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中国生物工程杂志

China Biotechnology
China Biotechnology
China Biotechnology  2021, Vol. 41 Issue (7): 50-57    DOI: 10.13523/j.cb.2103021
    
Enhanced Cyclic Adenosine Monophosphate Fermentation Production by Aminophylline and Citrate Coupling Addition
LI Zhi-gang1,2,GU Yang2,TAN Hai2,ZHANG Zhong-hua2,CHANG Jing-ling1,2,*()
1 Collaborative Innovation Center of Modem Biological Breeding of Henan Province, Xinxiang 453003, China
2 School of Life Science and Technology, Henan Institute of Science and Technology, Xinxiang 453003, China
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Abstract  

Objective: The aim of this study is to develop a novel cAMP fermentation process via inhibiting phosphodiesterase activity.Methods: cAMP fermentations with aminophylline addition were conducted in a 7 L fermenter and corresponding fermentation kinetics, key enzymes activities and energy metabolism levels were analyzed. Finally, a fermentation process with aminophylline and citrate coupling added was proposed and conducted for improving cAMP content.Results: Due to 5 mg/L aminophylline added, cAMP concentration was increased by 25.9% with a decrease of 41.6% for coproduct adenosine when compared with those of a control group. There were no obvious deviation for adenylate cyclase and succinyladenylate dehydrogenase activities in the two fermentation batches whereas the activities of phosphodiesterase and 5'-nucleotide enzyme were obvious lower than those of the control group. The energy metabolism analysis results showed that intracellular ATP/AMP were lower than control with an improved AMP level, indicating that ATP synthesis had been the main limited factor for product accumulation. Using the fermentation process with aminophylline and citrate coupling addition, cAMP content achieved 4.48 g/L with 22.1% and 13.8% increasement than those of aminophylline and citrate individual added, and adenosine content was 0.98 g/L with 51.7% and 25.3% lower than individual added.Conclusion: With aminophylline added, the activities of phosphodiesterase and 5'-nucleotide enzyme were inhibited and cAMP content was also improved obviously due to decreased cAMP decomposition and adenosine synthesis. However, the ATP synthesis level had been the main limiting factor for improving cAMP production. The fermentation process with aminophylline and citrate coupling addition could decrease cAMP decomposition and improve energy metabolism simultaneously, which further promoted cAMP fermentation synthesis.

Key words： Cyclic adenosine monophosphate      Phosphodiesterase      Aminophylline      Adenosine      Citrate     
Received: 11 March 2021      Published: 03 August 2021
ZTFLH:  Q819  
Corresponding Authors: Jing-ling CHANG     E-mail: changjl001@126.com
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Zhi-gang LI
Yang GU
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Cite this article:

LI Zhi-gang,GU Yang,TAN Hai,ZHANG Zhong-hua,CHANG Jing-ling. Enhanced Cyclic Adenosine Monophosphate Fermentation Production by Aminophylline and Citrate Coupling Addition. China Biotechnology, 2021, 41(7): 50-57.

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https://manu60.magtech.com.cn/biotech/10.13523/j.cb.2103021     OR     https://manu60.magtech.com.cn/biotech/Y2021/V41/I7/50

Fig.1 The effect of different aminophylline addition amounts on cAMP biosynthesis by Arthrobacter sp. CCTCC2013431 (a) The effect of aminophylline concetrations on cAMP production (b) The effect of aminophylline concetrations on PDE activity
Fig. 2 Aminophylline improved cAMP fermentation performance conducted in a 7 L bioreactor (a) Time courses of cell and glucose contents (b) Time courses of cAMP and adenosine contents ■/□ OD600, ●/○ glucose concentrations, ▲/△ cAMP concentrations, ◆/◇ adenosine concentrations; blank symbols: control, solid symbols: with 5 mg/L-broth aminophylline addition
Fig. 3 The effect of aminophylline on key enzymes activities existed in cAMP metabolic pathway (a) The effect of aminophylline on sAMPase activities (b) The effect of aminophylline on AC activities (c) The effect of aminophylline on 5-NT activities (d) The effect of aminophylline on PDE activities □: control, ■: with 5 mg/L-broth aminophylline addition, *: P<0.01
Fig.4 The effect of aminophylline on ATP biosynthesis and activity of respiratory chain dehydrogenase (a) The effect of aminophylline on ATP/AMP ratios (b) The effect of aminophylline on AMP contents (c) The effect of aminophylline on NADH/NAD+ ratios (d) The effect of aminophylline on respiratory chain dehydrogenase activities □: control, ■: with 5 mg/L-broth aminophylline addition, *: P<0.01
Fig. 5 Enhanced cAMP fermentation performance with aminophylline and citrate coupling addition (a) Time courses of cell and glucose contents (b) Time courses of cAMP and adenosine contents ■/□ OD600, ●/○ glucose concentrations, ▲/△ cAMP concentrations, ◆/◇ adenosine concentrations; blank symbols: with 3 g/L-broth sodium citrate addition, solid symbols: with 5 mg/L-broth aminophylline and 3 g/L-broth sodium citrate coupling addition
Fig.6 The effects of aminophylline and citrate coupling addition.on cells physiological state (a) The effect of novel fermentation process on PDE activities (b) The effect of novel fermentation process on ATP/AMP ratios □: with 3 g/L-broth sodium citrate addition, ■: with 5 mg/L-broth aminophylline and 3 g/L-broth sodium citrate coupling addition, *: P<0.01
Fig.7 The schematic diagram for cAMP efficient biosynthesis with aminophylline and citrate addition
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