Physiological Mechanisms for Enhanced Cyclic Adenosine Monophosphate Biosynthesis by Sodium Fluoride in Arthrobacter sp.

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

Abstract

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

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.

Figures/Tables 7

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

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

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

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

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

Fig.5 Effects of NaF on main intracellular metabolites contents

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

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