氟化钠促进节杆菌发酵合成环磷酸腺苷的生理机制

doi:10.13560/j.cnki.biotech.bull.1985.2020-1234

摘要/Abstract

摘要：

环磷酸腺苷（cyclic adenosine monophosphate,cAMP）发酵过程中存在严重的“碳溢流”现象并伴随副产物腺苷的大量积累,导致cAMP产量和糖苷转化率低下。为提高cAMP产量和转化率,向发酵液中添加糖酵解抑制剂氟化钠,并从发酵动力学、酶活性分析、能量代谢及关键代谢物水平等方面,对发酵性能得以提高的原因进行分析。结果表明,通过添加0.2 g/L氟化钠,cAMP产量在从头合成和补救合成发酵模式下分别达到3.36 g/L和4.35 g/L,与对应未添加氟化钠批次相比,分别提高了25.8%和27.9%,糖苷转化率也得到等比例提高,同时腺苷和有机酸合成量显著降低,发酵性能得到明显改善。关键酶活性测定结果表明,氟化钠一定程度上抑制了糖酵解途径,使碳流更多分配到磷酸戊糖途径,用于产物合成,而催化腺苷合成的5'-核苷酸酶活性则显著下降,减少了前体AMP的分解,促进了产物合成和糖苷转化率的提高。另外,由于氟化钠的添加,ATP/AMP、NADH/NAD⁺以及胞内天冬氨酸、谷氨酸等前体物质水平均得到明显提高,能量代谢和前体物质水平的提升也为产物合成提供有利条件。

关键词: 氟化钠, 环磷酸腺苷, 腺苷, 关键酶, 能量代谢

Abstract:

Due to severe carbon overflow and high accumulation of byproduct adenosine presence in cAMP（cyclic adenosine monophosphate）fermentation process,cAMP concentration and yield stayed at lower levels all the time. In order to increase cAMP concentration and yield,glycolysis inhibitor sodium fluoride（NaF）was fed into the broth,and the causes for the improved fermentation performance were investigated from fermentation kinetics,activities of enzymes,energy metabolism and key metabolites levels. By adding 0.2 g/L NaF,cAMP concentrations in de novo synthesis and salvage fermentation modes reached 3.36 g/L and 4.35 g/L,with increments of 25.8% and 27.9%,respectively,when compared with those without NaF addition;and the conversion rate of glycoside also increased. In addition,adenosine and organic acids contents in NaF addition batches decreased observably,meaning that the fermentation performance was significantly improved. The results of key enzymes activities indicated that more carbon flux was directed into the pentose phosphate pathway for cAMP synthesis due to the inhibited glycolysis pathway by NaF;while 5'-nucleotidase from catalyzing adenosine significantly decreased,resulting in the reduced decomposition of precursor AMP and the product synthesis and glycoside conversion improved. Moreover,because of the addition of sodium fluoride,the levels of ATP/AMP,NADH/NAD⁺ and intracellular aspartate,glutamate and other precursor substances were significantly improved,and the increase in the energy metabolism and precursor substances also provided favorable conditions for product synthesis.

Key words: sodium fluoride, cyclic adenosine monophosphate, adenosine, key enzymes, energy metabolism

顾阳, 谭海, 员林娜, 孙海彦, 常景玲, 李志刚. 氟化钠促进节杆菌发酵合成环磷酸腺苷的生理机制[J]. 生物技术通报, 2021, 37(5): 108-116.

GU Yang, TAN Hai, YUAN Lin-na, SUN Hai-yan, CHANG Jing-ling, LI Zhi-gang. Physiological Mechanisms for Enhanced Cyclic Adenosine Monophosphate Biosynthesis by Sodium Fluoride in Arthrobacter sp.[J]. Biotechnology Bulletin, 2021, 37(5): 108-116.

图/表 7

表1 氟化钠添加量对cAMP发酵合成的影响

Table 1 Effects of NaF adding amounts on cAMP fermentation synthesis in shake flasks

NaF adding amount/（g·L^-1）	DCW/（g·L^-1）	Glucose consumption/（g·L^-1）	cAMP concentration/（g·L^-1）	cAMP yield/（g·g^-1）
0.00	7.25±0.12a	31.51±0.25a	2.33±0.13d	0.073c
0.05	7.31±0.10a	33.42±0.27a	2.26±0.21d	0.067d
0.10	7.02±0.07b	30.23±0.19a	2.53±0.11c	0.084b
0.15	6.52±0.15c	28.54±0.31b	2.81±0.08b	0.098a
0.20	6.23±0.20c	30.65±0.28a	3.16±0.12a	0.103a
0.25	5.14±0.17d	26.82±0.18c	2.45±0.14c	0.090b
0.30	5.01±0.22d	27.46±0.34b	1.73±0.13e	0.063d

图1 氟化钠对cAMP从头合成发酵模式整体性能的影响

Fig.1 Effects of NaF on cAMP fermentation production by de novo synthesis fermentation mode

图2 氟化钠对cAMP补救合成发酵模式整体性能的影响

Fig.2 Effects of NaF on cAMP fermentation production by salvage synthesis fermentation mode

图3 氟化钠对cAMP合成相关途径中关键酶活性的影响

Fig.3 Effects of NaF on the activities of key enzymes presented in cAMP biosynthesis pathways

图4 氟化钠对腺苷酸环化酶和5'-核苷酸酶活性的影响

Fig.4 Effects of NaF on the activities of AC and 5-NT

图5 氟化钠对胞内关键代谢物含量的影响

Fig.5 Effects of NaF on main intracellular metabolites contents

图6 添加氟化钠对细胞内能量代谢的影响

Fig.6 Effects of NaF on NADH/NAD+ and ATP/AMP

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