BACKGROUND: Previous research proved that fermentation via dissolved oxygen (DO) step-wise reduction was favorable for vitamin B-12 productivity by Pseudomonas denitrificans, but the high-production mechanism is still unclear. Based on this fact, P. denitrificans metabolic characteristics under different DO supply models were investigated in a 5 L fermenter by assaying the activities of the key enzymes involved in central carbon metabolism and the vitamin B-12 biosynthetic pathway. RESULTS: Compared with fermentation processes under the DO-stat strategy and DO step-wise increase, it was found that DO step-wise reduction for P. denitrificans fermentation could significantly strengthen the Embden-Meyerhof-Parnas flux and weaken the tricarboxylic acid cycle and hexose monophosphate pathway, but also facilitate the activities of delta-aminolevulinic acid synthase and porphobilinogen synthase. CONCLUSIONS: When P. denitrificans fermentation was implemented using DO step-wise reduction, the metabolic characteristics indicated that more precursors for vitamin B-12 biosynthesis could be generated. As a result, a significant improvement in vitamin B-12 productivity was achieved (69.09+/-4.48mgL(-1)). (C) 2013 Society of Chemical Industry