Establishment And Application Of Isotopic Abundance Determination Method For Intracellular Metabolites Of Saccharopolyspora Erythraea

Saccharopolyspora erythraea（S.erythraea）is a widely used strain for industrial production of erythromycin.Precise determination of the ¹³C isotopic abundance of the intracellular metabolites is the prerequisite for deep investigation of the cell metabolism,which is crucial for comprehensively understanding the regulation mechanism of the cell metabolism,for guiding production technique optimization and novel strain construction.The thick cell wall,the narrow and long bacteria of S.erythraea hinders reliable harvest of the intracellular metabolites.In this paper,three extracting methods（liquid nitrogen grinding,liquid nitrogen freezing and thrawing,and boiling ethanol extraction）were evaluated for obtaining three types of intracellular metabolites.We found that boiling ethanol extraction was best for carboxylic acid,and liquid nitrogen grinding was best for phosphate sugars and CoA.The intracellular metabolites have the characteristic of multi category,fast turnover and low concentration.The introduction of isotope atoms leads to difficulties in accurately measuring the isotopic abundances.Based on ultra-high performance liquid chromatography（UPLC）-triple quadrupole mass spectrometry,we established a methods for measuring the ¹³C isotopic abundances of the intracellular metabolites of S.erythraea.We optimized the chromatographic condition of UPLC and the appropriate and unique tube lens voltage,collision energy and ion pairs of MS.The effects of different types of MS detecting methods were evaluated.The results showed that for the metabolites without ¹³C in daughter ions,like phosphate sugar,conventional "one to one" method meets the requirements;for the metabolites with short carbon chains containing ¹³C in daughter ions,like carboxylic acid,"one to many" method is better;for the metabolites with long carbon chains containing ¹³C in daughter ions,like CoA,SIM method is better.It was generally believed that n-propanol only served as a precursor for erythromycin synthesis.In this paper,the metabolic fate of n-propanol was inspected using the above methods.The dynamic changes of the isotopic abundances of carboxylic acids,phosphates and CoAs in the erythromycin fermentation were determined by adding isotope labeled sodium propionate into medium.The results showed that sodium propionate can be converted to succinyl-CoA,then be oxidized to carbon dioxide through TCA cycle.This unexpected fate not only wastes the high-value n-propanol,but also may limit the supply of erythromycin precursor,thus reducing the level of erythromycin production.The methods established in this paper help to deeply investigate the regulation mechanism of S.erythraea metabolism,to guide the production technique optimization and novel strain construction for achieving efficient production of erythromycin.