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The Optimization Of Erythromycin Fermentation Process By Genetically Engineered Strain Based On Dynamic Regulation Of Utilization Of Carbon, Nitrogen And Phosphate Sources And Preliminary Exploration Of Its Metabolic Mechanism Using 13C Isotope

Posted on:2014-08-16Degree:DoctorType:Dissertation
Country:ChinaCandidate:Y ChenFull Text:PDF
GTID:1221330485995051Subject:Fermentation engineering
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At present, the problems of low erythromycin titer, the low content of active component erythromycin A and the high fermentation broth viscosity seriously influence the international competitive power in erythromycin market in our country, which are urgent to be solved. In this paper, we systematically investigated the process regulation and control strategies in erythromycin fermentation. The dynamic regulation and control approaches for erythromycin fermentation with genetically engineered strain were established based on the utilization of carbon, nitrogen and phosphate. With these newly established strategies, erythromycin A was effectively enhanced meanwhile the broth viscosity was significantly decreased.Firstly, the fluctuation of erythromycin production in industrial fermentation resulting from the quality instability of corn steep liquor was solved by completely substituting corn steep liquor with a new easily-metabolized organic nitrogen source. The optimal medium was successfully scaled up to 25-ton fermenter and the erythromycin production was increased by 21.7%as compared to the control. The analysis of fermentation data indicated that with new nitrogen source, the activities of amylase and proteinase were increased as well as OUR level. Through comparing the extracellular amino acids and organic acids concentration between the fermentation with the new organic nitrogen source and the fermentation with corn steep liquor, it was found that with the new organic nitrogen source, the concentrations of extracellular precursor amino acids(glutamate, alanine, methionine, isoleucine and leucine) for erythromycin synthesis were relatively high and the concentrations of extracellular organic acids(pyruvate, propionate, succinate, citrate and acetate) were relatively low. The analysis of the mycelial morphology and the broth rheology indicated that the new easily-metabolized organic nitrogen source increased the length of hyphae and then the consistency index K.Secondly, the dynamic substrate-feeding strategies for erythromycin fermentation with gene engineering strain were established based on the utilization relation between glucose and propanol. With the quantitative metabolic flux analysis method, metabolic distribution under the different ratios of glucose feed rate to propanol feed rate with complex medium was analyzed. According to the multi-parameter correlation analysis, it was proposed to feed glucose according to RQ and feed propanol based on the dissolved oxygen and residual propanol, by which erythromycin production was increased by 8.3%. Furthermore, erythromycin titer was reached to 12491U/ml by dynamic regulation of glucose and propanol feed based on the equal sum of carbon atoms. The macro-metabolic analysis revealed that propionyl-CoA (-2.147mmol/(g-day)) and methylmalonyl-CoA (-1.708 mmol/(g-day)) were increased through modulating the glucose and propanol feed which led to the higher flux to erythromycin. It was also found that 45%-77% of the propanol went into the TCA cycle which provided the new clues for studying the metabolism of propanol with isotope labeling experiment and the strain improvement with metabolic engineering method. The energy metabolism analysis disclosed that the low ATP generation resulting from the low glucose feed rate almost did not affect erythromycin biosynthesis under the present existing condition and high NADPH should benefit erythromycin biosynthesis.Thirdly, through nitrogen and phosphate regulation, the broth viscosity and glucose consumption rate were significantly decreased. By controlling ammonium sulfate concentration, broth viscosity and glucose consumption were decreased by 18.2% and 61.6% respectively, whereas erythromycin biosynthesis was little affected.Furthermore, erythromycin A production was increased by 8.7% still with characteristics of low broth viscosity and glucose consumption through the rational regulations of phosphate, soybean meal and ammonium sulfate. It was elucidated that high concentration of ammonium sulfate decreased the release rate of organic phosphate in soybean meal and then led to the slow utilization of ammonium ion.The strain could consume the inorganic ammonium salt and the inorganic phosphate salt first for cell growth instead of organic nitrogen source. The pellets formation under the relatively high concentration of ammonium sulfate contributed much to the decrease of broth viscosity. The accumulation of extracellular propionate and succinate under the new regulation strategy indicated that higher propanol consumption might increase the concentration of methylmalonyl-CoA and propionyl-CoA and thus could increase the flux leading to erythromycin A. Ammonium nitrate could also decrease the broth viscosity and glucose feed rate, but the formation of pigment resulted in the low erythromycin production.Finally, it was found that the phosphate content in organic nitrogen exerted a significant impact on erythromycin fermentation. The soluble phosphate can be limited to a relatively low level by adopting low-phosphate-content nitrogen source. Feeding inorganic phosphate during exponential stage increased the OUR level and thus improved erythromycin biosynthesis. Hence, we proposed a method to control phosphate level, that is, to decrease the phosphate releasing rate from organic nitrogen and to increase phosphate level by feeding inorganic phosphate during exponential stage. By this method, erythromycin concentration was increased by 20.9%.In addition, the isotope labelling experiment with [1-13C] propionate sodium revealed that propanol flowed into pyruvate via succinate which supported the macro-metabolic analysis results. This finding rejected the hypothesis that propanol was only used for erythromycin precursor. It was found that proline was the key amino acid for S.erythraea growth during the selection of chemical defined medium. The isotope labeling experiment with [U-13C] glucose and natural labeled proline elucidated the pathways of proline metabolism. The proline went to TCA cycle via the formation of glutamate first and then pyruvate was formed afterwards. The building up of the isotope labelling experiment platform laid a solid foundation for further study of micro-metabolic mechanism for erythromycin biosynthesis regulation.
Keywords/Search Tags:Erythromycin, process optimization, gene engineering strain, feeding strategy, micro-metabolism
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