The Study Of Enhancing ATP Synthesis With Auxiliary Energy Substance To Improve CAMP Fermentation Level

Cyclic adenosine phosphate(cAMP)is a physiologically active substance existed in living cells,and plays an important role in metabolic regulation for sugar,fat,nucleic acid and protein synthesis widely used for plant growth regulation,clinical medicine and feed additives.For fermentation production by microorganisms,cAMP is synthesized from ATP under the catalysis of adenylate cyclase directly and enhanced energy metabolism is beneficial to cAMP synthesis.In this study,Arthrobacter.sp.CCTCC2013431 was used for cAMP fermentation production and auxiliary energy substance was added for enhancing energy metabolism further promoting cAMP synthesis,then,the fermentation kinetics,activities of key enzymes,the levels of cofactors and amino acids were analyzed to elucidate the physiological mechanism for improved cAMP synthesis when auxiliary energy substance was added.Moreover,by adding respiratory chain inhibitor rotenone,the insufficient ATP synthesis condition was simulated and then the fermentation performance was restored to the normal level equaled to control via auxiliary energy substance added.The main research contents and experiment results are as follows:(1)cAMP fermentation synthesis was promoted by enhanced energy metabolism caused by sodium pyruvate added.The optimal condition of 3 g/L-broth sodium pyruvate added at 24 h was determined in shaking flasks.Using the optimal condition in a 7 L fermentor,cAMP content reached the maximum of 3.69 g/L at 50 h,with a increment of 25.94%than that of control,and the productivity was also improved significantly.The activities of key enzymes in cAMP synthesis pathway were determined and the results showed that due to sodium pyruvate added,glycolysis pathway was weakened significantly while the metabolic intensity of pentose phosphate pathway was significantly increased,indicting that more carbon flux was distributed for product synthesis.In addition,pyruvate improved the metabolic intensity of tricarboxylate cycle significantly accompanied by enhanced NADH/NAD+,resulting in higher ATP/ADP ratios owing to efficient electronic respiratory chain activity,which supplied abundant energy for cAMP fermentation production.(2)An ATP synthesis system based on sodium hexametaphoshpate was constructed for promoting cAMP fermentation production.2 g/L-broth sodium hexametaphoshpate added at 24 h was determined as the optimal condition for cAMP fermentation via shaking flask experiment,then the optimal condition was conducted in a 7 L fermenter and cAMP content reached 3.56 g/L at 72 h,with a increment of 21.50%than that of control.The activities of key enzymes,energy metabolism and amino acid levels were assayed and the results indicated that the activities of PPK were improved significantly due to sodium hexamophosphate added,indicting that ATP synthesis system based on sodium hexametaphoshpate and PPK was constructed successfully.Based on the ATP synthesis system,a fermentation process with hexametaphoshpate and aspartic acid coupling added was developed which further improved cAMP production.(3)Adding auxiliary energy can effectively solve the insufficient energy supply problem commonly existed in cAMP fermentation process.The energy weakened culture condition was simulated successfully by adding 15 mg/L rotenone at 36 h,then the attempt with auxiliary energy substance added at appropriate conditions was conducted in 7 L fermentor.In addition,researches on fermentation kinetics,cell viability,energy metabolism and enzyme activities were carried out to explain the physiological mechanism.The results showed that rotenone addition reduced cell viability,ATP contents,and cAMP production to some extent which made insufficient energy supply problem more serious in cAMP fermentation,however,when sodium pyruvate and sodium hexametaphosphate were added,cell viability,ATP contents,and cAMP production were improved obviously compared with those of fermentation only with rotenone added,which were recovered to the levels of control.It provided practicable reference for solving insufficient energy problem caused by occasional cases.