Cloning And Prokaryotic Expression Of Pyrroloquinoline Quinine Gene And Its Quinoprotein Glucose Dehydrogenase

Pyrroloquinoline quinone (PQQ) is the third redox-cofactor vitamin indentified thus far after FMN and NAD, which is essential for some bacteria and mammals and has exhibited promising applications in the fields of food, medicine, and agriculture. Since PQQ-dependent glucose dehydrogenase can convert glucose to gluconic acid without recruiting dissolve oxygen as electron acceptor during glucose oxidation, PQQGDHs are thereby the industrially attractive enzymes in glucose sensors.A gene cluster (pqqA to pqqF) in Klebsiella pneumoniae has been verified to be responsible for PQQ biosynthesis. Here we report the construction of expression vectors of PQQ gene cluster from K. pneumoniae. Taking advantage of the inherent Sal I cutting sites in PQQ gene cluster, the entire PQQ gene cluster was digested into two fragments and linked to pET28a aiming to avoid non-specific amplification of large DNA segment. The productivity of PQQ in transformed BL21 was trebled according to NBT noenzymatic system assay. Besides, the gene gcd encoding PQQ-dependent glucose dehydrogenase (PQQGDH) was PCR cloned and an inducible expression vector pET28a-gcd was constructed and transformed into E. coli BL21. SDS-PAGE analysis showed the high expression of PQQGDH in recombinant strain upon IPTG induction, with nearly 20-fold increase of GDH compared with the control strain. This work set the stage for its applications in sensors and the forthcoming microbial production of PQQ.