脱氮假单胞杆菌系典型的维生素B12好氧合成菌株。但本项目组前期发现，其发酵过程限制性供氧反而更有利于维生素B12的合成。它是否会与大多数假单胞菌类一样，因对胞内氧化环境较敏感而弱化了高溶氧下的产物合成？为此，本项目拟开展脱氮假单胞杆菌在不同氧化状态下的代谢变化特征研究，以揭示限制性供氧促维生素B12高效合成的作用机理：1）直接从发酵过程供氧差异着手，通过考察中心代谢途径和产物合成途径中的关键生化参数变化特征，从细胞水平探索到菌体在不同溶氧水平下的代谢调节机制；2）借助外源添加氧化应激诱导剂，探究菌体在不同氧化应激状态下的能荷、NADH/NAD+、NADPH/NADP+等代谢辅因子变化规律，以及辅因子关键调节酶（NAD+激酶和NADP+磷酸酶）和菌体抗氧化系统主要酶（超氧化物歧化酶和过氧化氢酶）的酶活力变化特点，并辅以菌体胞内代谢物的代谢组学分析，阐明菌体抗氧化应激的生理响应特征及调节机制。

Pesudomonas denitrificans is a typical microorganism owning the aerobic pathway for vitamin B12 biosynthesis. However, compared to the high dissolved oxygen (DO) concentration for P. denitrificans fermentation, our previous study showed that the limiting oxygen supply was more beneficial to vitamin B12 biosynthesis. As one of microorganisms belonging to Pesudomonas sp., P. denitrificans also has the typical ED pathway. As a hypothesis, is P. denitrificans similar to the others Pesudomonas sp., being also sensitive to the intracellular oxidation environment? As a result, the vitamin B12 biosynthesis was highly weakened under the high DO level. Based on this hypothesis, this project aims to illuminate the action mechanism of efficient vitamin B12 biosynthesis caused by the limiting oxygen control level, through investigating the metabolic kinetics of P. denitrificans in the case of various oxidation states: 1) The fermentation processes of P. denitrificans are peformed with different oxygen supply levels, respectively, and it is expected to explore the metabolic regulation mechanism of P. denitrificans under these DO control conditions, by assaying and analyzing the dynamic characteristics of the key biochemical parameters involved in the central metabolic pathways and vitamin B12 biosynthetic pathway. 2) It is planned to improve the oxidation states of P. denitrificans through exogenously adding the oxygen-stress inducer to the fermentation broths. During these various fermentation processes, the following key parameters will detailedly be investigated to elucidate the physiological response characteristics and regulation mechansim of P. denitrificans on counteracting the oxidative stress, such as the change characteristics of the metabolic cofactors (energy change level, NADH/NAD+, NADPH/NADP+), the enzyme kinetics of NAD+ kinase, NADP+ phosphatase, superoxide dismutase and catalase, together with the metabolome analysis of intracellular metabolites.