科研之友
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摘要
Pyrroloquinoline quinone (PQQ) is a cofactor of glucose dehydrogenase (GDH) and thus participates in glucose utilization. In Klebsiella pneumoniae, glucose utilization involves PQQ-dependent direct oxidation pathway (DOP) and phosphoenolpyruvate-dependent transport system (PTS). It is challenging to overproduce PQQ, as its biosynthesis remains unclear. Here, we report that PQQ production can be enhanced by stimulating the metabolic demand for it. First, we developed CRISPR interference (CRISPRi) system to block PTS and thereby intensify DOP. In shake-flask cultivation, the strain with CRISPRi system (simultaneously inhibiting four PTS-related genes) produced 225 center dot 65 nmol l(-1) PQQ, which was 2 center dot 14 times that of wild type. In parallel, an exogenous soluble glucose dehydrogenase (sGDH) was overexpressed in K. pneumoniae. In the shake-flask cultivation, this sGDH-overexpressing strain accumulated 140 center dot 05 nmol l(-1) PQQ, which was 1 center dot 33 times that of wild type. To combine the above two strategies, we engineered a strain harbouring both CRISPRi vector and sGDH-overexpressing vector. In the shake-flask cultivation, this two-plasmid strain generated 287 center dot 01 nmol l(-1) PQQ, which was 2 center dot 72 times that of wild type. In bioreactor cultivation, this two-plasmid strain produced 2206 center dot 1 nmol l(-1) PQQ in 57 h, which was 7 center dot 69 times that in shake-flask cultivation. These results indicate that PQQ production can be enhanced by intensifying DOP, as the apo-enzyme GDH is intrinsically coupled with cofactor PQQ. This study provides a strategy for the production of cofactors whose biosynthesis mechanisms remain ambiguous.
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单位北京化工大学
