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.

Breeding of Hyphomicrobium denitrificans for high production of pyrroloquinoline quinone by adaptive directed domestication / 生物工程学报

Pyrroloquinoline quinone (PQQ) is widely distributed in organisms and has physiological functions such as boosting body growth, maintaining mitochondrial function, promoting synthesis of nerve growth factor and regulating free radical levels in the body. It has broad application prospects in the fields of medicine, food and cosmetics. In order to improve the PQQ production of Hyphamicrobium denitrificans FJNU-6, the high-concentration methanol was used as the antagonistic factor for laboratory adaptive domestication. The PQQ positive mutants were selected using rapid screening system by spectroscopy. After 6 rounds of adaptive domestication, about 10% mutants were acquired with a doubled yield, and over 90% positive mutation rate of each round of domestication was reached. Subsequently, the mutant strain FJNU-R8 was fermented by 5 L fermenter. Compared with the original strain, the expression of pqq and moxF gene clusters were higher at different methanol concentrations and similar to each other. Meanwhile, the methanol consumption rate and growth rate were slower than the original strain. Finally, the PQQ yield was increased by 1.42 times to 1 087.81 mg/L (143 h), indicating good industrial application potential. The adaptive domestication combined with rapid screening system described in this study can easily and rapidly obtain mutants with high yield of PQQ, which can be used as reference for high-throughput screening of other high-yield PQQ mutants of methylotrophic bacteria.

High-throughput screening of Methylobacterium extorquens for high production of pyrroloquinoline quinone / 生物工程学报

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.

Pyrroloquinoline quinone inhibited oxidative stress induced-apoptosis of Schwann cells via mitochondrial apoptotic signaling pathway in vitro / 中华整形外科杂志

Objective@#To investigate the mechanisms of Pyrroloquinoline quinone (PQQ) against oxidative stress induced apoptosis in Schwann cells (SCs).@*Methods@#SCs were cultured in vitro, identified by S-100 immunofluorence staining. SCs were divided into control group, H2O2 induced group, H2O2 + PQQ treated group. CCK-8 assay was used to detect cell proliferation. Apoptosis was detected by flow cytometry with Annecin V-FITC/PI staining, mitochondrial transmembrane potential was detected by flow cytometry with JC-1 labeled staining, cytochrome C (CytC), Bax and Caspase-9 protein levels was detected by Western blot analysis.@*Results@#In this study, the S-100 positive cells were more than 95%, cell proliferation was decreased in H2O2 induced SCs, apoptotic rate was increased, mitochondrial transmembrane potential was decreased, CytC, Bax and Caspase-9 protein levels were increased. After PQQ added, cell proliferation was increased, apoptotic rate decreased, mitochondrial transmembrane potential increased, CytC, Bax and Caspase-9 protein levels decreased.@*Conclusions@#PQQ protects SCs from oxidative induced apoptosis by inhibiting mitochondrial signaling pathway.

Effect of pyrroloquinoline quinone on learning and memory ability of apolexis rats / 中国食品卫生杂志

Objective This study aimed to investigate the role of pyrroloquinoline quinone (PQQ) in repairing oxidative nerve cells,and to study the antioxidant capacity of PQQ on the oxidative damage of rats caused by apolexis,as well as the effects on learning and memory abilities of apolexis rats.Methods Oxidative damage of PC12 was induced by H2O2,and the repairing rate of PQQ on oxidative PC12 cells was tested by methylthiazolyldiphenyl-tetrazolium bromide assay kit.The 18-month-old male SD rats were administered PQQ (0,10,20,40 mg/kg).After 4 weeks,Morris water maze test was used to test the learning and memory ability.After 6 weeks,serum and brain tissue related indicators and antioxidant capacity were recorded.Results The survival rate of PC12 cells increased from 59.1％ to 90.5％ with 200 nmol/L PQQ.Compared with the apolexis model group,the latency of the PQQ group (20,40 mg/kg) was shortened in the Morris water maze experiment,the swimming distance was reduced,pass-through counts were increased,and the first secure platform pass-through was reduced.Meanwhile,the levels of malondialdehyde and lipofuscin in serum and brain tissue of PQQ group decreased,the levels of superoxide dismutase,glutathione peroxidase vitality,antioxidant capacity of PQQ group (20,40 mg/kg) were enhanced.Conclusion PQQ could repair the oxidative damage of nerve cells,and it was confirmed that PQQ could play the same antioxidant effect in body and brain,and increase the learning and memory ability of apolexis rats.

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.

Bioinformatics based structural characterization of glucose dehydrogenase (gdh) gene and growth promoting activity of Leclercia sp. QAU-66

Glucose dehydrogenase (GDH; EC 1.1. 5.2) is the member of quinoproteins group that use the redox cofactor pyrroloquinoline quinoine, calcium ions and glucose as substrate for its activity. In present study, Leclercia sp. QAU-66, isolated from rhizosphere of Vigna mungo, was characterized for phosphate solubilization and the role of GDH in plant growth promotion of Phaseolus vulgaris. The strain QAU-66 had ability to solubilize phosphorus and significantly (p < 0.05) promoted the shoot and root lengths of Phaseolus vulgaris. The structural determination of GDH protein was carried out using bioinformatics tools like Pfam, InterProScan, I-TASSER and COFACTOR. These tools predicted the structural based functional homology of pyrroloquinoline quinone domains in GDH. GDH of Leclercia sp. QAU-66 is one of the main factor that involved in plant growth promotion and provides a solid background for further research in plant growth promoting activities.

Pyrroloquinoline-quinone and its versatile roles in biological processes.

Pyrroloquinoline-quinine (PQQ) was initially characterized as a redox cofactor for membrane-bound dehydrogenases in the bacterial system. Subsequently, PQQ was shown to be an antioxidant protecting the living cells from oxidative damage in vivo and the biomolecules from artificially produced reaction oxygen species in vitro. The presence of PQQ has been documented from different biological samples. It functions as a nutrient and vitamin for supporting the growth and protection of living cells under stress. Recently, the role of PQQ has also been shown as a bio-control agent for plant fungal pathogens, an inducer for proteins kinases involved in cellular differentiation of mammalian cells and as a redox sensor leading to development of biosensor. Recent reviews published on PQQ and enzymes requiring this cofactor have brought forth the case specific roles of PQQ. This review covers the comprehensive information on various aspects of PQQ known till date. These include the roles of PQQ in the regulation of cellular growth and differentiation in mammalian system, as a nutrient and vitamin in stress tolerance, in crop productivity through increasing the availability of insoluble phosphate and as a bio-control agent, and as a redox agent leading to the biosensor development. Most recent findings correlating the exceptionally high redox recycling ability of PQQ to its potential as anti-neurodegenerative, anticancer and pharmacological agents, and as a signalling molecule have been distinctly brought out. This review discusses different findings suggesting the versatility in PQQ functions and provides the most plausible intellectual basis to the ubiquitous roles of this compound in a large number of biological processes, as a nutrient and a perspective vitamin.

Effect of Pyrroloquinoline quinone on proliferation and expression of c-myc gene in cultured rat Schwann cells / 中华显微外科杂志

, which will provide enough seed cells for peripheral nerve tissus engineering research.

Effects of pyrroloquinoline quinone on proliferation and expression of NF-κB in cultured Schwann cells / 中国矫形外科杂志

[Objective] To investigate the effects of pyrroloquinoline quinine (PQQ) on proliferation and expression of NF - kB of Schwann cells. [Methods] Schwann cells were cultured from sciatic nerves of SD rats in vitro. The Schwann cells were identified and purified by immunofluorescence of S-100 and Ara-C. Experimental group with was 100 nmol/L of PQQ and control group were set up. The expression of NF-kB in Schwann cells was determined by RT-PCR and Western blotting. [ Results] The expression of NF - kB were up - ragulated by PQQ in cultured Schwann cells (P<0.05).[Conclusion ] PQQ could promote the proliferation of Schwann cells and up-regulation of NF-rd3 play an important role in this process.

Effect of Pyrroloquinoline Quinone on Aging of Rat Hippocampal Neurons in Vitro / 中国康复理论与实践

@#Objective To investigate the effect of pyrroloquinoline quinone(PQQ) on the aging of rat hippocampal neurons induced by D-galactose(D-gal).Methods Hippocampal neurons were cultured in vitro.The aging of the hippocampal neurons was induced by high dose D-gal,PQQ protection were used 30 min before D-gal.The metamorphosis of hippocampal neurons was observed under the microscope.The contents of free radical was measured.The incidence of apoptosis of hippocampus cells was tested with the flow cytometry.The expression of Bax was detected with immunohistochemical staining.Results After the cells cultured in vitro exposed to D-gal,the content of free radical and the expression of Bax of the hippocampal neurons increased.After pretreatment of the cultured neurons with PQQ,the contents of free radical and the expression of Bax decreased,the survival of hippocampal neurons increased.Conclusion PQQ may slow the aging progress of hippocamal neurons induced by D-gal.

Effects of Wnt/?-catenin signal pathway in proliferation of Schwann cells promoted by pyrroloquinoline quinine / 中国矫形外科杂志

[Objective]To investigate the effects of Wnt/?-catenin signal pathway on Schwann cells proliferation promoted by pyrroloquinoline quinine (PQQ) and its molecular mechanisms.[Method]Schwann cells were cultured and purified in vitro. The purity was identified by S-100. PQQ of 10 nmol/L and 100 nmol/L were added into culture medium for 24 hours,respectively. Then the morphological changes promoted by PQQ were observed by inverted microscope. The expression of ?-catenin was detected by RT-PCR and Western blot in Schwann cells promoted by PQQ of different concentration for 72 hours.[Result]Morphological change was observed in Schwann cells treated by PQQ of 10 nmol/L and 100 nmol/L. The most obvious morphological changes took place in the Schwann cells treated by 100 nmol/L of PQQ,the RT-PCR and Western blot results showed that PQQ of 1-1000 nmol/L could up-regulate the expression of ?-catenin,especially when Schwann cells was treated by PQQ of 100 nmol/L(P

Effects of pyrroloquinoline quinone on proliferation and expression of NF-?B in cultured Schwann cells / 中国矫形外科杂志

[Objective] To investigate the effects of pyrroloquinoline quinine(PQQ)on proliferation and expression of NF-?B of Schwann cells.[Methods]Schwann cells were cultured from sciatic nerves of SD rats in vitro.The Schwann cells were identified and purified by immunofluorescence of S-100 and Ara-C.Experimental group with was 100 nmol/L of PQQ and control group were set up.The expression of NF-?B in Schwann cells was determined by RT-PCR and Western blotting.[Results]The expression of NF-?B were up-ragulated by PQQ in cultured Schwann cells(P

Protective effect of pyrroloquinoline quinone on damage of hippocampal neurons induced by NMDA in rats / 中国康复理论与实践

@# ObjectiveTo investigate the protective effect of pyrroloquinoline quinone (PQQ) on damage of hippocampal neurons induced by NMDA.MethodsHippocampal neurons were cultured in vitro and NMDA was used to induce neurotoxicity 8 d after, while PQQ were used or not before. The metamorphosis of hippocampal neurons was observed under the microscope. Intracellular calcium levels was measured by confocal laser microscopy. ResultsThe intracellular Ca2+ level increased rapidly after exposure to 0.1 mmol/L NMDA and resulted in neuronal necrosis and apoptosis. Otherwise, pretreatment of the cultured neurons with PQQ reduced the increase of the intracellular Ca2+ level and the neuronal necrosis or apoptosis induced by NMDA.ConclusionPQQ can protect hippocampal neurons from damage induced by NMDA.