定量代谢物组学研究硫酸铵添加对高产红色糖多孢菌生理代谢的影响

Impacts of Ammonium Sulfate Addition on Physiological Metabolism of High-yielding Saccharopolyspora erythraea Based on Quantitative Metabonomics

为了考察添加硫酸铵对高产红色糖多孢菌生理代谢的影响,首先研究了硫酸铵添加对菌体量、红霉素效价、比葡萄糖消耗速率、比红霉素合成速率等表观生理参数的影响;并进一步采用^(13)C辅助的定量代谢物组学初步探索其调控机理,对比了硫酸铵添加后胞内有机酸、氨基酸等代谢物水平的变化情况。结果表明:在红霉素发酵后期的培养基中添加硫酸铵后,比红霉素合成速率和比葡萄糖消耗速率分别比对照组提高了30.0%、47.2%,表明菌体代谢活性得到提高。与对照组相比,红霉素效价从830μg/mL提高至1126μg/mL,红霉素A组分的比例从802.9μg/mL提高到956.1μg/mL,红霉素C的转化率在96 h达到99.2%。定量代谢物组学结果表明添加硫酸铵后胞内有机酸水平差异明显,其中α-酮戊二酸浓度的降低(4.15μmol/g DCW到1.05μmol/g DCW)有利于菌体生长维持。胞内菌体合成前体类和红霉素合成前体类氨基酸均有所提高,其中脯氨酸、丝氨酸、天冬氨酸、异亮氨酸浓度分别提高了60.4%、90.0%、204.2%、106.7%。利用定量代谢物组学研究红霉素发酵过程中氮源对胞内中心碳代谢影响大大加深了对红霉素工业生产菌代谢特性的认识,为后续的工业化生产提供了一定的指导意义。

In order to investigate the effect of the addition of ammonium sulfate on the physiological metabolism of Saccharopolyspora erythraea,the effects of ammonium sulfate on dry cell weight,erythromycin titer,specific glucose consumption rate,specific erythromycin synthesis rate,and other apparent physiological parameters were first investigated.Furthermore,13 C-assisted quantitative metabolomics was used to explore its regulation mechanism,and the changes of intracellular organic acids levels and amino acids levels after ammonium sulfate addition were compared.The results showed that the addition of ammonium sulfate to the medium in the late stage of erythromycin fermentation increased the specific erythromycin synthesis rate and specific glucose consumption rate by 30.0%and 47.2%respectively,indicating that the metabolic activity of the stains was improved.Compared with the control group,the erythromycin titer increased from 830μg/mL to 1126μg/mL,the proportion of erythromycin A component increased from 802.9μg/mL to 956.1μg/mL,the conversion rate of erythromycin C reached 99.2%at 96 h.Quantitative metabolomics results showed that the levels of intracellular organic acids were significantly different after the addition of ammonium sulfate,and the decrease in the concentration ofα-ketoglutarate(4.15μmol/g DCW to 1.05μmol/g DCW)was conducive to the maintenance of cell growth.Intracellular amino acids of cell synthesis precursors and erythromycin synthesis precursor were improved,for instance,the concentration of proline,serine,aspartic acid,isoleucine was increased by 60.4%,90.0%,204.2%,106.7%,respectively.It greatly deepened the understanding of the metabolic characteristics of erythromycin industrial production strains by using quantitative metabolomics to study the effect of nitrogen sources on intracellular central carbon metabolism during the fermentation of erythromycin,and provided certain guiding significance for the subsequent industrial production.