Research On The Metabolic Characteristics Of High-yield Vitamin B₁₂ Production By Pseudomonas Denitrificans

In this paper, the effects of different oxygen supply and respiratory chain inhibitors on the metabolic characteristics of Pseudomonas denitrificans were investigated in detail.Firstly, the influence of different oxygen supply on the metabolic processes of P. denitrificans was studied by using the shake-flask fermentation. It was discovered that, compared to the lower level of oxygen supply (180 rpm of revolution,40 ml of a medium volume in 250 ml shake-flask), the higher level of oxygen supply (240 rpm of revolution, 30ml of a medium volume in 250 ml shake-flask) could significantly accelerate the cell growth, but would cause lower vitamin B12 production.In order to accurately investigate the effects of dissolved oxygen (DO) concentration on the metabolic process of P. denitrificans, the two-stage oxygen supply strategies (A:the DO shifting from 10%(48 h) to 20%(49-96 h); B:the DO shifting from 10%(48 h) to 5% (49-96 h)) were implemented by regulating the agitation speed in 5-L fermenter. Based on the determination of the specific activities of the key enzymes involved in central metabolism pathway, it was found that, compared to the fermentation process under constant DO (10%), periodically decreasing the DO could significantly improve the metabolic flux of EMP pathway, but obviously reduce the metabolic flux of HMP and TC A pathway. In addition, the results obtained under the condition of periodically increasing DO were contrary to the fementation in the case of periodically decreasing the DO.In order to further explore the effects of the metabolic flux changes involved in sugar catabolic pathways on P. denitrificans fermentation processes, different kinds of respiratory chain inhibitors were added to the fermentation broths. As a result, although 5 mg/L of rotenone addition to the fermentation broths could slightly reduce the cell growth, it would significantly increase the specific activities of pyruvate kinase and phosphofructokinase involved in EMP pathway, and would reduce the specific activities of glucose-6-phosphate dehydrogenase involved in HMP pathway. Furthermore, the pyruvia acid concentrations and vitamin B12 biosynthesis were both significantly increased under 5 mg/L of rotenone addition. When 50 mg/L of iodoacetic acid was added to the fermentation broth, the cell growth of P. denitrificans was obviously reduced. Moreover, 50 mg/L of iodoacetic acid would seriously cause the specific activities of pyruvate kinase and phosphofructokinase decrease. As a result, the pyruvia acid concentrations and vitamin B12 biosynthesis were both significantly reduced.According to the above results, it could be concluded that, compared to the fermentation of lower-yield vitamin B12 production, the critical characteristics of higher-yield vitamin B12 production is that the metabolic flux of EMP pathway is relatively stronger, and the metabolic flux of EMP pathway is relatively weaker.