Expression Of Glucose Dehydrogenase And Biosynthesis Of Pyrroloquinoline Quinone

Glucose dehydrogenases (GDHs) are divided into NADP+-dependent GDH and quinoprotein glucose dehydrogenase. As a key enzyme in pentosephophate pathway, NADP+-dependent GDH shows broad applications in the fields of food and medicine. Pyrroloquinoline quinone (PQQ) is the cofactor of quinoprotein GDH. As a newly reported cofactor, PQQ has attracted much attentions for its biological functions in medicine. Although GDH has long been studied, its biological functions remain unclear, and industrial production of PQQ is far from commercialization. This study was to unravel GDH function and enhance PQQ fermentation.Relying on BLAST search over GeneBank, the NADP+-dependent GDHs coding genes from Klebsiella pneumoniae and Escherichia coli were subjected to engineer three recombinant strains. Detailed analysis of the biomass, glucose consumption and enzyme activity of three recombination strains revealed that, compared with control strain, both glucose consumption and biomass increased significantly. More importantly, the GDH activities in three strain increased by 8.5-,10.5- and 12 folds. Overall these results indicated that GDH overexpression benefited bacterial growth.The quinoprotein GDH gene from E. coli was amplified by PCR. Next, it was subcloned into vector and transformed into K. pneumoniae. PQQ concentration was measured by a HPLC, which equipped with a UV detector at a wavelength of 200 nm. The mobile phase was methanol:phosphate & water (10:90) at a flow rate of 0.8 mL/min. PQQ standard curve with R2 value of 0.999 was harnessed for determination of PQQ production in recombinant strains.Glutamic acid and tyrosine are the synthetic precursor of PQQ. To optimize the fermentation conditions, both single-factor experiment and orthogonal experiment were carried out. For single-factor experiment, addition of glutamic acid, tyrosine or their mixture into fermentation medium led to 1044.91 mg/L,626.7 mg/L and 1186.21 mg/L of PQQ, respectively. Through optimization of fermentation temperature, rotational speed, the concentrations of carbon source and nitrogen source, the PQQ titer reached up to 1226.37 mg/L. This study has provided basis for further enhancement of PQQ production.