| Erythromycin is a macrolide antibiotic produced by Saccharopolyspora erythraea.Because of its strong antibacterial activity and broad antibacterial spectrum,erythromycin is widely used in clinical applications.With the rapid development of molecular manipulation techniques,the molecular modification of erythromycin has attracted the attention of domestic and foreign researchers.A large number of erythromycin analogue drugs(azithromycin,clarithromycin,roxithromycin,etc.)enter the market and participate in clinical treatment.At present,some problems like low yield and high inpurity components are still encountered in erythromycin production area of our country,which seriously restricts the development of the erythromycin industry.In this study,the real-time dynamic optimizations of seed and fermentation medium were carried out to increase the yield and purity of erythromycin.In the medium optimization of seed,first of all,increase the proportion of glucose in the medium to reduce the pH at the later stage of seed tank,and to avoid the process of supplementing sugar in the fermentation process,to simplify the protocol Secondly,the addition of yeast powder to the medium resulted in a 17%increase in the PMV of the seed tank,a 50%increase in viscosity,and thus a high quality seed was obtained.In the medium and feeding technology optimization of genetically engineered strain,to solve the problem of low supplementation rate of corn steep liquor and n-propanol,the feed rates of these two substrates were increased in the new technology;Secondly,the proportion of glucose and soybean flour in the medium was adjusted.Because of the weak metabolic activity of the genetically engineered strain,it is necessary to increase glucose and reduce the proportion of soybean flour to sustain its metabolic function.Through the above optimization approach,the yield of erythromycin was increased by 30%,and the erythromycin A component approached to 11000 U/mL.In the medium optimization of industrial strain,first of all,the available organic nitrogen source yeast powder and corn steep liquor in the medium were adjusted,and the corn steep liquor and yeast powder were mixed and used in fermenter.Finally,the best concentration mix of corn steep liquor and yeast powder made the yield of erythromycin increase by 30%.And the erythromycin A component approached to 10000 U/mL.To explore the influence of nitrogen perturbations on the utilization of glucose and n-propanol by strain metabolic flux analysis was performed and compared under different conditions.The results of metabolic flux analysis showed that the flux of the pentose phosphate pathway under the condition with high erythromycin yield was increased.It indicated that pentose phosphate pathway of strain was strengthened under the condition of best combination of corn steep liquor and yeast powder.Furthermore,the proportion of NADPH involved in erythromycin synthesis was the highest in the total NADPH content.Thus,synthetic pathway leading to erythromycin had the highest flux. |
