Physiological Mechanism For Enhanced Cyclic Adenosine Monophosphate Fermentation Synthesis And High-yield Strategy Development Based On Sodium Citrate Addition

Cyclic adenosine monophosphate(cAMP)is an important physiological active ingredient in living cells for its regulation actions in various metabolic processes such as glycometabolism,lipometabolism,nucleotide synthesis and protein synthesis,which was applied in plant growth regulation,disease treatment and animal feed additives extensively.Due to the advantages of clean,high-efficient and environmentally friendly,microbial fermentation for cAMP production is considered as a potential synthesis method.In living cells,cAMP is synthesized from ATP via cyclization reaction directly under catalysis of adenylate cyclase,and higher ATP level and generation efficiency are beneficial for cAMP biosynthesis.In this study,(Arthrobacter sp.)A.sp01 was used for cAMP fermentation production with auxiliary energy substance addition to regulate metabolic synthesis process,and cAMP concentrations and conversion yields from glucose were enhanced significantly.Furthermore,the regulation mechanism of sodium citrate on cAMP biosynthesis was investigated,and two cAMP high-yield fermentation processes based on sodium citrate addition were proposed.The main results were showed as follows:(1)The regulation mechanism of sodium citrate on enhanced cAMP biosynthesis was investigated.The optimal condition of 3 g/L-broth sodium citrate added at 30 h was determined by fermentations conducted in shaking flasks,and cAMP production was increased by 54.3% when compared with control.When the optimal condition was used in a 7 L fermentor,cAMP content reached the maximum of 3.68 g/L at 50 h,with a increment of 11.2% than that of control(3.32 g/L at 72 h)and productivity was improved by 60.2% due to the shortened fermentation period.Analysis results of key enzymes activities,intermediate metabolites and amino acid contents showed that sodium citrate improved activities of enzymes existed in pentose phosphate pathway,TCA cycle and cAMP synthesis pathway significantly,and weakened metabolic intensity of glycolysis pathway.At same time,contents of intracellular amino acids,especially the precursor ones such as Asp,Glu and Gly,increased obviously.The results indicated that sodium citrate addition changed the distribution of metabolic flux and more carbon flow was distributed to cAMP synthesis pathway.In addition,the results of intracellular NADH and ATP content showed that as an auxiliary energy substance,sodium citrate promoted NADH and ATP obviously,providing energy basis for cAMP high-efficient synthesis.(2)Addition of precursor amino acids could promote cAMP synthesis effectively via de novo synthesis pathway when no purines exist in fermentation broth.The optimal combination of Asp 0.2 g/L,Gly 1.0 g/L and Glu 4.0 g/L was determined by orthogonal experiment conducted in shaking flask.Fermentation with the optimal combination condition was carried out in a 7 L fermentor,and the results showed that when amino acids mixture was added at 27 h,the fermentation period was shortened by 12 h and the production efficiency was increased by 21.7%.In order to further enhanced cAMP production,a fermentation process with precursor amino acids and sodium citrate coupling addition was proposed and performed in 7 L fermentor.The results showed that cAMP concentration and productivity achieved 4.31 g/L and 0.086 g/(L·h),which were 29.8% and 86.9% higher than those of control,respectively.(3)Hypoxanthine was proved as the best substance for cAMP salvage pathway and the optimal concentration was determined as 2 g/L by shake flask experiment.A fermentation batch with hypoxanthine added was carried out in 7 L fermentor and the results showed that hypoxanthine increased cAMP synthesis rate significantly.However,fermentation batch with hypoxanthine addition ceased at 51 h with a maximum concentration of 3.29 g/L,which was slightly lower than the final content of control,but productivity was increased by 39.1% caused by shortened fermentation period.To further improve cAMP production via salvage pathway,a cAMP fermentation process with sodium citrate and hypoxanthine coupling addition was proposed.A fermentation with 3 g/L-broth sodium citrate added at 24 h and hypoxanthine pulsed addition was conducted and cAMP concentration and productivity reached 4.42 g/L and 0.088 g/(L·h),which were 33.1% and 91.3% higher than those of control,respectively.