Metabolic Characteristics Investiagtion And Technique Optimization Of High Erythromycin-producing Engineered Saccharopolyspora Erythraea Via¹³C Labeling Experiment

Erythromycin has broad spectrum of antibacterial activity,and its semi-synthetic drugs,such as azithromycin,roxithromycin and clarithromycin,are widely used in the treatment of diseases.As the demand for erythromycin continues to expand,competition between production enterprises is becoming more intense.To enhance erythromycin production level and reduce production cost,in-depth understanding of the metabolism of industrial strain and then making adaptation between culture condtions and cell metabolism is necessary.In our previous study,an industrial erythromycin-producing strain S.erythraea E3 was used as starting strain.By deleting the sucC（SACE₆669）gene encoding succinyl-CoA synthetase,a novel engineered strain E3-ΔsucC was constructed,but the knowledge about its metabolism was limited.This paper comprehensively investigated the metabolism of E3-ΔsucC from macroscopic and microscopic perspectives,and optimized the culture conditions to further enhance erythromycin level.The engineered strain E3-ΔsucC and the starting strain E3 were cultured in a 5-L bioreactor using a synthetic medium,respectively.The experimental results showed that the growth rate of E3-ΔsucC significantly increased in 0-50 hours.During the erythromycin synthesis period,the erythromycin level in E3-ΔsucC increased from 392.93 mg/L to 754.56 mg/L,which was nearly doubled,indicating that the knock-out of succinyl-CoA synthetase gene was beneficial to erythromycin biosynthesis.13C isotope labeling experiments were performed in a 250 mL bioreactor.The concentrations and isotopic abundances of intracellular metabolites and the metabolic network model were used to calculate the central metabolic flux distribution.Metabolomic data showed that,during the erythromycin synthesis period,the phosphate saccharides in the upstream of glycolysis（EMP）pathway and most of the organic acids in the tricarboxylic acid（TCA）cycle showed a downward trend in both strains.However,most of the phosphates in the pentose phosphate（PP）pathway and propionyl-CoA and methylmalonyl-CoA,which are precursors of erythromycin synthesis,showed a rise in E3-ΔsucC and a decrease in E3.The metabolic flux analysis results showed that,compared with the starting strain E3,the flux of PP pathway in E3-ΔsucC increased significantly,the flux of TCA increased by 18%.The most significant difference located in the TCA cycle.The flux of the metabolic reaction catalyzed by succinyl-CoA synthetase in E3-ΔsucC was almost zero,while the glyoxylate bypass flux significantly increased.These results indicated that the knock-out of succinyl-CoA synthetase gene effectively reduced the conversion of succinyl-CoA to succinic acid,and more succinyl-CoA was directed to methylmalonyl-CoA,which was beneficial to erythromycin biosynthesis.During the culture process of E3-ΔsucC,the cells were found to be autolyzed prematurely in the late fermentation stage.According to this phenomenon,the culture technique was optimized.By adding ammonium sulfate during erythromycin synthesis period,the erythromycin level improved by 43%,and the proportion of erythromycin active ingredient A increased from 85%to 98%.The chemometrics-based metabolic flux analysis results showed that the EMP pathway flux increased by 10%,the TCA cycle reaction flux increased by 5%,the PP pathway reaction flux decreased by 23%,and the glyoxylate cycle flux increased by 15%.The results of metabolic analysis of energy and cofactor showed that the total synthesis rate of NADH increased to 31.41 from 27.78 mmol/g DCW/day,and the ATP synthesis also increased,which played an important role in cell activity maintenance,while the NADPH synthesis rate decreased by 21.4%,whics was consistent with the low erythromycin synthesis rate.These results indicate that,by adding ammonium sulfate,although the erythromycin synthesis ability per unit cell reduced,it was still beneficial to improve erythromycin level due to high cell density.The research results in this paper have deepened the understanding of the metabolic characteristics of industrial erythromycin-producing strains,and have important significance for accelerating the industrial application of engineered strain.