Effects of Pyrroloquinoline Quinone (PQQ) and Coenzyme Q10 on Mitochondrial Genes, MitomiRs and Cellular Properties in HepG2 Cell Line.

Our study aimed to reveal the effects and changes, antioxidant metabolism (Oxidative Stress), inflammatory response, mitochondrial biogenesis and mitochondrial dysfunction characteristics in hepatocellular carcinoma cell line HepG2; that occur in genes (NRF-1, NRF-2, NFκB and PGC-1α) and miRNAs (miR15-a, miR16-1, miR181-c) that can control related features. To investigate the effects of Pyrroloquinoline quinone (PQQ) and Coenzyme Q10 (CoQ10) in HepG2, and their effects on cell viability, lateral cell migration, gene expression and miRNA expression levels were investigated. If the data we have obtained are evaluated in terms of anti-cancer effectiveness, the most effective use of CoQ10 can be defined as the use alone rather than the combined use. According to the results of the wound healing experiment, we determined that Pyrroloquinoline quinone and combined drug application increased the wound closure area and cell proliferation compared to the control group, while CoQ10 application decreased it. We found that Pyrroloquinoline quinone and Coenzyme Q10 exposure in the HepG2 cell line increased Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) expression but not NRF-1 gene expression. We reported only a small increase in expression of the NRF-2 gene in the Pyrroloquinoline quinone application compared to the control group. We found that only Pyrroloquinoline quinone and CoQ10 application caused more expression increase in the Nuclear Factor kappa B (NFκB) gene compared to combined application. Pyrroloquinoline quinone and CoQ10 administration down-regulated the expression levels of miR16-1, miR15a and miR181c. The use of Pyrroloquinoline quinone and CoQ10 is effective on epigenetic factors, miR-15a, miR-16-1 and miR181c are important candidate biomarkers in hepatocellular carcinoma and diseases accompanied by mitochondrial dysfunction.

Pyrroloquinoline Quinone Chemistry, Biology, and Biosynthesis.

The widely distributed, essential redox factor pyrroloquinoline quinone (PQQ, methoxatin) (1) was discovered in the mid-1960s. The breadth and depth of its biological effects are steadily being revealed, and understanding its biosynthesis at the genomic level is a continuing process. In this review, aspects of the chemistry, biology, biosynthesis, and commercial production of 1 at the gene level, and some applications, are presented from discovery through to mid-2021.

Understanding the biological role of PqqB in Pseudomonas stutzeri using molecular dynamics simulation approach.

Phosphate solubilization is an important and widely studied plant growth promoting trait exhibited by many bacteria. Pyrroloquinoline quinone (PQQ), a redox cofactor of methanol and glucose dehydrogenases has been well established as essential for phosphate solubilization. PQQ operon has been well studied in growth promoting rhizobacteria like Pseudomonas spp., Gluconobacter oxydans, Klebsiella pneumoniae, etc. However, the role of PqqB is quite ambiguous as its functional role has been contradicted in many studies. In the present study, we selected Pseudomonas stutzeri - a well-known P solubilizing bacterium as a representative species of the Pseudomonas genus on the basis of phylogenetic and statistical analyses of PqqB proteins. A 3 D model was generated for this protein. Docking of PqqB with PQQ showed good interaction with a theoretical binding affinity of -7.4 kcal/mol. On the other hand, docking of PqqC with 3a-(2-amino-2-carboxy-ethyl)-4,5-dioxo-4,5,6,7,8,9-hexahydro-quinoline-7,9-dicarboxylic acid (AHQQ, immediate precursor of PQQ) showed strong interaction (-10.4 kcal/mol) but the same was low with PQQ (-6.4 kcal/mol). Molecular dynamic simulation of both the complexes showed stable conformation. The binding energy of PqqB-PQQ complex (-182.710 ± 16.585 kJ/mol) was greater than PqqC-PQQ complex (-166.114 ± 12.027 kJ/mol). The results clearly indicated that kinetically there is a possibility that after cyclization of AHQQ to PQQ by PqqC, PQQ can be taken up by PqqB and transported to periplasm for the oxidation of glucose. To the best of our knowledge, this is the first attempt to understand the biological role of PqqB on the basis of molecular interactions and dynamics.Communicated by Ramaswamy H. Sarma.

Generation of a Gluconobacter oxydans knockout collection for improved extraction of rare earth elements.

Bioleaching of rare earth elements (REEs), using microorganisms such as Gluconobacter oxydans, offers a sustainable alternative to environmentally harmful thermochemical extraction, but is currently not very efficient. Here, we generate a whole-genome knockout collection of single-gene transposon disruption mutants for G. oxydans B58, to identify genes affecting the efficacy of REE bioleaching. We find 304 genes whose disruption alters the production of acidic biolixiviant. Disruption of genes underlying synthesis of the cofactor pyrroloquinoline quinone (PQQ) and the PQQ-dependent membrane-bound glucose dehydrogenase nearly eliminates bioleaching. Disruption of phosphate-specific transport system genes enhances bioleaching by up to 18%. Our results provide a comprehensive roadmap for engineering the genome of G. oxydans to further increase its bioleaching efficiency.

Bioconversion of recombinantly produced precursor peptide pqqA into pyrroloquinoline quinone (PQQ) using a cell-free in vitro system.

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.

Combinational expression of D-sorbitol dehydrogenase and pyrroloquinoline quinone increases 6-(N-hydroxyethyl)-amino-6-deoxy-α-L-sorbofuranose production by Gluconobacter oxydans through cofactor manipulation.

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 ±â€¯3.0 g/L of 6NSL production with 94.5 ±â€¯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.

[Synthesis of pyrroloquinoline quinone by recombinant Gluconobacter oxydans].

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.

Engineering CRISPR interference system to enhance the production of pyrroloquinoline quinone in Klebsiella pneumonia.

Pyrroloquinoline quinone (PQQ) is a cofactor of glucose dehydrogenase (GDH) and thus participates in glucose utilization. In Klebsiella pneumoniae, glucose utilization involves PQQ-dependent direct oxidation pathway (DOP) and phosphoenolpyruvate-dependent transport system (PTS). It is challenging to overproduce PQQ, as its biosynthesis remains unclear. Here, we report that PQQ production can be enhanced by stimulating the metabolic demand for it. First, we developed CRISPR interference (CRISPRi) system to block PTS and thereby intensify DOP. In shake-flask cultivation, the strain with CRISPRi system (simultaneously inhibiting four PTS-related genes) produced 225·65 nmol l-1 PQQ, which was 2·14 times that of wild type. In parallel, an exogenous soluble glucose dehydrogenase (sGDH) was overexpressed in K. pneumoniae. In the shake-flask cultivation, this sGDH-overexpressing strain accumulated 140·05 nmol l-1 PQQ, which was 1·33 times that of wild type. To combine the above two strategies, we engineered a strain harbouring both CRISPRi vector and sGDH-overexpressing vector. In the shake-flask cultivation, this two-plasmid strain generated 287·01 nmol l-1 PQQ, which was 2·72 times that of wild type. In bioreactor cultivation, this two-plasmid strain produced 2206·1 nmol l-1 PQQ in 57 h, which was 7·69 times that in shake-flask cultivation. These results indicate that PQQ production can be enhanced by intensifying DOP, as the apo-enzyme GDH is intrinsically coupled with cofactor PQQ. This study provides a strategy for the production of cofactors whose biosynthesis mechanisms remain ambiguous. SIGNIFICANCE AND IMPACT OF THE STUDY: Pyrroloquinoline quinone (PQQ) is an economically important chemical, which typically serves as a cofactor of glucose dehydrogenase (GDH) and thus participates in glucose metabolism. Klebsiella pneumoniae can naturally synthesize PQQ, but current yield constrains its commercialization. In this study, the PQQ level was improved by stimulating metabolic demand for PQQ, instead of overexpressing PQQ synthetic genes, as the synthetic mechanism remains ambiguous.

Improved Production of Pyrroloquinoline Quinone by Simultaneous Augmentation of Its Synthesis Gene Expression and Glucose Metabolism in Klebsiella pneumoniae.

Klebsiella pneumoniae can naturally synthesize pyrroloquinoline quinone (PQQ), but current low yield restricts its commercialization. Here, we reported that PQQ production can be improved by simultaneously intensifying PQQ gene expression and glucose metabolism. Firstly, tandem repetitive tac promoters were constructed to overexpress PQQ synthesis genes. Results showed that when three repeats of tac promoter were recruited to overexpress PQQ synthesis genes, the recombinant strain generated 1.5-fold PQQ relative to the strain recruiting only one tac promoter. Quantitative real-time PCR (qRT-PCR) revealed the increased transcription levels of PQQ synthesis genes. Next, fermentation parameters were optimized to augment the glucose direct oxidation pathway (GDOP) mediated by PQQ-dependent glucose dehydrogenase (PQQ-GDH). Results demonstrated that the cultivation conditions of sufficient glucose (≥ 32 g/L), low pH (5.8), and limited potassium (0.7 nmol/L) significantly promoted the biosynthesis of gluconic acid, 2-ketogluconic acid, and PQQ. In optimum shake flask fermentation conditions, the K. pneumoniae strain overexpressing PQQ synthesis genes under three repeats of tac promoter generated 363.3 nmol/L of PQQ, which was 2.6-fold of that in original culture conditions. In bioreactor cultivation, this strain produced 2371.7 nmol/L of PQQ. To our knowledge, this is the highest PQQ titer reported so far using K. pneumoniae as a host strain. Overall, simultaneous intensification of pqq gene expression and glucose metabolism is effective to improve PQQ production.

Rapid Detection of Phosphate-Solubilizing Bacteria from Agricultural Areas in Erzurum.

In this study, the newly designed pqq gene-specific primer sets were used for determination of phosphate-solubilizing capabilities of bacterial isolates from the agricultural regions of Erzurum. The specificity of newly designed primer sets (PqqA2F/PqqA2R, Pqq5F/Pqq5R, PqqF2/PqqF2R) were tested against ten isolates, whose phosphate-solubilizing activities were initially proved by the conventional methods. Non-phosphate-solubilizing bacteria were also chosen as negative control. According to the results, five of ten phosphate-solubilizing bacteria with PqqA2F/PqqA2R, two of ten phosphate-solubilizing bacteria with Pqq5F/Pqq5R primer set, and one of ten phosphate solubilizing with PqqF2F/PqqF2R bacteria were successfully amplificated in the PCR assay and none of the non-phosphate-solubilizing bacteria was amplificated. Then, the molecular characterization of the active phosphate-solubilizing strains was done based on the partial 16S ribosomal RNA gene region sequence analysis method. Two isolates of Enterobacter sp., 1 Rhizobium sp., 1 Enterococcus sp., 1 Bacillus cereus, 1 Bacillus atrophaeus, 1 Bacillus aryabhattai, 1 Acinetobacter sp., 1 Pseudomonas japonica, and 1 Enterobacter cloacae were identified as active phosphate-solubilizing strains. Consequently, the results showed that this specific primer sets could be used as an economic, rapid, and useful tool for the detection of phosphate-solubilizing strains in the agricultural researches.