis of an environmentally significant cofactor produced by a promising biocontrol and biological fertilizer strain.

Pyrroloquinoline quinone (PQQ) is a versatile quinone cofactor participating in numerous biological processes. Klebsiella pneumoniae can naturally synthesize PQQ for harboring intact PQQ synthesis genes. Previous metabolic engineering of K. pneumoniae failed to overproduce PQQ due to the employment of strong promoter in expression vector. Here we report that a moderate rather than strong promoter is efficient for PQQ production. To screen an appropriate promoter, a total of four distinct promoters-lac promoter, pk promoter of glycerol dehydratase gene (dhaB1), promoter of kanamycin resistance gene, and T7 promoter (as the control)-were individually used for overexpressing the endogenous PQQ genes in K. pneumoniae along with heterologous expression in Escherichia coli. We found that all recombinant K. pneumoniae strains produced more PQQ than recombinant E. coli strains that carried corresponding vectors, indicating that K. pneumoniae is superior to E. coli for the production of PQQ. Particularly, the recombinant K. pneumoniae recruiting the promoter of kanamycin resistance gene produced the highest PQQ (1,700 nmol), revealing that a moderate rather than strong promoter is efficient for PQQ production. Furthermore, PQQ production was roughly proportional to glucose concentration increasing from 0.5 to 1.5 g/L, implying the synergism between PQQ biosynthesis and glucose utilization. This study not only provides a feasible strategy for production of PQQ in K. pneumoniae, but also reveals the exquisite synchronization among PQQ biosynthesis, glucose metabolism, and cell proliferation.

Inflammation of the gastrointestinal tract is associated with reactive oxygen species (ROS) genesis. Alleviation of oxidative stress is achieved by using antioxidants and probiotics. Present study investigates a synergistic effect of the probiotic Escherichia coli CFR 16 containing Vitreoscilla haemoglobin gene (vgb), green fluorescent protein (gfp) gene and pyrroloquinoline quinone (pqq) gene cluster on oxidative stress induced by 1,2-dimethylhydrazine (DMH). Adult virgin Charles foster male rats (3-4 months) weighing 200-250 g were administered with DMH (25 mg/kg body weight, s.c.) twice a week for eight consecutive weeks. Rats receiving only DMH dose showed increased lipid peroxidation in liver and intestinal tissues with reduced activity of antioxidant enzymes, i.e. superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx). Oral dose of E. coli CFR 16::vgb-gfp harbouring pqq gene cluster increased rat faecal PQQ concentration by twofold, reduced lipid peroxidation and retained SOD, CAT and GPx activities close to normal levels in liver and colonic tissues following DMH treatment. In addition, significant protection was found in colonic histological sections of these rat groups. This study demonstrates a protective efficacy in the following order: E. coli CFR 16 < E. coli CFR 16::vgb-gfp < vitamin C = PQQ < E. coli CFR 16::vgb-gfp (pqq).

Pyrroloquinoline quinone (PQQ) is a small, redox active molecule that serves as a cofactor for several bacterial dehydrogenases, introducing pathways for carbon utilization that confer a growth advantage. Early studies had implicated a ribosomally translated peptide as the substrate for PQQ production. This study presents a sequence- and structure-based analysis of the components of the pqq operon. We find the necessary components for PQQ production are present in 126 prokaryotes, most of which are Gram-negative and a number of which are pathogens. A total of five gene products, PqqA, PqqB, PqqC, PqqD, and PqqE, are identified as being obligatory for PQQ production. Three of the gene products in the pqq operon, PqqB, PqqC, and PqqE, are members of large protein superfamilies. By combining evolutionary conservation patterns with information from three-dimensional structures, we are able to differentiate the gene products involved in PQQ biosynthesis from those with divergent functions. The observed persistence of a conserved gene order within analyzed operons strongly suggests a role for protein-protein interactions in the course of cofactor biosynthesis. These studies propose previously unidentified roles for several of the gene products, as well as identifying possible new targets for antibiotic design and application.

Many root-colonizing pseudomonads are able to promote plant growth by increasing phosphate availability in soil through solubilization of poorly soluble rock phosphates. The major mechanism of phosphate solubilization by pseudomonads is the secretion of gluconic acid, which requires the enzyme glucose dehydrogenase and its cofactor pyrroloquinoline quinone (PQQ). The main aim of this study was to evaluate whether a PQQ biosynthetic gene is suitable to study the phylogeny of phosphate-solubilizing pseudomonads. To this end, two new primers, which specifically amplify the pqqC gene of the Pseudomonas genus, were designed. pqqC fragments were amplified and sequenced from a Pseudomonas strain collection and from a natural wheat rhizosphere population using cultivation-dependent and cultivation-independent approaches. Phylogenetic trees based on pqqC sequences were compared to trees obtained with the two concatenated housekeeping genes rpoD and gyrB. For both pqqC and rpoD-gyrB, similar main phylogenetic clusters were found. However, in the pqqC but not in the rpoD-gyrB tree, the group of fluorescent pseudomonads producing the antifungal compounds 2,4-diacetylphloroglucinol and pyoluteorin was located outside the Pseudomonas fluorescens group. pqqC sequences from isolated pseudomonads were differently distributed among the identified phylogenetic groups than pqqC sequences derived from the cultivation-independent approach. Comparing pqqC phylogeny and phosphate solubilization activity, we identified one phylogenetic group with high solubilization activity. In summary, we demonstrate that the gene pqqC is a novel molecular marker that can be used complementary to housekeeping genes for studying the diversity and evolution of plant-beneficial pseudomonads.

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., (© 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

PQQ is an exogenous, tricyclic, quino-cofactor for a number of bacterial dehydrogenases. The final step of PQQ formation is catalyzed by PqqC, a cofactorless oxidase. This study focuses on the activation of molecular oxygen in an enzyme active site without metal or cofactor and has identified a specific oxygen binding and activating pocket in PqqC. The active site variants H154N, Y175F,S, and R179S were studied with the goal of defining the site of O(2) binding and activation. Using apo-glucose dehydrogenase to assay for PQQ production, none of the mutants in this "O(2) core" are capable of PQQ/PQQH(2) formation. Spectrophotometric assays give insight into the incomplete reactions being catalyzed by these mutants. Active site variants Y175F, H154N, and R179S form a quinoid intermediate (Figure 1) anaerobically. Y175S is capable of proceeding further from quinoid to quinol, whereas Y175F, H154N, and R179S require O(2) to produce the quinol species. None of the mutations precludes substrate/product binding or oxygen binding. Assays for the oxidation of PQQH(2) to PQQ show that these O(2) core mutants are incapable of catalyzing a rate increase over the reaction in buffer, whereas H154N can catalyze the oxidation of PQQH(2) to PQQ in the presence of H(2)O(2) as an electron acceptor. Taken together, these data indicate that none of the targeted mutants can react fully to form quinone even in the presence of bound O(2). The data indicate a successful separation of oxidative chemistry from O(2) binding. The residues H154, Y175, and R179 are proposed to form a core O(2) binding structure that is essential for efficient O(2) activation.

pqqD is one of six genes required for PQQ production in Klebsiella pneumoniae. Herein, we demonstrate that PqqD interacts specifically with the radical SAM enzyme PqqE, causing a perturbation in the electronic environment around the [4Fe-4S](+) clusters. This interaction redirects the role for PqqD in PQQ biosynthesis.

Objective: To confirm the involvement of pqqL gene of Escherichia coli in PQQ biosynthesis, a pqqL deletion mutant of E. coli DH5alpha was constructed and investigated., Methods: pqqL and kan gene were cloned and a linear targeting fragment pqqL-kan-pqqL was constructed in vitro. Then pqqL gene was knocked out and DH5alphadeltapqqL mutant was constructed by Red homologous recombination. The PQQ biosynthesis was compared between the mutant and its parential strain by detection of bio-activity of sorbose dehydrogenase with DCIP method., Results: The DH5alphadeltapqqL deletion mutant was successfully constructed, and the result indicated that PQQ would be synthesized in pBCP162/DH5alphadeltapqqL and pMD19T Simple-pqqABCDE/DH5alpha, but not in pMD19T Simple-pqqABCDE/DH5alphadeltapqqL., Conclusion: The function of pqqL gene in Escherichia coli is the same to that of pqqF gene.

Phosphate-solubilizing bacteria (PSBs) were isolated from different plant rhizosphere soils of various agroecological regions of India. These isolates showed synthesis of pyrroloquinoline quinone (PQQ), production of gluconic acid, and release of phosphorus from insoluble tricalcium phosphate. The bacterial isolates synthesizing PQQ also showed higher tolerance to ultraviolet C radiation and mitomycin C as compared to Escherichia coli but were less tolerant than Deinococcus radiodurans. Unlike E. coli, PSB isolates showed higher tolerance to DNA damage when grown in the absence of inorganic phosphate. Higher tolerance to ultraviolet C radiation and oxidative stress in these PSBs grown under PQQ synthesis inducible conditions, namely phosphate starvation, might suggest the possible additional role of this redox cofactor in the survival of these isolates under extreme abiotic stress conditions.

Objective: To isolate PQQ biosynthesis gene cluster from Gluconobacter oxydans H24 based on sorbose-dehydrogenase activity., Methods: A library of Gluconobacter oxydans H24 genomic DNA was constructed with host strains Escherichia coli JM109s, which was integrated of sdh gene at the ptsG site on the chromosome of JM109. By detecting sorbose-dehydrogenase activity, clone of PQQ biosynthesis was isolated and subcloned., Results: A positive clone was isolated from Gluconobacter oxydans H24 genomic DNA library. Within the 5,400-base-pair DNA fragment five reading frames are presented, corresponding to five of the pqq genes (pqqABCDE). The nucleotide and amino acid sequence showed highly homology to pqq genes of other bacteria., Conclusion: The pqqABCDE gene cluster was successfully isolated from Gluconobacter oxydans H24 by sorbose dehydrogenase activity.

We have expressed the pqqABCDE gene cluster from Gluconobacter oxydans, which is involved in pyrroloquinoline quinone (PQQ) biosynthesis, in Escherichia coli, resulting in PQQ accumulation in the medium. Since the gene cluster does not include the tldD gene needed for PQQ production, this result suggests that the E. coli tldD gene, which shows high homology to the G. oxydans tldD gene, carries out that function. The synthesis of PQQ activated d-glucose dehydrogenase in E. coli and the growth of the recombinant was improved. In an attempt to increase the production of PQQ, which acts as a vitamin or growth factor, we transformed E. coli with various recombinant plasmids, resulting in the overproduction of the PQQ synthesis enzymes and, consequently, PQQ accumulation--up to 6 mM--in the medium. This yield is 21.5-fold higher than that obtained in previous studies.

Gene PA1990 of Pseudomonas aeruginosa, located downstream of pqqE and encoding a putative peptidase, was shown to be involved in excretion of PQQ into the culture supernatant. This gene is cotranscribed with the pqqABCDE cluster and was named pqqH. A PA1990::Km(r) mutant (VK3) did not show any effect in growth behaviour; however, in contrast to the wild-type, no excretion of PQQ into the culture supernatant was observed. The putative pqqF gene of P. aeruginosa was shown to be essential for PQQ biosynthesis. A pqqF::Km(r) mutant did not grow aerobically on ethanol, because of its inability to produce PQQ. Transcription of the pqqABCDEH operon was induced upon aerobic growth on ethanol, 1-propanol, 1,2-propanediol and 1-butanol, while on glycerol, succinate and acetate, transcription was low. Transcription of the pqqABCDEH operon was also found upon anoxic growth on ethanol with nitrate as electron acceptor, but no PQQ was produced. Expression of the pqqABCDEH operon is regulated at the transcriptional level. In contrast, the pqqF operon appeared to be transcribed constitutively at a very low level under all growth conditions studied.

Rahnella aquatilis HX2, a biocontrol agent for grapevine crown gall caused by Agrobacterium vitis, produces an antibacterial substance that inhibits the growth of A. vitis in vitro. In this study, we show that MH15 and MH16, two Tn5-induced mutants of HX2, have lost their abilities to inhibit A. vitis and have reduced biocontrol activities; they grow in logarithmic phase at a rate similar to that of the wild type and have single Tn5 insertions. They are also impaired in producing pyrroloquinoline quinone (PQQ) or glucose dehydrogenase (GDH). Complementation of MH15 and MH16 with cosmid clones of CP465 and CP104 from an HX2 DNA library restored the antibiosis, biocontrol, and PQQ or GDH production phenotypes. A 6.7-kb BamHI fragment from CP465 that fully restored the MH15-affected phenotypes was cloned and sequenced. Sequence analysis of the mutated DNA region resulted in the identification of seven open reading frames (ORFs), six of which share significant homology with PQQ-synthesizing genes in other bacteria, designated pqqA through pqqF. Meanwhile, A 5.5-kb PstI fragment from CP104 fully complemented the MH16 mutant and contained a single ORF highly similar to that of genes coding for GDHs. An in-frame gdh deletion mutant has the same phenotypes as the Tn5 mutant of MH16. Complementation of both deletion and Tn5 gdh mutants restored the affected phenotypes to wild-type levels. Our results suggest that an antibacterial substance plays a role in biocontrol of A. vitis by HX2.

Biogenesis of pyrroloquinoline quinone (PQQ) in Klebsiella pneumoniae requires the expression of six genes (pqqA-F). One of these genes (pqqE) encodes a 43 kDa protein (PqqE) that plays a role in the initial steps in PQQ formation [Veletrop, J. S., et al. (1995) J. Bacteriol. 177, 5088-5098]. PqqE contains two highly conserved cysteine motifs at the N- and C-termini, with the N-terminal motif comprised of a CX(3)CX(2)C consensus sequence that is unique to a family of proteins known as radical S-adenosyl-l-methionine (SAM) enzymes [Sofia, H. J., et al. (2001) Nucleic Acids Res. 29, 1097-1106]. PqqE from K. pneumoniae was cloned into Escherichia coli and expressed as the native protein and with an N-terminal His(6) tag. Anaerobic expression and purification of the His(6)-tagged PqqE results in an enzyme with a brownish-red hue indicative of Fe-S cluster formation. Spectroscopic and physical analyses indicate that PqqE contains a mixture of Fe-S clusters, with the predominant form of the enzyme containing two [4Fe-4S] clusters. PqqE isolated anaerobically yields an active enzyme capable of cleaving SAM to methionine and 5'-deoxyadenosine in an uncoupled reaction (k(obs) = 0.011 +/- 0.001 min(-1)). In this reaction, the 5'-deoxyadenosyl radical either abstracts a hydrogen atom from a solvent accessible position in the enzyme or obtains a proton and electron from buffer. The putative PQQ substrate PqqA has not yet been shown to be modified by PqqE, implying that PqqA must be modified before becoming the substrate for PqqE and/or that another protein in the biosynthetic pathway is critical for the initial steps in PQQ biogenesis.

Background: The biosynthesis pathway of Pyrroloquinoline quinone, a bacterial redox active cofactor for numerous alcohol and aldose dehydrogenases, is largely unknown, but it is proven that at least six genes in Klebsiella pneumoniae (PqqA-F) are required, all of which are located in the PQQ-operon., Results: New structural data of some PQQ biosynthesis proteins and their homologues provide new insights and functional assignments of the proteins in the pathway. Based on sequence analysis and homology models we propose the role and catalytic function for each enzyme involved in this intriguing biosynthesis pathway., Conclusion: PQQ is derived from the two amino acids glutamate and tyrosine encoded in the precursor peptide PqqA. Five reactions are necessary to form this quinone cofactor. The PqqA peptide is recognised by PqqE, which links the C9 and C9a, afterwards it is accepted by PqqF which cuts out the linked amino acids. The next reaction (Schiff base) is spontaneous, the following dioxygenation is catalysed by an unknown enzyme. The last cyclization and oxidation steps are catalysed by PqqC. Taken together the known facts of the different proteins we assign a putative function to all six proteins in PQQ biosynthesis pathway.

Pseudomonas fluorescens B16 is a plant growth-promoting rhizobacterium. To determine the factors involved in plant growth promotion by this organism, we mutagenized wild-type strain B16 using OmegaKm elements and isolated one mutant, K818, which is defective in plant growth promotion, in a rockwool culture system. A cosmid clone, pOK40, which complements the mutant K818, was isolated from a genomic library of the parent strain. Tn3-gusA mutagenesis of pOK40 revealed that the genes responsible for plant growth promotion reside in a 13.3-kb BamHI fragment. Analysis of the DNA sequence of the fragment identified 11 putative open reading frames, consisting of seven known and four previously unidentified pyrroloquinoline quinone (PQQ) biosynthetic genes. All of the pqq genes showed expression only in nutrient-limiting conditions in a PqqH-dependent manner. Electrospray ionization-mass spectrometry analysis of culture filtrates confirmed that wild-type B16 produces PQQ, whereas mutants defective in plant growth promotion do not. Application of wild-type B16 on tomato (Solanum lycopersicum) plants cultivated in a hydroponic culture system significantly increased the height, flower number, fruit number, and total fruit weight, whereas none of the strains that did not produce PQQ promoted tomato growth. Furthermore, 5 to 1,000 nm of synthetic PQQ conferred a significant increase in the fresh weight of cucumber (Cucumis sativus) seedlings, confirming that PQQ is a plant growth promotion factor. Treatment of cucumber leaf discs with PQQ and wild-type B16 resulted in the scavenging of reactive oxygen species and hydrogen peroxide, suggesting that PQQ acts as an antioxidant in plants.

Pyrroloquinoline quinone (PQQ) is a cofactor of some oxido-reductases with many important physiological effects and potential pharmaceutical applications. The glucose dehydrogenase of Escherichia coli, being a candidate for enzymic detection of PQQ, is known to be a quinoprotein which is obligately dependant on PQQ as cofactor. The gdh gene of E. coli was amplified and cloned into plasmid pET28a. The recombinant GDH was overexpressed in soluble form in E. coli BL21 (DE3). A bioassay method was established for determination of PQQ by the purified GDH. A screening model was set up for the enrichment of methylotrophic bacteria. Together with the above bioassay method, over 2000 soil samples were screened for the isolation of high-yielding PQQ producing strains. A methylotrophic strain, named MP606, was thus isolated. The PQQ production of MP606 is determined to be 113mg/L without conditional optimization and genetic breeding. The PQQ crystal was obtained from the culture supernatant which has been identified by HPLC, absorption spectra assay, and enzymatic analysis. The 16S rDNA of MP606 was amplified and sequenced. According to the comparison of 16S rDNA sequences, overall similarity value between strain MP606 and 12 typical methylotrophic bacteria is above 95% . The highest value is with two strains of Methylovorus, which reached at 99%.

Pyrroloquinoline quinone [4,5-dihydro-4,5-dioxo-1H-pyrrolo[2,3-f]quinoline-2,7,9-tricarboxylic acid (PQQ)] is a bacterial vitamin that serves as a cofactor in numerous alcohol dehydrogenases. Its biosynthesis in Klebsiella pneumoniae is facilitated by six genes, pqqABCDEF, and proceeds by an unknown pathway. The protein encoded by pqqC catalyzes the final step of PQQ formation, which involves a ring closure and an overall eight-electron oxidation of 3a-(2-amino-2-carboxyethyl)-4,5-dioxo-4,5,6,7,8,9-hexahydroquinoline-7,9-dicarboxylic acid (AHQQ) in the absence of a redox-active metal or cofactor. A recent crystal structure has implicated numerous PQQ-PqqC interactions [Magnusson et al. (2004) Proc. Natl. Acad. Sci. U.S.A. 101, 7913-7918]. To investigate the mechanism of the PqqC reaction, the active site residue His84 has been mutated to H84A and H84N, and the kinetic and spectroscopic properties have been compared to each other and the wild-type enzyme using aerobic and anaerobic conditions. Both mutants form PQQ under aerobic conditions with rate constants of 0.09 min-1 and 0.056 min-1 relative to 0.34 min-1 for the wild-type enzyme. In addition to the initial E-AHQQ complex (532-536 nm) and the product E-PQQ complex (346-366 nm), a number of spectral intermediates are observed between 316 and 344 nm. The anaerobic reaction is particularly informative, showing that while mixing of H84N with AHQQ leads to a 344 nm intermediate, this is unable to proceed to a final 318 nm species; by contrast H84A forms the 344 nm species as a precursor to the 318 nm species. In the context of the proposed chemical mechanism for PqqC [Magnusson et al. (2004) Proc. Natl. Acad. Sci. U.S.A. 101, 7913-7918], we assign the 344 nm intermediate to a quinoid species and the 318 nm intermediate to an initial quinol species. The proposed role of H84 is as a proton donor to the oxyanion of the quinoid species such that subsequent C-H bond cleavage can occur to form a monoanionic quinol. In the absence of a proton donor (as occurs in H84N), the normal reaction path is precluded as this would require formation of an unstable, dianionic species. Unlike H84N, H84A appears to be small enough to allow entry of active site water, which is postulated to adopt the role of active site proton donor.

The biosynthesis of pyrroloquinoline quinone (PQQ), a vitamin and redox cofactor of quinoprotein dehydrogenases, is facilitated by an unknown pathway that requires the expression of six genes, pqqA to -F. PqqC, the protein encoded by pqqC, catalyzes the final step in the pathway in a reaction that involves ring cyclization and eight-electron oxidation of 3a-(2-amino-2-carboxyethyl)-4,5-dioxo-4,5,6,7,8,9-hexahydroquinoline-7,9-dicarboxylic-acid to PQQ. Herein, we describe the crystal structures of PqqC and its complex with PQQ and determine the stoichiometry of H2O2 formation and O2 uptake during the reaction. The PqqC structure(s) reveals a compact seven-helix bundle that provides the scaffold for a positively charged active site cavity. Product binding induces a large conformational change, which results in the active site recruitment of amino acid side chains proposed to play key roles in the catalytic mechanism. PqqC is unusual in that it transfers redox equivalents to molecular oxygen without the assistance of a redox active metal or cofactor. The structure of the enzyme-product complex shows additional electron density next to R179 and C5 of PQQ, which can be modeled as O2 or H2O2, indicating a site for oxygen binding. We propose a reaction sequence that involves base-catalyzed cyclization and a series of quinone-quinol tautomerizations that are followed by cycles of O2/H2O2-mediated oxidations.

Pyrroloquinoline quinone (PQQ) is a tricyclic o-quinone, which serves as a cofactor in several enzyme-catalyzed redox reactions in certain bacteria. PQQ is also important for human health, and its role as a vitamin in mammals has recently been suggested. Although much is known about the function of enzymes that use PQQ as cofactor, relatively little is known about the biosynthesis of this coenzyme. Six gene products in Klebsiella pneumoniae (PqqA-F) are involved in PQQ biosynthesis, and PqqC has been shown to catalyze the last step in the pathway. The chemical structure of the substrate for PqqC has remained elusive and has hampered our understanding of the nature of this reaction. In this report we describe the purification and structure of the substrate as deduced by a number of spectroscopic and chemical methods. The substrate is 3a-(2-amino-2-carboxyethyl)-4,5-dioxo-4,5,6,7,8,9-hexahydroquinoline-7,9-dicarboxylic acid-a fully reduced derivative of PQQ, which has not undergone ring cyclization. These results show that PqqC catalyzes a novel reaction, which involves ring closure and an amazing eight-electron oxidation of the substrate.

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 DH5alpha. 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 DH5alpha 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 DH5alpha expressing the pqq cluster activates an endogenous glucose dehydrogenase to permit gluconic acid secretion that solubilizes the insoluble phosphate.