Synthesis of pyrroloquinoline quinone by recombinant Gluconobacter oxydans / 生物工程学报

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.

Mutagenesis of Methylobacterium extorquens AM1 for increasing pyrroloquinoline quinone production by atmospheric and room temperature plasma / 生物工程学报

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.

The treatment effects and mechanisms of pyrroloquinoline quinone on defective teeth and mandible in Bmi-1 knockout mice / 中华口腔医学杂志

<p><b>OBJECTIVE</b>To investigate the treatment effects and mechanisms of pyrroloquinoline quinine(PQQ) on defective teeth and mandible in Bmi-1 knockout mice.</p><p><b>METHODS</b>Male and female Bmi1(+/-) mice were paired with each other from the same nest. At the age of 7 weeks, the mice were divided into three groups, the wild type mice received normal diet(10 mice, WT group), Bmi1(-/-) mice received normal diet (10 mice, BKO group), and the Bmi1(-/-) mice received normal diet and PQQ diet(10 mice, BKO+PQQ group). X-ray and micro- CT were used to detect mandible and dental size and bone mineral density. HE staining, histochemical and immunohistochemical methods were respectively used to detect alveolar bone thickness of cortical bone, predentin thickness of mandibular first molar, mandibular osteoblast number and osteoclast number. Flow cytometry was used to detect reactive oxygen species(ROS) levels of various organs(femur, thymus and liver). The data were statistically analyzed with one-way ANOVA and t test.</p><p><b>RESULTS</b>Compared with BKO mice, BKO+PQQ mice partially rescued total body phenotype, increased body weight and prolonged survival time. X- ray and micro- CT showed the size of the mandible and teeth and bone mineral density of PQQ+BKO mice increased compared with BKO mice. In PQQ+BKO mice, mandibular alveolar bone cortical thickness [(68.65 ± 0.25) µm] was significantly different from that in BKO mice [(42.45 ± 0.35) µm] (P<0.01). There was significant difference in predentin thickness of mandibular first molar between PQQ+BKO mice [(4.25 ± 0.15) µm] and BKO mice [(31.55 ± 0.35) µm] (P<0.001). The number of osteoblasts in the mandible of BKO+PQQ mice [(38.45 ± 0.25) cell/mm³] was significantly higher than that in the BKO mice [(18.15 ± 0.55) cell/mm³] (P<0.01). However, the number of osteoclasts in the BKO+PQQ mice [(9.45 ± 0.25) cell/mm³] was significantly lower than that in the BKO group [(14.25 ± 0.35) cell/mm³] (P<0.01). Compared with the BKO group, ROS levels of the femur, thymus and liver in the BKO+PQQ mice were significantly decreased (P<0.01).</p><p><b>CONCLUSIONS</b>The results indicate that PQQ may have treatment effects on defective teeth and mandible through promoting osteoblast bone formation and reducing osteoclast bone resorption, scavenging ROS and reducing DNA damage.</p>

Effects of pyrroloquinoline quinone on proliferation and expression of c-fos, c-jun, CREB and PCNA in cultured Schwann cells / 中华整形外科杂志

<p><b>OBJECTIVE</b>To investigate the effects of pyrroloquinoline quinine (PQQ) on proliferation and expression of c-fos, c-jun, CREB and PCNA in cultured Schwann cells.</p><p><b>METHODS</b>Schwann cells were cultured and purified in vitro. The purity of Schwann cells was identified by immunofluorescence of S-100. After synchronization of cell cycle by serum-free medium, different concentration of PQQ (0,1, 10, 100, 1,000, 10,000 nmol/L) were added into culture medium for 72 h. Flow cytometry was used to determine cell cycle. The content of c-fos, c-jun, and CREB mRNA were detected by RT-PCR, and the expression of PCNA protein was detected by Western blot.</p><p><b>RESULTS</b>After PQQ treatment, the percentage of cells in G0/G1 phase decreased and the percentage of cells in S and G2/M phase increased. After treated by PQQ at concentration of 1-10,000nmol/L, content of c-fos,c-jun,CREB mRNA was increased by 0.33,0.42 and 0. 52 fold (P < 0. 05). However, at concentration of 1 000 nmol/L, there was no difference in mRNAs content when compare to control (P >0.05). And it showed a decline at concentration of 10,000 nmol/L (P < 0.05). PCNA protein expression was up-regulated at PQQ concentration of 1-100 nmol/L. At 100 nmol/L, the expression increased by 1.17 fold (P < 0.05); However, at 1,000 nmol/L, there was no difference in PCNA expression when compared to control. And 10,000 nmol/L of PQQ inhibited the expression of PCNA (P < 0.05).</p><p><b>CONCLUSIONS</b>When treated with PQQ at concentration of 10-100 nmol/L, the proliferation of Schwann cells increased and the expression of c-fos,c-jun, CREB and PCNA was up-regulated.</p>

The roles of PI3K/Akt pathway in proliferation of Schwann cells promoted by pyrroloquinoline quinone / 中华整形外科杂志

<p><b>OBJECTIVE</b>To investigate the roles of PI3K/Akt signal pathway in Schwann cells proliferation promoted by pyrroloquinoline quinone (PQQ).</p><p><b>METHODS</b>Schwann cells were cultured and purified in vitro. The purity was identified by immunofluorescence of S-100; the expression of Akt and phosphorylated-Akt (p-Akt) were detected by western blot, and the expression of p-Akt after blocking the PI3K signal transduction pathway by PI3K inhibitor wortmannin was detected by western blot.</p><p><b>RESULTS</b>Morphological change was observed in PQQ-treated Schwann cells, p-Akt was detected 30 min after PQQ treated, reached the peak at 4 h, and disappeared 12 h later. 1-100 nmol/L PQQ could up-regulate the expression of p-Akt; this up-regulated expression of p-Akt was inhibited by wortmannin (P<0.05).</p><p><b>CONCLUSIONS</b>PQQ could affect morphology of Schwann cells and activation of Akt. It indicates that PI3K/Akt signal pathway might be involved in Schwann cells proliferation promoted by PQQ.</p>

Pyrroloquinoline quinone enhances regeneration of transected sciatic nerve in rats / 中华创伤杂志(英文版)

<p><b>OBJECTIVE</b>To investigate the effect of pyrroloquinoline quinone (PQQ) on nerve regeneration of transected sciatic nerve in animal models.</p><p><b>METHODS</b>Forty SD rats weighing 220-240 g were randomized into a PQQ group (n = 20) and a control group (n = 20). Each animal underwent sciatic nerve transection operation. After the operation, PQQ 0.5 ml (250 microg/Kg) was injected at the operation site in the PQQ group, while the same volume of normal saline was delivered in the control group. Nerve functional evaluation, electrophysiological index recording were carried out according to the experimental design. Newly generated nerve specimens were harvested 12 weeks postoperatively for morphological studies.</p><p><b>RESULTS</b>In the PQQ group there was a good nerve regeneration and the sciatic nerve function, sciatic nerve function index, electrophysiological index and morphological appearance were superior to the control group (P < 0.05).</p><p><b>CONCLUSIONS</b>PQQ has a remarkable effect in enhancing nerve regeneration of transected peripheral nerve.</p>