| Pyrroloquinoline quinone(PQQ)is a redox active aromatic,tricyclic o-quinone that invloves in redox reactions by serving as a cofactor for dehydrogenases,and electron transport in respiratory chain by collaborating coenzyme Q.In general,ortho-quinone cofactors is the third family of cofactors following pyridine nucleotide-and flavin-dependent cofactors.PQQ is widely distributed in biological organisms,and it is an unique physiologically active substance with the function of supporting body growth,protection against liver injury,maintenance of mitochondrial function,promotion of nerve growth factor synthesis,regulation of free radicals level,prevention of skin aging,and neuroprotective function.Because of the broad application in cosmetics,food and pharmaceutical industry,it is of great significance to breed high-yield PQQ strain and establish efficient fermentation control and purification process.1?Directional breeding of methylotrophic bacteria with high-yield PQQ production:Rapid screening system for PQQ producing bacteria was established by combining spectroscopy with liquid chromatography according to the absorption spectrum properties of PQQ.The methylotrophic strain Hyphomicrobium denitrificans FJNU-6 with high-yield PQQ and low protein secretion was isolated from chemical sewage,which was identified by morphological,physiological,biochemical and phylogenetic analysis.Ahigh-yield PQQ mutant FJNU-R8 was acquired through eight-times' directional domestication with a high concentration of methanol as the antagonistic factor by alternate UV and NTG mutagenesis according to the secretion mechanism.PQQ production of mutant FJNU-R8 reached 65.73±1.65 mg/L.Compared with the original strain FJNU-6,PQQ production impoved 1.17-fold with the methanol consumption accelerating albeit at a rate reduced in the growth.2?The establishment of DO-stat and pH-stat fed-batch fermentation process with methanol segmented control:Firstly,optimization of PQQ production from FJNU-R8 in shake flask culture system by single factor experimental design,factorial and central composite design,PQQ production increased to 135.67 mg/L after optimization.Secondly,PQQ production from FJNU-R8 was proved to be growth-partial ly coupled fermentation based on the kinetic model,which implied lower methanol concentration in favor of PQQ generation.Then PQQ production reached 1.18 g/L with 52.17 mg/g DCW efficiency by applying methanol segmented control,DO-stat and pH-stat fed-batch fermentation process in 5 L fermenter batch culture.Furthmore,fermentation scale-up(5L-50L-1T)by applying the criterion "the same power was consumed per liquid volume" was conducted.As a result,2.05 g/L PQQ with 77.82 mg/g DCW efficiency was achieved in 1 T fermenter,which increased 49.17%than in 5 L fermenter.3?The establishment of crystalline preparation process by the combination of centrifugation,membrane filtration with adsorption chromatography coupled chromatographic separation:Firstly,all cells and 95%peptides PQQ fermentation broth was removed from fermentation broth by utilizing 15%ammonium sulfate together with centrifugation and membrane filtration technology,which could prevent the fermentation broth from turning green,and improve the stability of PQQ.Secondly,the macroporous resin DK-6 and appropriate buffers were selected as the separation medium and parsing agent for the purification and enrichment of PQQ by static adsorption experiments.Thirdly,the dynamical study showed that the adsorption using resin DK-6 belonged favorable adsorption,so the process was an exothermic process,and the adsorption rate is controlled by internal/external diffusion.Furthermore,the dynamic adsorption experiments determined the conditions of the chromatography:sample concentration of 1.5 g/L(pH 3.0),6 BV sample with the flow rate of 5 BV/h,multiple linear amplification by volume.As a result,PQQ prodution was concentrated up to 3.3 times with the recovery rate above 90%.After the factorial design,PQQ crystallization yield within 3h was more than 90%when the PQQ initial concentration was more than or equal to 3.5 g/L(pH<3.0)at 4?,meanwhile,PQQ residues was no more than 100 mg/L.After the second crystallization,the purity of PQQ crystal increased from 90.36 to 99.32%.Finally,more than 70%PQQ produciton yield with 99%purity was achieved according to the parameters of the separation and purification process4?The analysis of genome sequencing data from the high-yield PQQ mutant FJNU-R8:The genome of Hyphomicrobium denitrificans FJNU-R8 consists of a single circular chromosome of 3.54 M bp with a G+C content of 60.78%.There are 3498 putative open reading frames by Glimmer,and 46 tRNA-encoding genes and 3 rRNA-encoding operons were identified.The genes related to the methanol oxidation,serine pathway,tricarboxylic acid pathway,glyoxylate regeneration pathway and nitrate reduction are invovled in the genome.In addition,the genome contains six methanol dehydrogenase encoding genes and 11 genes associated with PQQ synthesis.Each gene involved in PQQ biosynthetic gene cluster was transcribed separately,and the expression of pqqA genes was significant difference with the highest expression level from pqqA3 gene.5?Improvement of PQQ synthesis by enhanced PqqA and Fe-S cluster expression in Escherichia coli:The E.coli BW25113 harboring pqqABCDE gene cluster under arabinose-inducible araBAD promoter was constructed for PQQ production.Therefore,4.09 ± 0.21 mg/L PQQ with 2.16±0.06 mg/g DCW efficiency was produced by the recombinant E.coli.When IscR(iron-sulfur cluster regulator)knockout strain was used as a host,3.13±0.07 mg/g DCW productivity was acquired.The pqqA3 gene was the most efficient for PQQ production among the five pqqA genes.The over-expression of pqqA3 alone resulted in 4.52±0.05 mg/g DCW efficiency in the recombinant ?IscR strain.When five concatenate pqqA3 genes was expressed,PQQ productivity reached 5.48 ±0.09 mg/g DCW,on the contrary,only 4.78±0.05 mg/g DCW efficiency was achieved by fusion expression of five pqqA3 gene.The results implied that the spatial structure was as important as the conserved domain for transfomation of PqqA to PQQ.In summary,a high-yield PQQ production strain Hyphomicrobium denitrificans FJNU-R8 was acquired by directional breeding.After optimization of the culture system,DO-stat and pH-stat fed-batch fermentation process with methanol segmented control as well as crystalline preparation process by chromatographic purification were established to achieve PQQ with high purity.Furthermore,genome sequencing of FJNU-R8 and profound analysis of metabolic pathway,especially for PQQ synthetic pathway,were conducted.Finally,PQQ productivity was improved by over-expression of a variety of pqqA genes in recombinant E.coli. |
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