Causes Analysis For Limited CAMP Synthesis Via Salvage Pathway And Development Of High Yield Fermentation Strategy

Cyclic adenosine monophosphate（cAMP）is an important physiologically active substance in organisms and possesses various regulation function on cell metabolism.It is mainly used for clinic treatment of many diseases,such as cardiovascular and cerebrovascular diseases,leukemia,liver and kidney function injury,diabetes and skin diseases,etc.There are three main methods for cAMP synthesis,including microbial fermentation,chemical synthesis and enzymatic synthesis.Compared to other two methods,microbial fermentation possesses characteristics of environmental friendliness,mild reaction conditions and low cost beneficial for industrial production.Microorganisms can synthesize cAMP via either de novo synthesis pathway or salvage pathway and the salvage pathway possesses shorter metabolic process and higher energy efficiency than those by de novo synthesis pathway beneficial for the achievement of metabolism regulation.Salvage pathway can be activated for cAMP biosynthesis when purines are added into the broth and maintained at appropriate concentration,moreover,it has been proved that hypoxanthine is the optimum activator.However,hypoxanthine can also induce xanthine oxidase（XOD）production by which hypoxanthine is decomposed into uric acid and reactive oxygen species（ROS）generate in abundance unfavourable for cell metabolism and cAMP production.In this study,Arthrobacter sp.CCTCC 2013431 was used for cAMP fermentation production and hypoxanthine was added into broth to activate the salvage pathway.We analyzed the fermentation kinetic parameter and cell metabolism to explore the physiological mechanism for cAMP limited synthesis.In addition,XOD inhibitors and reductants were screened and applied for developing novel fermentation process based on salvage synthesis.The main results are as follows:（1）Fermentation with 1 g/L-broth hypoxanthine fed at 18 h and 36 h,respectively was conducted in a 7 L bioreactor and the feeding condition had been tested in shake flasks.cAMP concentration was only3.97 g/L in hypoxanthine feeding batch,while in control it achieved 4.36 g/L,suggesting that there were some limiting factors for cAMP synthesis when hypoxanthine was fed.The analysis results showed that cells viability and key enzymes activities for cAMP synthesis decreased rapidly,and energy metabolism weakened severe as well when compared to those of control.Intracellular ROS and malondialdehyde（MDA）levels increased significantly,with increments of 25.55% and 34.15% at 50 h,respectively,when compared with control.In addition,hypoxanthine feeding resulted in higher XOD activities and uric acid contents.The results indicated that salvage pathway was activated for cAMP synthesis due to hypoxanthine feeding,meanwhile,a mass of XOD was induced and produced,bywhich hypoxanthine was decomposed into uric acid and ROS generated in abundence resulting in cell damage and lower cell metabolism level,finally,cAMP synthesis was blocked severely.（2）For suppressing XOD activity,12 mg/L-broth quercetin added at 18 h was determined as the optimum inhibitor addition condition.Due to quercetin addition,cAMP concentration reached 5.326 g/L,with an increment of 34.16%,hypoxanthine conversion ratio was also improved by 29.92% at 36 h,when compared with control（without quercetin addition）.The analysis results showed that quercetin could inhibit the XOD activities effectively,at the same time,the contents of uric acid and xanthine（products derived from XOD）together with intracellular ROS and MDA levels were decreased significantly.Then,key enzymes activities and cells viability were also improved significantly beneficial for cAMP production.（3）Fermentation with 200 mg/L glutathione（GSH）added at 24 h was conducted in a 7 L bioreactor for maintaining appropriate redox condition.Due to GSH addition,cAMP concentration reached7.38 g/L,with a increment of 85.89% when compared with that of control（without GSH）and intracellular ROS and MDA contents were reduced obviously and kept at a lower level.Meanwhile,cells viability and metabolic intensity were also enhanced significantly as well.It suggested that GSH could remove intracellular ROS and MDA effectively and decrease cells damage,further metabolic intensity and cAMP synthesis would be improved.We tried to develop a novel cAMP fermentation process with GSH and quercetin coupling addition and cAMP content was reduced by 10.16% compared with that of control（with GSH addition alone）.The cells viability and respiration intensity also decreased slightly.Therefore,fermentation process with GSH addition alone had more advantage for cAMP production than GSH and quercetin coupling addition process.