The Effect Of Ammonium Sulfate On The Synthesis Of Erythromycin Based On Metabolomics

Erythromycin,as a broad-spectrum macrolide antibiotic,has important application value in the treatment of diseases.Genetic engineering technology was used to modify industrial erythromycin-producing strains to obtain high performance recombinant strains.The establishment of appropriate culture technology is great significance for the recombinant strains to increase erythromycin level and reduce the cost.In our previous study,an industrial erythromycin-producing strain Saccharopolyspora erythraea（S.erythraea）E3 was used as original strain.By deleting the sucC（SACE₆669）gene encoding succinyl-CoA synthetase,a novel engineered strain E3-ΔsucC was constructed.In this study,the metabolic characteristics of the engineered strains were investigated to find the key technological regulation affecting erythromycin level and optimize the culture process.Finally,the mechanism of the process regulation was preliminarily expounded through metabonomics and metabolic flux analysis.The original strain E3 and engineered bacteria E3-ΔsucC were cultured by using synthetic medium and original culture technology simultaneously.The results showed that the erythromycin level and specific synthesis rate of erythromycin were 651.1 μg/mL and 0.005 mmol/gDCW/h,respectively,were 76.9%and 117.0%higher than the original strain.These indicated that the knockout gene sucC was beneficial to erythromycin synthesis.At the same time,it was found that,the specific growth rate,the specific glucose consumption rate and the specific amino nitrogen consumption rate of the engineered strain were higher than the original strain in the growth period.Thus,nutrients were consumed too early and there was autolysis in the late fermentation,which was not conducive to the production of erythromycin.It was found that adding ammonium sulfate at the late stage of fermentation could not only avoid autolysis of engineered strain,maintain the biomass,but also increase the specific synthesis rate of erythromycin,through fermentation transfer experiment.Therefore,three different ammonium sulfate feeding rates were investigated.When the feeding rate was 0.02 g/L/h,the level and specific rate of erythromycin were 1311.1 μg/mL and 0.008 mmol/gDCW/h,were 101.3%and 41.0%higher than the original culture process,and the proportion of erythromycin A increased from 83.2%to 99.5%.The results of non-targeted metabolome analysis showed that the content of some nitrogenous compounds increased significantly,such as glutamate,S-adenosine L-methionine and S-adenosine homocysteine,while the content of some sugars and organic acids decreased obviously,such as D-ribose-5-phosphate,citric acid and oxaloacetic acid after adding ammonium sulfate.Targeted metabonomics analysis showed that the intracellular amino acid metabolism pool increased after adding ammonium sulfate,which ensured the supply of precursor of organic acid and Co-A,and provided sufficient precursor for the maintenance of bacteria and the synthesis of erythromycin.The results of metabolic flux analysis showed that the metabolic flux was increased under the three different ammonium sulfate feeding rates.When the rate was 0.02 g/L/h,the reaction flux through EMP pathway increased by 55.7%,and the reaction flux through TCA cycle increased by 67.5%,indicated that the growth activity of strain was improved.Moreover,the reaction flux of PP pathway and erythromycin synthesis increased by 55.5%,and the synthesis rate of NADPH increased by 48.1%,which was conducive to improving the synthesis rate of erythromycin.This study deepened the understanding of the metabolic characteristics of industrial erythromycin-producing strains and the influencing mechanism of ammonium sulfate,which was of great significance to accelerate the industrial application of genetically engineered strain.