限制性供氧促脱氮假单胞杆菌高效合成维生素B12的代谢机理研究

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.

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。

脱氮假单胞杆菌系典型的维生素B12好氧合成菌株，但是限制性供氧反而更利于产物合成。脱氮假单胞杆菌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）阐释了不同供氧水平下的菌体代谢调节机制：相对于较高的供氧发酵条件（5%、10%和20%溶氧浓度），尽管限制性供氧水平（2%）会显著降低HMP、TCA和ED途径的代谢通量致使菌体生长受到一定抑制，但是会显著提高维生素B12合成途径关键酶酶活及产物合成量。.（2）阐释了氧化应激状态下的菌体代谢特征：外源添加活性氧O2-产生剂——甲萘醌会对脱氮假单胞杆菌形成氧化胁迫作用，致使菌体胞内ATP浓度和能荷水平出现显著下降，同时菌体生长和产物合成也受到相应抑制。同时，为了抵御胞内氧化胁迫作用，脱氮假单胞杆菌一方面通过增强与NADPH合成相关的调节酶（HMP途径关键酶葡萄糖-6-磷酸脱氢酶和6-磷酸葡萄糖酸脱氢酶，ED代谢途径关键酶2-酮3-脱氧-6-磷酸葡萄糖酸醛缩酶，NAD +激酶）的比活力，减弱与NADH合成相关的TCA途径关键酶尤其是ICDH-NAD+的比活力，增加了胞内NADPH 的供给并限制NADH的合成，以促进胞内氧化还原平衡；另一方面，菌体会激活超氧化物歧化酶（SOD）和过氧化氢酶（CAT）等抗氧化酶系酶活，以对抗氧化胁迫。.（3）菌体在不同氧化应激状态下的代谢组和蛋白组学分析：代谢组学分析结果表明，甲萘醌导致脱氮假单胞杆菌代谢轮廓产生显著改变，尤其是对细胞膜前体物质——脂肪酸和乙醇胺的代谢产生显著的负作用；蛋白组学分析结果显示，甲萘醌导致菌体蛋白表达水平出现整体下调，其中以核糖体蛋白的下调最为严重。.由此可见，脱氮假单胞杆菌对胞内的氧化环境较为敏感，尤其是氧化胁迫会导致菌体细胞膜和蛋白质合成遭到破坏，最终影响了菌体的正常代谢。本项目的研究可为基于氧调控的维生素B12工业化发酵工艺的进一步优化提供理论指导。